Or Gozani

All Publications

• Epigenetics and beyond: targeting writers of protein lysine methylation to treat disease. Nature reviews. Drug discovery Bhat, K. P., Ümit Kaniskan, H. n., Jin, J. n., Gozani, O. n. 2021

  Abstract

  Protein lysine methylation is a crucial post-translational modification that regulates the functions of both histone and non-histone proteins. Deregulation of the enzymes or 'writers' of protein lysine methylation, lysine methyltransferases (KMTs), is implicated in the cause of many diseases, including cancer, mental health disorders and developmental disorders. Over the past decade, significant advances have been made in developing drugs to target KMTs that are involved in histone methylation and epigenetic regulation. The first of these inhibitors, tazemetostat, was recently approved for the treatment of epithelioid sarcoma and follicular lymphoma, and several more are in clinical and preclinical evaluation. Beyond chromatin, the many KMTs that regulate protein synthesis and other fundamental biological processes are emerging as promising new targets for drug development to treat diverse diseases.

  View details for DOI 10.1038/s41573-020-00108-x

  View details for PubMedID 33469207

• Elevated NSD3 histone methylation activity drives squamous cell lung cancer. Nature Yuan, G. n., Flores, N. M., Hausmann, S. n., Lofgren, S. M., Kharchenko, V. n., Angulo-Ibanez, M. n., Sengupta, D. n., Lu, X. n., Czaban, I. n., Azhibek, D. n., Vicent, S. n., Fischle, W. n., Jaremko, M. n., Fang, B. n., Wistuba, I. I., Chua, K. F., Roth, J. A., Minna, J. D., Shao, N. Y., Jaremko, Ł. n., Mazur, P. K., Gozani, O. n. 2021

  Abstract

  Amplification of chromosomal region 8p11-12 is a common genetic alteration that has been implicated in the aetiology of lung squamous cell carcinoma (LUSC)1-3. The FGFR1 gene is the main candidate driver of tumorigenesis within this region4. However, clinical trials evaluating FGFR1 inhibition as a targeted therapy have been unsuccessful5. Here we identify the histone H3 lysine 36 (H3K36) methyltransferase NSD3, the gene for which is located in the 8p11-12 amplicon, as a key regulator of LUSC tumorigenesis. In contrast to other 8p11-12 candidate LUSC drivers, increased expression of NSD3 correlated strongly with its gene amplification. Ablation of NSD3, but not of FGFR1, attenuated tumour growth and extended survival in a mouse model of LUSC. We identify an LUSC-associated variant NSD3(T1232A) that shows increased catalytic activity for dimethylation of H3K36 (H3K36me2) in vitro and in vivo. Structural dynamic analyses revealed that the T1232A substitution elicited localized mobility changes throughout the catalytic domain of NSD3 to relieve auto-inhibition and to increase accessibility of the H3 substrate. Expression of NSD3(T1232A) in vivo accelerated tumorigenesis and decreased overall survival in mouse models of LUSC. Pathological generation of H3K36me2 by NSD3(T1232A) reprograms the chromatin landscape to promote oncogenic gene expression signatures. Furthermore, NSD3, in a manner dependent on its catalytic activity, promoted transformation in human tracheobronchial cells and growth of xenografted human LUSC cell lines with amplification of 8p11-12. Depletion of NSD3 in patient-derived xenografts from primary LUSCs containing NSD3 amplification or the NSD3(T1232A)-encoding variant attenuated neoplastic growth in mice. Finally, NSD3-regulated LUSC-derived xenografts were hypersensitive to bromodomain inhibition. Thus, our work identifies NSD3 as a principal 8p11-12 amplicon-associated oncogenic driver in LUSC, and suggests that NSD3-dependency renders LUSC therapeutically vulnerable to bromodomain inhibition.

  View details for DOI 10.1038/s41586-020-03170-y

  View details for PubMedID 33536620

• Molecular basis of nucleosomal H3K36 methylation by NSD methyltransferases. Nature Li, W., Tian, W., Yuan, G., Deng, P., Sengupta, D., Cheng, Z., Cao, Y., Ren, J., Qin, Y., Zhou, Y., Jia, Y., Gozani, O., Patel, D. J., Wang, Z. 2020

  Abstract

  Histone methyltransferases of the nuclear receptor-binding SET domain protein (NSD) family, including NSD1, NSD2 and NSD3, have crucial roles in chromatin regulation and are implicated in oncogenesis1,2. NSD enzymes exhibit an autoinhibitory state that is relieved by binding to nucleosomes, enabling dimethylation of histone H3 at Lys36 (H3K36)3-7. However, the molecular basis that underlies this mechanism is largely unknown. Here we solve the cryo-electron microscopy structures of NSD2 and NSD3 bound to mononucleosomes. We find that binding of NSD2 and NSD3 to mononucleosomes causes DNA near the linker region to unwrap, which facilitates insertion of the catalytic core between the histone octamer and the unwrapped segment of DNA. A network of DNA- and histone-specific contacts between NSD2 or NSD3 and the nucleosome precisely defines the position of the enzyme on the nucleosome, explaining the specificity of methylation to H3K36. Intermolecular contacts between NSD proteins and nucleosomes are altered by several recurrent cancer-associated mutations in NSD2 and NSD3. NSDs that contain these mutations are catalytically hyperactive in vitro and in cells, and their ectopic expression promotes the proliferation of cancer cells and the growth of xenograft tumours. Together, our research provides molecular insights into the nucleosome-based recognition and histone-modification mechanisms of NSD2 and NSD3, which could lead to strategies for therapeutic targeting of proteins of the NSD family.

  View details for DOI 10.1038/s41586-020-03069-8

  View details for PubMedID 33361816

• SETD5-Coordinated Chromatin Reprogramming Regulates Adaptive Resistance to Targeted Pancreatic Cancer Therapy. Cancer cell Wang, Z. n., Hausmann, S. n., Lyu, R. n., Li, T. M., Lofgren, S. M., Flores, N. M., Fuentes, M. E., Caporicci, M. n., Yang, Z. n., Meiners, M. J., Cheek, M. A., Howard, S. A., Zhang, L. n., Elias, J. E., Kim, M. P., Maitra, A. n., Wang, H. n., Bassik, M. C., Keogh, M. C., Sage, J. n., Gozani, O. n., Mazur, P. K. 2020

  Abstract

  Molecular mechanisms underlying adaptive targeted therapy resistance in pancreatic ductal adenocarcinoma (PDAC) are poorly understood. Here, we identify SETD5 as a major driver of PDAC resistance to MEK1/2 inhibition (MEKi). SETD5 is induced by MEKi resistance and its deletion restores refractory PDAC vulnerability to MEKi therapy in mouse models and patient-derived xenografts. SETD5 lacks histone methyltransferase activity but scaffolds a co-repressor complex, including HDAC3 and G9a. Gene silencing by the SETD5 complex regulates known drug resistance pathways to reprogram cellular responses to MEKi. Pharmacological co-targeting of MEK1/2, HDAC3, and G9a sustains PDAC tumor growth inhibition in vivo. Our work uncovers SETD5 as a key mediator of acquired MEKi therapy resistance in PDAC and suggests a context for advancing MEKi use in the clinic.

  View details for DOI 10.1016/j.ccell.2020.04.014

  View details for PubMedID 32442403

• METTL13 Methylation of eEF1A Increases Translational Output to Promote Tumorigenesis CELL Liu, S., Hausmann, S., Carlson, S., Fuentes, M., Francis, J., Pillai, R., Lofgren, S., Hulea, L., Tandoc, K., Lu, J., Li, A., Nicholas Dang Nguyen, Caporicci, M., Kim, M., Maitra, A., Wang, H., Wistuba, I., Porco, J., Bassik, M., Elias, J., Song, J., Topisirovic, I., Van Rechem, C., Mazur, P., Gozani, O. 2019; 176 (3): 491-+

  View details for DOI 10.1016/j.cell.2018.11.038

  View details for Web of Science ID 000456526100011

• SETD3 is an actin histidine methyltransferase that prevents primary dystocia NATURE Wilkinson, A. W., Diep, J., Dai, S., Liu, S., Ooi, Y., Song, D., Li, T., Horton, J. R., Zhang, X., Liu, C., Trivedi, D. V., Ruppel, K. M., Vilches-Moure, J. G., Casey, K. M., Mak, J., Cowan, T., Elias, J. E., Nagamine, C. M., Spudich, J. A., Cheng, X., Carette, J. E., Gozani, O. 2019; 565 (7739): 372-+

  View details for DOI 10.1038/s41586-018-0821-8

  View details for Web of Science ID 000455781600044

• Enterovirus pathogenesis requires the host methyltransferase SETD3. Nature microbiology Diep, J. n., Ooi, Y. S., Wilkinson, A. W., Peters, C. E., Foy, E. n., Johnson, J. R., Zengel, J. n., Ding, S. n., Weng, K. F., Laufman, O. n., Jang, G. n., Xu, J. n., Young, T. n., Verschueren, E. n., Kobluk, K. J., Elias, J. E., Sarnow, P. n., Greenberg, H. B., Hüttenhain, R. n., Nagamine, C. M., Andino, R. n., Krogan, N. J., Gozani, O. n., Carette, J. E. 2019

  Abstract

  Enteroviruses (EVs) comprise a large genus of positive-sense, single-stranded RNA viruses whose members cause a number of important and widespread human diseases, including poliomyelitis, myocarditis, acute flaccid myelitis and the common cold. How EVs co-opt cellular functions to promote replication and spread is incompletely understood. Here, using genome-scale CRISPR screens, we identify the actin histidine methyltransferase SET domain containing 3 (SETD3) as critically important for viral infection by a broad panel of EVs, including rhinoviruses and non-polio EVs increasingly linked to severe neurological disease such as acute flaccid myelitis (EV-D68) and viral encephalitis (EV-A71). We show that cytosolic SETD3, independent of its methylation activity, is required for the RNA replication step in the viral life cycle. Using quantitative affinity purification-mass spectrometry, we show that SETD3 specifically interacts with the viral 2A protease of multiple enteroviral species, and we map the residues in 2A that mediate this interaction. 2A mutants that retain protease activity but are unable to interact with SETD3 are severely compromised in RNA replication. These data suggest a role of the viral 2A protein in RNA replication beyond facilitating proteolytic cleavage. Finally, we show that SETD3 is essential for in vivo replication and pathogenesis in multiple mouse models for EV infection, including CV-A10, EV-A71 and EV-D68. Our results reveal a crucial role of a host protein in viral pathogenesis, and suggest targeting SETD3 as a potential mechanism for controlling viral infections.

  View details for DOI 10.1038/s41564-019-0551-1

  View details for PubMedID 31527793

• Histone lysine methyltransferases in biology and disease. Nature structural & molecular biology Husmann, D. n., Gozani, O. n. 2019; 26 (10): 880–89

  Abstract

  The precise temporal and spatial coordination of histone lysine methylation dynamics across the epigenome regulates virtually all DNA-templated processes. A large number of histone lysine methyltransferase (KMT) enzymes catalyze the various lysine methylation events decorating the core histone proteins. Mutations, genetic translocations and altered gene expression involving these KMTs are frequently observed in cancer, developmental disorders and other pathologies. Therapeutic compounds targeting specific KMTs are currently being tested in the clinic, although overall drug discovery in the field is relatively underdeveloped. Here we review the biochemical and biological activities of histone KMTs and their connections to human diseases, focusing on cancer. We also discuss the scientific and clinical challenges and opportunities in studying KMTs.

  View details for DOI 10.1038/s41594-019-0298-7

  View details for PubMedID 31582846

• The PZP Domain of AF10 Senses Unmodified H3K27 to Regulate DOT1L-Mediated Methylation of H3K79. Molecular cell Chen, S., Yang, Z., Wilkinson, A. W., Deshpande, A. J., Sidoli, S., Krajewski, K., Strahl, B. D., Garcia, B. A., Armstrong, S. A., Patel, D. J., Gozani, O. 2015; 60 (2): 319-327

  Abstract

  AF10, a DOT1L cofactor, is required for H3K79 methylation and cooperates with DOT1L in leukemogenesis. However, the molecular mechanism by which AF10 regulates DOT1L-mediated H3K79 methylation is not clear. Here we report that AF10 contains a "reader" domain that couples unmodified H3K27 recognition to H3K79 methylation. An AF10 region consisting of a PHD finger-Zn knuckle-PHD finger (PZP) folds into a single module that recognizes amino acids 22-27 of H3, and this interaction is abrogated by H3K27 modification. Structural studies reveal that H3 binding triggers rearrangement of the PZP module to form an H3(22-27)-accommodating channel and that the unmodified H3K27 side chain is encased in a compact hydrogen-bond acceptor-lined cage. In cells, PZP recognition of H3 is required for H3K79 dimethylation, expression of DOT1L-target genes, and proliferation of DOT1L-addicted leukemic cells. Together, our results uncover a pivotal role for H3K27-via readout by the AF10 PZP domain-in regulating the cancer-associated enzyme DOT1L.

  View details for DOI 10.1016/j.molcel.2015.08.019

  View details for PubMedID 26439302

  View details for PubMedCentralID PMC4609290

• SMYD3 links lysine methylation of MAP3K2 to Ras-driven cancer. Nature Mazur, P. K., Reynoird, N., Khatri, P., Jansen, P. W., Wilkinson, A. W., Liu, S., Barbash, O., Van Aller, G. S., Huddleston, M., Dhanak, D., Tummino, P. J., Kruger, R. G., Garcia, B. A., Butte, A. J., Vermeulen, M., Sage, J., Gozani, O. 2014; 510 (7504): 283-287

  Abstract

  Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling.

  View details for DOI 10.1038/nature13320

  View details for PubMedID 24847881

• A general molecular affinity strategy for global detection and proteomic analysis of lysine methylation. Molecular cell Moore, K. E., Carlson, S. M., Camp, N. D., Cheung, P., James, R. G., Chua, K. F., Wolf-Yadlin, A., Gozani, O. 2013; 50 (3): 444-456

  Abstract

  Lysine methylation of histone proteins regulates chromatin dynamics and plays important roles in diverse physiological and pathological processes. However, beyond histone proteins, the proteome-wide extent of lysine methylation remains largely unknown. We have engineered the naturally occurring MBT domain repeats of L3MBTL1 to serve as a universal affinity reagent for detecting, enriching, and identifying proteins carrying a mono- or dimethylated lysine. The domain is broadly specific for methylated lysine ("pan-specific") and can be applied to any biological system. We have used our approach to demonstrate that SIRT1 is a substrate of the methyltransferase G9a both in vitro and in cells, to perform proteome-wide detection and enrichment of methylated proteins, and to identify candidate in-cell substrates of G9a and the related methyltransferase GLP. Together, our results demonstrate a powerful new approach for global and quantitative analysis of methylated lysine, and they represent the first systems biology understanding of lysine methylation.

  View details for DOI 10.1016/j.molcel.2013.03.005

  View details for PubMedID 23583077

  View details for PubMedCentralID PMC3660009

• The BAH domain of ORC1 links H4K20me2 to DNA replication licensing and Meier-Gorlin syndrome NATURE Kuo, A. J., Song, J., Cheung, P., Ishibe-Murakami, S., Yamazoe, S., Chen, J. K., Patel, D. J., Gozani, O. 2012; 484 (7392): 115-?

  Abstract

  The recognition of distinctly modified histones by specialized 'effector' proteins constitutes a key mechanism for transducing molecular events at chromatin to biological outcomes. Effector proteins influence DNA-templated processes, including transcription, DNA recombination and DNA repair; however, no effector functions have yet been identified within the mammalian machinery that regulate DNA replication. Here we show that ORC1--a component of ORC (origin of replication complex), which mediates pre-DNA replication licensing--contains a bromo adjacent homology (BAH) domain that specifically recognizes histone H4 dimethylated at lysine 20 (H4K20me2). Recognition of H4K20me2 is a property common to BAH domains present within diverse metazoan ORC1 proteins. Structural studies reveal that the specificity of the BAH domain for H4K20me2 is mediated by a dynamic aromatic dimethyl-lysine-binding cage and multiple intermolecular contacts involving the bound peptide. H4K20me2 is enriched at replication origins, and abrogating ORC1 recognition of H4K20me2 in cells impairs ORC1 occupancy at replication origins, ORC chromatin loading and cell-cycle progression. Mutation of the ORC1 BAH domain has been implicated in the aetiology of Meier-Gorlin syndrome (MGS), a form of primordial dwarfism, and ORC1 depletion in zebrafish results in an MGS-like phenotype. We find that wild-type human ORC1, but not ORC1-H4K20me2-binding mutants, rescues the growth retardation of orc1 morphants. Moreover, zebrafish depleted of H4K20me2 have diminished body size, mirroring the phenotype of orc1 morphants. Together, our results identify the BAH domain as a novel methyl-lysine-binding module, thereby establishing the first direct link between histone methylation and the metazoan DNA replication machinery, and defining a pivotal aetiological role for the canonical H4K20me2 mark, via ORC1, in primordial dwarfism.

  View details for DOI 10.1038/nature10956

  View details for Web of Science ID 000302343400045

  View details for PubMedID 22398447

  View details for PubMedCentralID PMC3321094

• Methylation of H4 lysines 5, 8 and 12 by yeast Set5 calibrates chromatin stress responses NATURE STRUCTURAL & MOLECULAR BIOLOGY Green, E. M., Mas, G., Young, N. L., Garcia, B. A., Gozani, O. 2012; 19 (3): 361-363

  Abstract

  Methylation of histones is central to chromatin regulation, and thus previously unknown mechanisms regulating genome function can be revealed through the discovery of new histone methyl marks. Here we identify Set5 as the first histone H4 methyltransferase, which monomethylates the critical H4 lysine residues 5, 8 and 12 in budding yeast. Set5's enzymatic activity functions together with the global chromatin-modifying complexes COMPASS and NuA4 to regulate cell growth and stress responses.

  View details for DOI 10.1038/nsmb.2252

  View details for Web of Science ID 000301181900015

  View details for PubMedID 22343720

  View details for PubMedCentralID PMC3334815

• NSD2 Links Dimethylation of Histone H3 at Lysine 36 to Oncogenic Programming MOLECULAR CELL Kuo, A. J., Cheung, P., Chen, K., Zee, B. M., Kioi, M., Lauring, J., Xi, Y., Park, B. H., Shi, X., Garcia, B. A., Li, W., Gozani, O. 2011; 44 (4): 609-620

  Abstract

  The histone lysine methyltransferase NSD2 (MMSET/WHSC1) is implicated in diverse diseases and commonly overexpressed in multiple myeloma due to a recurrent t(4;14) chromosomal translocation. However, the precise catalytic activity of NSD2 is obscure, preventing progress in understanding how this enzyme influences chromatin biology and myeloma pathogenesis. Here, we show that dimethylation of histone H3 at lysine 36 (H3K36me2) is the principal chromatin-regulatory activity of NSD2. Catalysis of H3K36me2 by NSD2 is sufficient for gene activation. In t(4;14)-positive myeloma cells, the normal genome-wide and gene-specific distribution of H3K36me2 is obliterated, creating a chromatin landscape that selects for a transcription profile favorable for myelomagenesis. Catalytically active NSD2 confers xenograft tumor formation upon t(4;14)-negative cells and promotes oncogenic transformation of primary cells in an H3K36me2-dependent manner. Together, our findings establish H3K36me2 as the primary product generated by NSD2 and demonstrate that genomic disorganization of this canonical chromatin mark by NSD2 initiates oncogenic programming.

  View details for DOI 10.1016/j.molcel.2011.08.042

  View details for Web of Science ID 000297387800012

  View details for PubMedID 22099308

  View details for PubMedCentralID PMC3222870

• Lysine methylation of the NF-kappa B subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-kappa B signaling NATURE IMMUNOLOGY Levy, D., Kuo, A. J., Chang, Y., Schaefer, U., Kitson, C., Cheung, P., Espejo, A., Zee, B. M., Liu, C. L., Tangsombatvisit, S., Tennen, R. I., Kuo, A. Y., Tanjing, S., Cheung, R., Chua, K. F., Utz, P. J., Shi, X., Prinjha, R. K., Lee, K., Garcia, B. A., Bedford, M. T., Tarakhovsky, A., Cheng, X., Gozani, O. 2011; 12 (1): 29-U47

  Abstract

  Signaling via the methylation of lysine residues in proteins has been linked to diverse biological and disease processes, yet the catalytic activity and substrate specificity of many human protein lysine methyltransferases (PKMTs) are unknown. We screened over 40 candidate PKMTs and identified SETD6 as a methyltransferase that monomethylated chromatin-associated transcription factor NF-κB subunit RelA at Lys310 (RelAK310me1). SETD6-mediated methylation rendered RelA inert and attenuated RelA-driven transcriptional programs, including inflammatory responses in primary immune cells. RelAK310me1 was recognized by the ankryin repeat of the histone methyltransferase GLP, which under basal conditions promoted a repressed chromatin state at RelA target genes through GLP-mediated methylation of histone H3 Lys9 (H3K9). NF-κB-activation-linked phosphorylation of RelA at Ser311 by protein kinase C-ζ (PKC-ζ) blocked the binding of GLP to RelAK310me1 and relieved repression of the target gene. Our findings establish a previously uncharacterized mechanism by which chromatin signaling regulates inflammation programs.

  View details for DOI 10.1038/ni.1968

  View details for Web of Science ID 000285465100010

  View details for PubMedID 21131967

  View details for PubMedCentralID PMC3074206

• ING4 Mediates Crosstalk between Histone H3 K4 Trimethylation and H3 Acetylation to Attenuate Cellular Transformation MOLECULAR CELL Hung, T., Binda, O., Champagne, K. S., Kuo, A. J., Johnson, K., Chang, H. Y., Simon, M. D., Kutateladze, T. G., Gozani, O. 2009; 33 (2): 248-256

  Abstract

  Aberrations in chromatin dynamics play a fundamental role in tumorigenesis, yet relatively little is known of the molecular mechanisms linking histone lysine methylation to neoplastic disease. ING4 (Inhibitor of Growth 4) is a native subunit of an HBO1 histone acetyltransferase (HAT) complex and a tumor suppressor protein. Here we show a critical role for specific recognition of histone H3 trimethylated at lysine 4 (H3K4me3) by the ING4 PHD finger in mediating ING4 gene expression and tumor suppressor functions. The interaction between ING4 and H3K4me3 augments HBO1 acetylation activity on H3 tails and drives H3 acetylation at ING4 target promoters. Further, ING4 facilitates apoptosis in response to genotoxic stress and inhibits anchorage-independent cell growth, and these functions depend on ING4 interactions with H3K4me3. Together, our results demonstrate a mechanism for brokering crosstalk between H3K4 methylation and H3 acetylation and reveal a molecular link between chromatin modulation and tumor suppressor mechanisms.

  View details for DOI 10.1016/j.molcel.2008.12.016

  View details for PubMedID 19187765

• RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination NATURE Matthews, A. G., Kuo, A. J., Ramon-Maiques, S., Han, S., Champagne, K. S., Ivanov, D., Gallardo, M., Carney, D., Cheung, P., Ciccone, D. N., Walter, K. L., Utz, P. J., Shi, Y., Kutateladze, T. G., Yang, W., Gozani, O., Oettinger, M. A. 2007; 450 (7172): 1106-U18

  Abstract

  Nuclear processes such as transcription, DNA replication and recombination are dynamically regulated by chromatin structure. Eukaryotic transcription is known to be regulated by chromatin-associated proteins containing conserved protein domains that specifically recognize distinct covalent post-translational modifications on histones. However, it has been unclear whether similar mechanisms are involved in mammalian DNA recombination. Here we show that RAG2--an essential component of the RAG1/2 V(D)J recombinase, which mediates antigen-receptor gene assembly--contains a plant homeodomain (PHD) finger that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3). The high-resolution crystal structure of the mouse RAG2 PHD finger bound to H3K4me3 reveals the molecular basis of H3K4me3-recognition by RAG2. Mutations that abrogate RAG2's recognition of H3K4me3 severely impair V(D)J recombination in vivo. Reducing the level of H3K4me3 similarly leads to a decrease in V(D)J recombination in vivo. Notably, a conserved tryptophan residue (W453) that constitutes a key structural component of the K4me3-binding surface and is essential for RAG2's recognition of H3K4me3 is mutated in patients with immunodeficiency syndromes. Together, our results identify a new function for histone methylation in mammalian DNA recombination. Furthermore, our results provide the first evidence indicating that disrupting the read-out of histone modifications can cause an inherited human disease.

  View details for DOI 10.1038/nature06431

  View details for Web of Science ID 000251579900092

  View details for PubMedID 18033247

  View details for PubMedCentralID PMC2988437

• Modulation of p53 function by SET8-mediated methylation at lysine 382 MOLECULAR CELL Shi, X., Kachirskaia, L., Yamaguchi, H., West, L. E., Wen, H., Wang, E. W., Dutta, S., Appella, E., Gozani, O. 2007; 27 (4): 636-646

  Abstract

  Reversible covalent methylation of lysine residues on histone proteins constitutes a principal molecular mechanism that links chromatin states to diverse biological outcomes. Recently, lysine methylation has been observed on nonhistone proteins, suggesting broad cellular roles for the enzymes generating and removing methyl moieties. Here we report that the lysine methyltransferase enzyme SET8/PR-Set7 regulates the tumor suppressor protein p53. We find that SET8 specifically monomethylates p53 at lysine 382 (p53K382me1). This methylation event robustly suppresses p53-mediated transcription activation of highly responsive target genes but has little influence on weak targets. Further, depletion of SET8 augments the proapoptotic and checkpoint activation functions of p53, and accordingly, SET8 expression is downregulated upon DNA damage. Together, our study identifies SET8 as a p53-modifying enzyme, identifies p53K382me1 as a regulatory posttranslational modification of p53, and begins to dissect how methylation may contribute to a dynamic posttranslational code that modulates distinct p53 functions.

  View details for DOI 10.1016/j.molcel.2007.07.012

  View details for Web of Science ID 000249050200014

  View details for PubMedID 17707234

  View details for PubMedCentralID PMC2693209

• ING2 PHD domain links histone H3 lysine 4 methylation to active gene repression NATURE Shi, X., Hong, T., Walter, K. L., Ewalt, M., Michishita, E., Hung, T., Carney, D., Pena, P., Lan, F., Kaadige, M. R., Lacoste, N., Cayrou, C., Davrazou, F., Saha, A., Cairns, B. R., Ayer, D. E., Kutateladze, T. G., Shi, Y., Cote, J., Chua, K. F., Gozani, O. 2006; 442 (7098): 96-99

  Abstract

  Dynamic regulation of diverse nuclear processes is intimately linked to covalent modifications of chromatin. Much attention has focused on methylation at lysine 4 of histone H3 (H3K4), owing to its association with euchromatic genomic regions. H3K4 can be mono-, di- or tri-methylated. Trimethylated H3K4 (H3K4me3) is preferentially detected at active genes, and is proposed to promote gene expression through recognition by transcription-activating effector molecules. Here we identify a novel class of methylated H3K4 effector domains--the PHD domains of the ING (for inhibitor of growth) family of tumour suppressor proteins. The ING PHD domains are specific and highly robust binding modules for H3K4me3 and H3K4me2. ING2, a native subunit of a repressive mSin3a-HDAC1 histone deacetylase complex, binds with high affinity to the trimethylated species. In response to DNA damage, recognition of H3K4me3 by the ING2 PHD domain stabilizes the mSin3a-HDAC1 complex at the promoters of proliferation genes. This pathway constitutes a new mechanism by which H3K4me3 functions in active gene repression. Furthermore, ING2 modulates cellular responses to genotoxic insults, and these functions are critically dependent on ING2 interaction with H3K4me3. Together, our findings establish a pivotal role for trimethylation of H3K4 in gene repression and, potentially, tumour suppressor mechanisms.

  View details for DOI 10.1038/nature04835

  View details for Web of Science ID 000238724500044

  View details for PubMedID 16728974

  View details for PubMedCentralID PMC3089773

• Multivalent tumor suppressor adenomatous polyposis coli promotes Axin biomolecular condensate formation and efficient beta-catenin degradation. Scientific reports Li, T., Ren, J., Husmann, D., Coan, J. P., Gozani, O., Chua, K. F. 2020; 10 (1): 17425

  Abstract

  The tumor suppressor adenomatous polyposis coli (APC) is frequently mutated in colorectal cancers. APC and Axin are core components of a destruction complex that scaffolds GSK3beta and CK1 to earmark beta-catenin for proteosomal degradation. Disruption of APC results in pathologic stabilization of beta-catenin and oncogenesis. However, the molecular mechanism by which APC promotes beta-catenin degradation is unclear. Here, we find that the intrinsically disordered region (IDR) of APC, which contains multiple beta-catenin and Axin interacting sites, undergoes liquid-liquid phase separation(LLPS) in vitro. Expression of the APC IDR in colorectal cells promotes Axin puncta formation and beta-catenin degradation. Our results support the model that multivalent interactions between APC and Axin drives the beta-catenin destruction complex to form biomolecular condensates in cells, which concentrate key components to achieve high efficient degradation of beta-catenin.

  View details for DOI 10.1038/s41598-020-74080-2

  View details for PubMedID 33060621

• Direct readout of heterochromatic H3K9me3 regulates DNMT1-mediated maintenance DNA methylation. Proceedings of the National Academy of Sciences of the United States of America Ren, W., Fan, H., Grimm, S. A., Guo, Y., Kim, J. J., Yin, J., Li, L., Petell, C. J., Tan, X., Zhang, Z., Coan, J. P., Gao, L., Cai, L., Detrick, B., Cetin, B., Cui, Q., Strahl, B. D., Gozani, O., Wang, Y., Miller, K. M., O'Leary, S. E., Wade, P. A., Patel, D. J., Wang, G. G., Song, J. 2020

  Abstract

  In mammals, repressive histone modifications such as trimethylation of histone H3 Lys9 (H3K9me3), frequently coexist with DNA methylation, producing a more stable and silenced chromatin state. However, it remains elusive how these epigenetic modifications crosstalk. Here, through structural and biochemical characterizations, we identified the replication foci targeting sequence (RFTS) domain of maintenance DNA methyltransferase DNMT1, a module known to bind the ubiquitylated H3 (H3Ub), as a specific reader for H3K9me3/H3Ub, with the recognition mode distinct from the typical trimethyl-lysine reader. Disruption of the interaction between RFTS and the H3K9me3Ub affects the localization of DNMT1 in stem cells and profoundly impairs the global DNA methylation and genomic stability. Together, this study reveals a previously unappreciated pathway through which H3K9me3 directly reinforces DNMT1-mediated maintenance DNA methylation.

  View details for DOI 10.1073/pnas.2009316117

  View details for PubMedID 32675241

• Methyltransferase-like 21C (METTL21C) methylates alanine tRNA synthetase at Lys-943 in muscle tissue. The Journal of biological chemistry Zoabi, M., Zhang, L., Li, T., Elias, J. E., Carlson, S. M., Gozani, O. 2020

  Abstract

  Protein lysine methylation is a common posttranslational modification (PTM) throughout the human proteome that plays important roles in diverse biological processes. In humans, there are >100 known and candidate protein lysine methyltransferases (PKMTs), many of which are linked to human diseases. Methyltransferase-like protein 21C (METTL21C) is a PKMT implicated in muscle biology that has been reported to methylate valosin-containing protein/p97 (VCP) and heat shock 70kDa protein 8 (HSPA8). However, a clear in vitro methyltransferase activity for METTL21C remains yet to be demonstrated, and whether it is an active enzyme that directly methylates substrate/s in vivo is unclear. Here, we used an unbiased biochemistry-based screening assay coupled to MS, which identified alanine-tRNA-synthetase 1 (AARS1) as a direct substrate of METTL21C. We found that METTL21C catalyzes methylation of Lys-943 of AARS1 (AARS1-K943me) both in vitro and in vivo. In vitro METTL21C-mediated AARS1 methylation was independent of ATP or tRNA molecules. Unlike for AARS1, and in conflict with previous reports, we did not detect METTL21C methylation of VCP and HSPA8. AARS1-K943 methylation in HEK293T cells is dependent upon METTL21C levels. Finally, METTL2C was almost exclusively expressed in muscle tissue, and, accordingly, we detected METTL21C-catalyzed methylation of AARS1 in mouse skeletal muscle tissue. These results reveal that AARS1 is a bona fide in vitro substrate of METTL21C and suggest a role for the METTL21C-AARS1 axis in the regulation of protein synthesis in muscle tissue. Moreover, our study describes a straightforward protocol for elucidating the physiological substrates of poorly characterized or uncharacterized PKMTs.

  View details for DOI 10.1074/jbc.RA120.014505

  View details for PubMedID 32611769

• An engineered variant of SETD3 methyltransferase alters target specificity from histidine to lysine methylation. The Journal of biological chemistry Dai, S. n., Horton, J. R., Wilkinson, A. W., Gozani, O. n., Zhang, X. n., Cheng, X. n. 2020

  Abstract

  Most characterized SET domain (SETD) proteins are protein lysine methyltransferases, but SETD3 was recently demonstrated to be a protein (i.e.actin) histidine-N3methyltransferase. Human SETD3 shares a high structural homology with two known protein lysine methyltransferases-human SETD6 and the plant LSMT-but differs in the residues constituting the active site. In the SETD3 active site, Asn-255 engages in a unique hydrogen-bonding interaction with the target histidine of actin that likely contributes to its >1300-fold greater catalytic efficiency (kcat/Km) on histidine than on lysine. Here, we engineered active-site variants to switch the SETD3 target specificity from histidine to lysine.  Substitution of Asn-255 with phenylalanine (N255F), together with substitution of Trp-273 with alanine (W273A), generated an active site mimicking that of known lysine methyltransferases. The doubly substituted SETD3 variant exhibited a 13-fold preference for lysine over histidine. We show, by means of X-ray crystallography, that the two target nitrogen atoms-the N3atom of histidine and the terminal e-amino nitrogen of lysine-occupy the same position and point towards and are within a short distance of the incoming methyl group of SAM for a direct methyl transfer during catalysis. In contrast, SETD3 and its Asn-255 substituted derivatives did not methylate glutamine (another potentially methylated amino acid). However, the glutamine-containing peptide competed with the substrate peptide, and glutamine bound in the active site, but too far away from SAM to be methylated. Our results provide insight into the structural parameters defining the target amino acid specificity of SET enzymes.

  View details for DOI 10.1074/jbc.RA119.012319

  View details for PubMedID 31911441

• Binding to medium and long chain fatty acyls is a common property of HEAT and ARM repeat modules. Scientific reports Li, T., Coan, J. P., Krajewski, K., Zhang, L., Elias, J. E., Strahl, B. D., Gozani, O., Chua, K. F. 2019; 9 (1): 14226

  Abstract

  Covalent post-translational modification (PTM) of proteins with acyl groups of various carbon chain-lengths regulates diverse biological processes ranging from chromatin dynamics to subcellular localization. While the YEATS domain has been found to be a prominent reader of acetylation and other short acyl modifications, whether additional acyl-lysine reader domains exist, particularly for longer carbon chains, is unclear. Here, we employed a quantitative proteomic approach using various modified peptide baits to identify reader proteins of various acyl modifications. We discovered that proteins harboring HEAT and ARM repeats bind to lysine myristoylated peptides. Recombinant HEAT and ARM repeats bind to myristoylated peptides independent of the peptide sequence or the position of the myristoyl group. Indeed, HEAT and ARM repeats bind directly to medium- and long-chain free fatty acids (MCFA and LCFA). Lipidomic experiments suggest that MCFAs and LCFAs interact with HEAT and ARM repeat proteins in mammalian cells. Finally, treatment of cells with exogenous MCFAs and inhibitors of MCFA-CoA synthases increase the transactivation activity of the ARM repeat protein beta-catenin. Taken together, our results suggest an unappreciated role for fatty acids in the regulation of proteins harboring HEAT or ARM repeats.

  View details for DOI 10.1038/s41598-019-50817-6

  View details for PubMedID 31578417

• Structural basis for the target specificity of actin histidine methyltransferase SETD3. Nature communications Dai, S., Horton, J. R., Woodcock, C. B., Wilkinson, A. W., Zhang, X., Gozani, O., Cheng, X. 2019; 10 (1): 3541

  Abstract

  SETD3 is an actin histidine-N3 methyltransferase, whereas other characterized SET-domain enzymes are protein lysine methyltransferases. We report that in a pre-reactive complex SETD3 binds the N3-protonated form (N3-H) of actin His73, and in a post-reactive product complex, SETD3 generates the methylated histidine in an N1-protonated (N1-H) and N3-methylated form. During the reaction, the imidazole ring of His73 rotates ~105°, which shifts the proton from N3 to N1, thus ensuring that the target atom N3 is deprotonated prior to the methyl transfer. Under the conditions optimized for lysine deprotonation, SETD3 has weak lysine methylation activity on an actin peptide in which the target His73 is substituted by a lysine. The structure of SETD3 with Lys73-containing peptide reveals a bent conformation of Lys73, with its side chain aliphatic carbons tracing along the edge of imidazole ring and the terminal epsilon-amino group occupying a position nearly identical to the N3 atom of unmethylated histidine.

  View details for DOI 10.1038/s41467-019-11554-6

  View details for PubMedID 31388018

• HeartBioPortal. Circulation. Genomic and precision medicine Khomtchouk, B. B., Vand, K. A., Koehler, W. C., Tran, D. T., Middlebrook, K., Sudhakaran, S., Nelson, C. S., Gozani, O., Assimes, T. L. 2019; 12 (4): e002426

  View details for DOI 10.1161/CIRCGEN.118.002426

  View details for PubMedID 31294639

• METTL13 Methylation of eEF1A Increases Translational Output to Promote Tumorigenesis. Cell Liu, S., Hausmann, S., Carlson, S. M., Fuentes, M. E., Francis, J. W., Pillai, R., Lofgren, S. M., Hulea, L., Tandoc, K., Lu, J., Li, A., Nguyen, N. D., Caporicci, M., Kim, M. P., Maitra, A., Wang, H., Wistuba, I. I., Porco, J. A., Bassik, M. C., Elias, J. E., Song, J., Topisirovic, I., Van Rechem, C., Mazur, P. K., Gozani, O. 2018

  Abstract

  Increased protein synthesis plays an etiologic role in diverse cancers. Here, we demonstrate that METTL13 (methyltransferase-like 13) dimethylation of eEF1A (eukaryotic elongation factor 1A) lysine 55 (eEF1AK55me2) is utilized by Ras-driven cancers to increase translational output and promote tumorigenesis invivo. METTL13-catalyzed eEF1A methylation increases eEF1A's intrinsic GTPase activity invitro and protein production in cells. METTL13 and eEF1AK55me2 levels are upregulated in cancer and negatively correlate with pancreatic and lung cancer patient survival. METTL13 deletion and eEF1AK55me2 loss dramatically reduce Ras-driven neoplastic growth in mouse models and in patient-derived xenografts (PDXs) from primary pancreatic and lung tumors. Finally, METTL13 depletion renders PDX tumors hypersensitive to drugs thattarget growth-signaling pathways. Together, our work uncovers a mechanism by which lethal cancers become dependent on the METTL13-eEF1AK55me2 axis to meet their elevated protein synthesis requirement and suggests that METTL13 inhibition may constitute a targetable vulnerability of tumors driven by aberrant Ras signaling.

  View details for PubMedID 30612740

• SETD3 is an actin histidine methyltransferase that prevents primary dystocia. Nature Wilkinson, A. W., Diep, J. n., Dai, S. n., Liu, S. n., Ooi, Y. S., Song, D. n., Li, T. M., Horton, J. R., Zhang, X. n., Liu, C. n., Trivedi, D. V., Ruppel, K. M., Vilches-Moure, J. G., Casey, K. M., Mak, J. n., Cowan, T. n., Elias, J. E., Nagamine, C. M., Spudich, J. A., Cheng, X. n., Carette, J. E., Gozani, O. n. 2018

  Abstract

  For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur1. Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism.

  View details for PubMedID 30626964

• RBM25 is a global splicing factor promoting inclusion of alternatively spliced exons and is itself regulated by lysine mono-methylation JOURNAL OF BIOLOGICAL CHEMISTRY Carlson, S. M., Soulette, C. M., Yang, Z., Elias, J. E., Brooks, A. N., Gozani, O. 2017; 292 (32): 13381–90

  Abstract

  In eukaryotes, precursor mRNA (pre-mRNA) splicing removes non-coding intron sequences to produce mature mRNA. This removal is controlled in part by RNA-binding proteins that regulate alternative splicing decisions through interactions with the splicing machinery. RNA binding motif protein 25 (RBM25) is a putative splicing factor strongly conserved across eukaryotic lineages. However, the role of RBM25 in global splicing regulation and its cellular functions are unknown. Here we show that RBM25 is required for the viability of multiple human cell lines, suggesting that it could play a key role in pre-mRNA splicing. Indeed, transcriptome-wide analysis of splicing events demonstrated that RBM25 regulates a large fraction of alternatively spliced exons throughout the human genome. Moreover, proteomic analysis indicated that RBM25 interacts with components of the early spliceosome and regulators of alternative splicing. Previously, we identified an RBM25 species that is mono-methylated at lysine 77 (RBM25K77me1), and here we used quantitative mass spectrometry to show that RBM25K77me1 is abundant in multiple human cell lines. We also identified a region of RBM25 spanning Lys-77 that binds with high affinity to serine- and arginine-rich splicing factor 2 (SRSF2), a crucial protein in exon definition, but only when Lys-77 is unmethylated. Together, our findings uncover a pivotal role for RBM25 as an essential regulator of alternative splicing and reveal a new potential mechanism for regulation of pre-mRNA splicing by lysine methylation of a splicing factor.

  View details for PubMedID 28655759

  View details for PubMedCentralID PMC5555197

• Characterization of H3.3K36M as a tool to study H3K36 methylation in cancer cells EPIGENETICS Sankaran, S. M., Gozani, O. 2017; 12 (11): 917–22

  Abstract

  Recurrent mutations at key lysine residues in the histone variant H3.3 are thought to play an etiologic role in the development of distinct subsets of pediatric gliomas and bone and cartilage cancers. H3.3K36M is one such mutation that was originally identified in chondroblastomas, and its expression in these tumors contributes to oncogenic reprogramming by triggering global depletion of dimethylation and trimethylation at H3K36 with a concomitant increase in the levels of H3K27 trimethylation. H3.3K36M expression can also cause epigenomic changes in cell types beyond chondrocytic cells. Here we show that expression of H3.3K36M in HT1080 fibrosarcoma cancer cells severely impairs cellular proliferation, which contrasts its role in promoting transformation of chondrocytic cells. H3.3K36M-associated cellular toxicity phenocopies the specific depletion of H3K36me2, but not loss of H3K36me3. We further find that the H3K36me2-associated toxicity is largely independent of changes in H3K27me3. Together, our findings lend support to the argument that H3K36me2 has distinct roles in cancer cells independent of H3K36me3 and H3K27me3, and highlight the use of H3.3K36M as an epigenetic tool to study H3K36 and H3K27 methylation dynamics in diverse cell types.

  View details for PubMedID 28933651

  View details for PubMedCentralID PMC5788406

• Nonhistone Lysine Methylation in the Regulation of Cancer Pathways. Cold Spring Harbor perspectives in medicine Carlson, S. M., Gozani, O. 2016; 6 (11)

  Abstract

  Proteins are regulated by an incredible array of posttranslational modifications (PTMs). Methylation of lysine residues on histone proteins is a PTM with well-established roles in regulating chromatin and epigenetic processes. The recent discovery that hundreds and likely thousands of nonhistone proteins are also methylated at lysine has opened a tremendous new area of research. Major cellular pathways involved in cancer, such as growth signaling and the DNA damage response, are regulated by lysine methylation. Although the field has developed quickly in recent years many fundamental questions remain to be addressed. We review the history and molecular functions of lysine methylation. We then discuss the enzymes that catalyze methylation of lysine residues, the enzymes that remove lysine methylation, and the cancer pathways known to be regulated by lysine methylation. The rest of the article focuses on two open questions that we suggest as a roadmap for future research. First is understanding the large number of candidate methyltransferase and demethylation enzymes whose enzymatic activity is not yet defined and which are potentially associated with cancer through genetic studies. Second is investigating the biological processes and cancer mechanisms potentially regulated by the multitude of lysine methylation sites that have been recently discovered.

  View details for DOI 10.1101/cshperspect.a026435

  View details for PubMedID 27580749

• Molecular and Neural Functions of Rai1, the Causal Gene for Smith-Magenis Syndrome. Neuron Huang, W., Guenthner, C. J., Xu, J., Nguyen, T., Schwarz, L. A., Wilkinson, A. W., Gozani, O., Chang, H. Y., Shamloo, M., Luo, L. 2016; 92 (2): 392-406

  Abstract

  Haploinsufficiency of Retinoic Acid Induced 1 (RAI1) causes Smith-Magenis syndrome (SMS), which is associated with diverse neurodevelopmental and behavioral symptoms as well as obesity. RAI1 encodes a nuclear protein but little is known about its molecular function or the cell types responsible for SMS symptoms. Using genetically engineered mice, we found that Rai1 preferentially occupies DNA regions near active promoters and promotes the expression of a group of genes involved in circuit assembly and neuronal communication. Behavioral analyses demonstrated that pan-neural loss of Rai1 causes deficits in motor function, learning, and food intake. These SMS-like phenotypes are produced by loss of Rai1 function in distinct neuronal types: Rai1 loss in inhibitory neurons or subcortical glutamatergic neurons causes learning deficits, while Rai1 loss in Sim1(+) or SF1(+) cells causes obesity. By integrating molecular and organismal analyses, our study suggests potential therapeutic avenues for a complex neurodevelopmental disorder.

  View details for DOI 10.1016/j.neuron.2016.09.019

  View details for PubMedID 27693255

• Systematic Analysis of Known and Candidate Lysine Demethylases in the Regulation of Myoblast Differentiation. Journal of molecular biology Munehira, Y., Yang, Z., Gozani, O. 2016

  Abstract

  Histone methylation dynamics plays a critical role in cellular programming during development. For example, specific lysine methyltransferases (KMTs) and lysine demethylases (KDMs) have been implicated in the differentiation of mesenchymal stem cells into various cell lineages. However, a systematic and functional analysis for an entire family of KMT or KDM enzymes has not been performed. Here, we test the function of all the known and candidate KDMs in myoblast and osteoblast differentiation using the C2C12 cell differentiation model system. Our analysis identified that LSD1 is the only KDM required for myogenic differentiation and that KDM3B, KDM6A, and KDM8 are the candidate KDMs required for osteoblast differentiation. We find that LSD1, via H3K4me1 demethylation, represses the master regulator of osteoblast differentiation RUNX2 to promote myogenesis in the C2C12 model system. Finally, MLL4 is required for efficient osteoblast differentiation in part by countering LSD1 H3K4me1 demethylation at the RUNX2 enhancer. Together, our findings provide additional mechanisms by which lysine methylation signaling impacts on cell fate decisions.

  View details for DOI 10.1016/j.jmb.2016.10.004

  View details for PubMedID 27732873

• ASH1L Links Histone H3 Lysine 36 Dimethylation to MLL Leukemia. Cancer discovery Zhu, L., Li, Q., Wong, S. H., Huang, M., Klein, B. J., Shen, J., Ikenouye, L., Onishi, M., Schneidawind, D., Buechele, C., Hansen, L., Duque-Afonso, J., Zhu, F., Martin, G. M., Gozani, O., Majeti, R., Kutateladze, T. G., Cleary, M. L. 2016; 6 (7): 770-783

  Abstract

  Numerous studies in multiple systems support that histone H3 lysine 36 dimethylation (H3K36me2) is associated with transcriptional activation; however, the underlying mechanisms are not well defined. Here, we show that the H3K36me2 chromatin mark written by the ASH1L histone methyltransferase is preferentially bound in vivo by LEDGF, a mixed-lineage leukemia (MLL)-associated protein that colocalizes with MLL, ASH1L, and H3K36me2 on chromatin genome wide. Furthermore, ASH1L facilitates recruitment of LEDGF and wild-type MLL proteins to chromatin at key leukemia target genes and is a crucial regulator of MLL-dependent transcription and leukemic transformation. Conversely, KDM2A, an H3K36me2 demethylase and Polycomb group silencing protein, antagonizes MLL-associated leukemogenesis. Our studies are the first to provide a basic mechanistic insight into epigenetic interactions wherein placement, interpretation, and removal of H3K36me2 contribute to the regulation of gene expression and MLL leukemia, and suggest ASH1L as a novel target for therapeutic intervention.Epigenetic regulators play vital roles in cancer pathogenesis and represent a new frontier in therapeutic targeting. Our studies provide basic mechanistic insight into the role of H3K36me2 in transcription activation and MLL leukemia pathogenesis and implicate ASH1L histone methyltransferase as a promising target for novel molecular therapy. Cancer Discov; 6(7); 770-83. ©2016 AACR.See related commentary by Balbach and Orkin, p. 700This article is highlighted in the In This Issue feature, p. 681.

  View details for DOI 10.1158/2159-8290.CD-16-0058

  View details for PubMedID 27154821

  View details for PubMedCentralID PMC4930721

• ASH1L Links Histone H3 Lysine 36 Dimethylation to MLL Leukemia CANCER DISCOVERY Zhu, L., Li, Q., Wong, S. K., Huang, M., Klein, B. J., Shen, J., Ikenouye, L., Onishi, M., Schneidawind, D., Buechele, C., Hansen, L., Duque-Afonso, J., Zhu, F., Martin, G., Gozani, O., Majeti, R., Kutateladze, T. G., Cleary, M. L. 2016; 6 (7): 770–83

  View details for DOI 10.1158/2159-8290.CD-16-0058

  View details for Web of Science ID 000383354500027

• A PWWP Domain of Histone-Lysine N-Methyltransferase NSD2 Binds to Dimethylated Lys-36 of Histone H3 and Regulates NSD2 Function at Chromatin JOURNAL OF BIOLOGICAL CHEMISTRY Sankaran, S. M., Wilkinson, A. W., Elias, J. E., Gozani, O. 2016; 291 (16): 8465-8474

  Abstract

  The readout of histone modifications plays a critical role in chromatin-regulated processes. Dimethylation at Lys-36 on histone H3 (H3K36me2) is associated with actively transcribed genes, and global up-regulation of this modification is associated with several cancers. However, the molecular mechanism by which H3K36me2 is sensed and transduced to downstream biological outcomes remains unclear. Here we identify a PWWP domain within the histone lysine methyltransferase and oncoprotein NSD2 that preferentially binds to nucleosomes containing H3K36me2. In cells, the NSD2 PWWP domain interaction with H3K36me2 plays a role in stabilizing NSD2 at chromatin. Furthermore, NSD2's ability to induce global increases in H3K36me2 via its enzymatic activity, and consequently promote cellular proliferation, is compromised by mutations within the PWWP domain that specifically abrogate H3K36me2-recognition. Together, our results identify a pivotal role for NSD2 binding to its catalytic product in regulating its cellular functions, and suggest a model for how this interaction may facilitate epigenetic spreading and propagation of H3K36me2.

  View details for DOI 10.1074/jbc.M116.720748

  View details for Web of Science ID 000374773200013

  View details for PubMedID 26912663

  View details for PubMedCentralID PMC4861420

• Coordination of stress signals by the lysine methyltransferase SMYD2 promotes pancreatic cancer GENES & DEVELOPMENT Reynoird, N., Mazur, P. K., Stellfeld, T., Flores, N. M., Lofgren, S. M., Carlson, S. M., Brambilla, E., Hainaut, P., Kaznowska, E. B., Arrowsmith, C. H., Khatri, P., Stresemann, C., Gozani, O., Sage, J. 2016; 30 (7): 772-785

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) is a lethal form of cancer with few therapeutic options. We found that levels of the lysine methyltransferase SMYD2 (SET and MYND domain 2) are elevated in PDAC and that genetic and pharmacological inhibition of SMYD2 restricts PDAC growth. We further identified the stress response kinase MAPKAPK3 (MK3) as a new physiologic substrate of SMYD2 in PDAC cells. Inhibition of MAPKAPK3 impedes PDAC growth, identifying a potential new kinase target in PDAC. Finally, we show that inhibition of SMYD2 cooperates with standard chemotherapy to treat PDAC cells and tumors. These findings uncover a pivotal role for SMYD2 in promoting pancreatic cancer.

  View details for DOI 10.1101/gad.275529.115

  View details for Web of Science ID 000373194300003

  View details for PubMedCentralID PMC4826394

• Coordination of stress signals by the lysine methyltransferase SMYD2 promotes pancreatic cancer. Genes & development Reynoird, N., Mazur, P. K., Stellfeld, T., Flores, N. M., Lofgren, S. M., Carlson, S. M., Brambilla, E., Hainaut, P., Kaznowska, E. B., Arrowsmith, C. H., Khatri, P., Stresemann, C., Gozani, O., Sage, J. 2016; 30 (7): 772-785

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) is a lethal form of cancer with few therapeutic options. We found that levels of the lysine methyltransferase SMYD2 (SET and MYND domain 2) are elevated in PDAC and that genetic and pharmacological inhibition of SMYD2 restricts PDAC growth. We further identified the stress response kinase MAPKAPK3 (MK3) as a new physiologic substrate of SMYD2 in PDAC cells. Inhibition of MAPKAPK3 impedes PDAC growth, identifying a potential new kinase target in PDAC. Finally, we show that inhibition of SMYD2 cooperates with standard chemotherapy to treat PDAC cells and tumors. These findings uncover a pivotal role for SMYD2 in promoting pancreatic cancer.

  View details for DOI 10.1101/gad.275529.115

  View details for PubMedID 26988419

  View details for PubMedCentralID PMC4826394

• Histone H4 Lysine 20 (H4K20) Methylation, Expanding the Signaling Potential of the Proteome One Methyl Moiety at a Time. Molecular & cellular proteomics van Nuland, R., Gozani, O. 2016; 15 (3): 755-764

  Abstract

  Covalent post-translational modifications (PTMs) of proteins can regulate the structural and functional state of a protein in the absence of primary changes in the underlying sequence. Common PTMs include phosphorylation, acetylation, and methylation. Histone proteins are critical regulators of the genome and are subject to a highly abundant and diverse array of PTMs. To highlight the functional complexity added to the proteome by lysine methylation signaling, here we will focus on lysine methylation of histone proteins, an important modification in the regulation of chromatin and epigenetic processes. We review the signaling pathways and functions associated with a single residue, H4K20, as a model chromatin and clinically important mark that regulates biological processes ranging from the DNA damage response and DNA replication to gene expression and silencing.

  View details for DOI 10.1074/mcp.R115.054742

  View details for PubMedID 26598646

  View details for PubMedCentralID PMC4813698

• Structural Basis for the Unique Multivalent Readout of Unmodified H3 Tail by Arabidopsis ORC1b BAH-PHD Cassette STRUCTURE Li, S., Yang, Z., Du, X., Liu, R., Wilkinson, A. W., Gozani, O., Jacobsen, S. E., Patel, D. J., Du, J. 2016; 24 (3): 486-494

  Abstract

  DNA replication initiation relies on the formation of the origin recognition complex (ORC). The plant ORC subunit 1 (ORC1) protein possesses a conserved N-terminal BAH domain with an embedded plant-specific PHD finger, whose function may be potentially regulated by an epigenetic mechanism. Here, we report structural and biochemical studies on the Arabidopsis thaliana ORC1b BAH-PHD cassette which specifically recognizes the unmodified H3 tail. The crystal structure of ORC1b BAH-PHD cassette in complex with an H3(1-15) peptide reveals a strict requirement for the unmodified state of R2, T3, and K4 on the H3 tail and a novel multivalent BAH and PHD readout mode for H3 peptide recognition. Such recognition may contribute to epigenetic regulation of the initiation of DNA replication.

  View details for DOI 10.1016/j.str.2016.01.004

  View details for Web of Science ID 000373568300016

  View details for PubMedID 26876097

  View details for PubMedCentralID PMC4775424

• SMYD3 links lysine methylation of MAP3K2 to ras-driven lung cancer Reynoird, N., Mazur, P., Sage, J., Gozani, O. EUROPEAN RESPIRATORY SOC JOURNALS LTD. 2015

  View details for DOI 10.1183/13993003.congress-2015.PA4256

  View details for Web of Science ID 000451979406146

• SMYD3 links methylation of MAP3K2 to Ras-driven tumors Mazur, P. K., Reynoird, N., Khatri, P., Butte, A. J., Wilkinson, A., Garcia, B., Liu, S., Vermeulen, M., Jansen, P. C., Tummino, P. J., Kruger, R. G., Van Aller, G. S., Barbash, O., Huddleston, M., Gozani, O., Sage, J. AMER ASSOC CANCER RESEARCH. 2015

  View details for DOI 10.1158/1538-7445.PANCA2014-A23

  View details for Web of Science ID 000371263900033

• A Proteomic Strategy Identifies Lysine Methylation of Splicing Factor snRNP70 by the SETMAR Enzyme JOURNAL OF BIOLOGICAL CHEMISTRY Carlson, S. M., Moore, K. E., Sankaran, S. M., Reynoird, N., Elias, J. E., Gozani, O. 2015; 290 (19): 12040-12047

  Abstract

  The lysine methyltransferase (KMT) SETMAR is implicated in the response to and repair of DNA damage, but its molecular function is not clear. SETMAR has been associated with dimethylation of histone H3 lysine 36 (H3K36) at sites of DNA damage. However, SETMAR does not methylate H3K36 in vitro. This and the observation that SETMAR is not active on nucleosomes suggest that H3K36 methylation is not a physiologically relevant activity. To identify potential non-histone substrates, we utilized a strategy on the basis of quantitative proteomic analysis of methylated lysine. Our approach identified lysine 130 of the mRNA splicing factor snRNP70 as a SETMAR substrate in vitro, and we show that the enzyme primarily generates monomethylation at this position. Furthermore, we show that SETMAR methylates snRNP70 Lys-130 in cells. Because snRNP70 is a key early regulator of 5' splice site selection, our results suggest a model in which methylation of snRNP70 by SETMAR regulates constitutive and/or alternative splicing. In addition, the proteomic strategy described here is broadly applicable and is a promising route for large-scale mapping of KMT substrates.

  View details for DOI 10.1074/jbc.M115.641530

  View details for Web of Science ID 000354388600019

  View details for PubMedID 25795785

  View details for PubMedCentralID PMC4424340

• A Meier-Gorlin Syndrome Mutation Impairs the ORC1-Nucleosome Association ACS CHEMICAL BIOLOGY Zhang, W., Sankaran, S., Gozani, O., Song, J. 2015; 10 (5): 1176-1180

  Abstract

  Recent studies have identified several genetic mutations within the BAH domain of human Origin Recognition Complex subunit 1 (hORC1BAH), including the R105Q mutation, implicated in Meier-Gorlin Syndrome (MGS). However, the pathological role of the hORC1 R105Q mutation remains unclear. In this study, we have investigated the interactions of the hORC1BAH domain with histone H4K20me2, DNA, and the nucleosome core particle labeled with H4Kc20me2, a chemical analog of H4K20me2. Our study revealed a nucleosomal DNA binding site for hORC1BAH. The R105Q mutation reduces the hORC1BAH-DNA binding affinity, leading to impaired hORC1BAH-nucleosome interaction, which likely influences DNA replication initiation and MGS pathogenesis. This study provides an etiologic link between the hORC1 R105Q mutation and MGS.

  View details for DOI 10.1021/cb5009684

  View details for Web of Science ID 000354907400003

  View details for PubMedID 25689043

  View details for PubMedCentralID PMC4654454

• An unexpected journey: Lysine methylation across the proteome BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS Moore, K. E., Gozani, O. 2014; 1839 (12): 1395-1403

  Abstract

  The dynamic modification of histone proteins by lysine methylation has emerged over the last decade as a key regulator of chromatin functions. In contrast, our understanding of the biological roles for lysine methylation of non-histone proteins has progressed more slowly. Though recently it has attracted less attention, ε-methyl-lysine in non-histone proteins was first observed over 50 years ago. In that time, it has become clear that, like the case for histones, non-histone methylation represents a key and common signaling process within the cell. Recent work suggests that non-histone methylation occurs on hundreds of proteins found in both the nucleus and the cytoplasm, and with important biomedical implications. Technological advances that allow us to identify lysine methylation on a proteomic scale are opening new avenues in the non-histone methylation field, which is poised for dramatic growth. Here, we review historical and recent findings in non-histone lysine methylation signaling, highlight new methods that are expanding opportunities in the field, and discuss outstanding questions and future challenges about the role of this fundamental post-translational modification (PTM).

  View details for DOI 10.1016/j.bbagrm.2014.02.008

  View details for Web of Science ID 000347129100006

  View details for PubMedID 24561874

  View details for PubMedCentralID PMC4139483

• Emerging Technologies to Map the Protein Methylome JOURNAL OF MOLECULAR BIOLOGY Carlson, S. M., Gozani, O. 2014; 426 (20): 3350-3362

  Abstract

  Protein methylation plays an integral role in cellular signaling, most notably by modulating proteins bound at chromatin and increasingly through regulation of non-histone proteins. One central challenge in understanding how methylation acts in signaling is identifying and measuring protein methylation. This includes locus-specific modification of histones, on individual non-histone proteins, and globally across the proteome. Protein methylation has been studied traditionally using candidate approaches such as methylation-specific antibodies, mapping of post-translational modifications by mass spectrometry, and radioactive labeling to characterize methylation on target proteins. Recent developments have provided new approaches to identify methylated proteins, measure methylation levels, identify substrates of methyltransferase enzymes, and match methylated proteins to methyl-specific reader domains. Methyl-binding protein domains and improved antibodies with broad specificity for methylated proteins are being used to characterize the "protein methylome". They also have the potential to be used in high-throughput assays for inhibitor screens and drug development. These tools are often coupled to improvements in mass spectrometry to quickly identify methylated residues, as well as to protein microarrays, where they can be used to screen for methylated proteins. Finally, new chemical biology strategies are being used to probe the function of methyltransferases, demethylases, and methyl-binding "reader" domains. These tools create a "system-level" understanding of protein methylation and integrate protein methylation into broader signaling processes.

  View details for DOI 10.1016/j.jmb.2014.04.024

  View details for Web of Science ID 000343617400004

  View details for PubMedCentralID PMC4177301

• Orchestrated recruitment of histone methyltransferases to DNA double strand breaks facilitates 53BP1 binding and proficient repair Tuzon, C. T., Spektor, T. M., Congdon, L. M., Kong, X., Cheung, P., Kuo, A. J., Yokomori, K., Gozani, O., Rice, J. C. AMER ASSOC CANCER RESEARCH. 2014

  View details for DOI 10.1158/1538-7445.AM2014-LB-133

  View details for Web of Science ID 000349910205167

• A molecular threading mechanism underlies Jumonji lysine demethylase KDM2A regulation of methylated H3K36. Genes & development Cheng, Z., Cheung, P., Kuo, A. J., Yukl, E. T., Wilmot, C. M., Gozani, O., Patel, D. J. 2014; 28 (16): 1758-1771

  Abstract

  The dynamic reversible methylation of lysine residues on histone proteins is central to chromatin biology. Key components are demethylase enzymes, which remove methyl moieties from lysine residues. KDM2A, a member of the Jumonji C domain-containing histone lysine demethylase family, specifically targets lower methylation states of H3K36. Here, structural studies reveal that H3K36 specificity for KDM2A is mediated by the U-shaped threading of the H3K36 peptide through a catalytic groove within KDM2A. The side chain of methylated K36 inserts into the catalytic pocket occupied by Ni(2+) and cofactor, where it is positioned and oriented for demethylation. Key residues contributing to K36me specificity on histone H3 are G33 and G34 (positioned within a narrow channel), P38 (a turn residue), and Y41 (inserts into its own pocket). Given that KDM2A was found to also bind the H3K36me3 peptide, we postulate that steric constraints could prevent α-ketoglutarate from undergoing an "off-line"-to-"in-line" transition necessary for the demethylation reaction. Furthermore, structure-guided substitutions of residues in the KDM2A catalytic pocket abrogate KDM2A-mediated functions important for suppression of cancer cell phenotypes. Together, our results deduce insights into the molecular basis underlying KDM2A regulation of the biologically important methylated H3K36 mark.

  View details for DOI 10.1101/gad.246561.114

  View details for PubMedID 25128496

• Histone-binding domains: strategies for discovery and characterization. Biochimica et biophysica acta Wilkinson, A. W., Gozani, O. 2014; 1839 (8): 669-675

  Abstract

  Chromatin signaling dynamics fundamentally regulate eukaryotic genomes. The reversible covalent post-translational modification (PTM) of histone proteins by chemical moieties such as phosphate, acetyl and methyl groups constitutes one of the primary chromatin signaling mechanisms. Modular protein domains present within chromatin-regulatory activities recognize or "read" specifically modified histone species and transduce these modified species into distinct downstream biological outcomes. Thus, understanding the molecular basis underlying PTM-mediated signaling at chromatin requires knowledge of both the modification and the partnering reader domains. Over the last ten years, a number of innovative approaches have been developed and employed to discover reader domain binding events with histones. Together, these studies have provided crucial insight into how chromatin pathways influence key cellular programs. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.

  View details for DOI 10.1016/j.bbagrm.2014.01.007

  View details for PubMedID 24525102

  View details for PubMedCentralID PMC4099300

• Histone-binding domains: Strategies for discovery and characterization BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS Wilkinson, A. W., Gozani, O. 2014; 1839 (8): 669-675

  Abstract

  Chromatin signaling dynamics fundamentally regulate eukaryotic genomes. The reversible covalent post-translational modification (PTM) of histone proteins by chemical moieties such as phosphate, acetyl and methyl groups constitutes one of the primary chromatin signaling mechanisms. Modular protein domains present within chromatin-regulatory activities recognize or "read" specifically modified histone species and transduce these modified species into distinct downstream biological outcomes. Thus, understanding the molecular basis underlying PTM-mediated signaling at chromatin requires knowledge of both the modification and the partnering reader domains. Over the last ten years, a number of innovative approaches have been developed and employed to discover reader domain binding events with histones. Together, these studies have provided crucial insight into how chromatin pathways influence key cellular programs. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.

  View details for DOI 10.1016/j.bbagrm.2014.01.007

  View details for Web of Science ID 000339696900006

  View details for PubMedID 24525102

  View details for PubMedCentralID PMC4099300

• Nuclear PI5P, Uhrf1, and the road not taken. Molecular cell Reynoird, N., Gozani, O. 2014; 54 (6): 901-903

  Abstract

  In this issue of Molecular Cell, Gelato et al. (2014) identify the signaling molecule phosphatidylinositol 5-phosphate (PI5P) as an allosteric regulator that determines the mode of chromatin binding for the DNA methylation maintenance factor Uhrf1. This work links nuclear lipids to chromatin signaling in the maintenance of DNA methylation and epigenetic regulation.

  View details for DOI 10.1016/j.molcel.2014.06.012

  View details for PubMedID 24950375

  View details for PubMedCentralID PMC4123760

• Set5 and Set1 cooperate to repress gene expression at telomeres and retrotransposons. Epigenetics Martín, G. M., King, D. A., Green, E. M., Garcia-Nieto, P. E., Alexander, R., Collins, S. R., Krogan, N. J., Gozani, O. P., Morrison, A. J. 2014; 9 (4): 513-522

  Abstract

  A complex interplay between multiple chromatin modifiers is critical for cells to regulate chromatin structure and accessibility during essential DNA-templated processes such as transcription. However, the coordinated activities of these chromatin modifiers in the regulation of gene expression are not fully understood. We previously determined that the budding yeast histone H4 methyltransferase Set5 functions together with Set1, the H3K4 methyltransferase, in specific cellular contexts. Here, we sought to understand the relationship between these evolutionarily conserved enzymes in the regulation of gene expression. We generated a comprehensive genetic interaction map of the functionally uncharacterized Set5 methyltransferase and expanded the existing genetic interactome of the global chromatin modifier Set1, revealing functional overlap of the two enzymes in chromatin-related networks, such as transcription. Furthermore, gene expression profiling via RNA-Seq revealed an unexpected synergistic role of Set1 and Set5 in repressing transcription of Ty transposable elements and genes located in subtelomeric regions. This study uncovers novel pathways in which the methyltransferase Set5 participates and, more importantly, reveals a partnership between Set1 and Set5 in transcriptional repression near repetitive DNA elements in budding yeast. Together, our results define a new functional relationship between histone H3 and H4 methyltransferases, whose combined activity may be implicated in preserving genomic integrity.

  View details for DOI 10.4161/epi.27645

  View details for PubMedID 24442241

  View details for PubMedCentralID PMC4121362

• Proteome-wide enrichment of proteins modified by lysine methylation. Nature protocols Carlson, S. M., Moore, K. E., Green, E. M., Martín, G. M., Gozani, O. 2014; 9 (1): 37-50

  Abstract

  We present a protocol for using the triple malignant brain tumor domains of L3MBTL1 (3xMBT), which bind to mono- and di-methylated lysine with minimal sequence specificity, in order to enrich for such methylated lysine from cell lysates. Cells in culture are grown with amino acids containing light or heavy stable isotopic labels. Methylated proteins are enriched by incubating cell lysates with 3xMBT, or with the binding-null D355N mutant as a negative control. Quantitative liquid chromatography and tandem mass spectrometry (LC-MS/MS) are then used to identify proteins that are specifically enriched by 3xMBT pull-down. The addition of a third isotopic label allows the comparison of protein lysine methylation between different biological conditions. Unlike most approaches, our strategy does not require a prior hypothesis of candidate methylated proteins, and it recognizes a wider range of methylated proteins than any available method using antibodies. Cells are prepared by growing in isotopic labeling medium for about 7 d; the process of enriching methylated proteins takes 3 d and analysis by LC-MS/MS takes another 1-2 d.

  View details for DOI 10.1038/nprot.2013.164

  View details for PubMedID 24309976

• Nuclear phosphatidylinositol-5-phosphate regulates ING2 stability at discrete chromatin targets in response to DNA damage SCIENTIFIC REPORTS Bua, D. J., Martin, G. M., Binda, O., Gozani, O. 2013; 3

  Abstract

  ING2 (inhibitor of growth family member 2) is a component of a chromatin-regulatory complex that represses gene expression and is implicated in cellular processes that promote tumor suppression. However, few direct genomic targets of ING2 have been identified and the mechanism(s) by which ING2 selectively regulates genes remains unknown. Here we provide evidence that direct association of ING2 with the nuclear phosphoinositide phosphatidylinositol-5-phosphate (PtdIns(5)P) regulates a subset of ING2 targets in response to DNA damage. At these target genes, the binding event between ING2 and PtdIns(5)P is required for ING2 promoter occupancy and ING2-associated gene repression. Moreover, depletion of PtdIns(5)P attenuates ING2-mediated regulation of these targets in the presence of DNA damage. Taken together, these findings support a model in which PtdIns(5)P functions as a sub-nuclear trafficking factor that stabilizes ING2 at discrete genomic sites.

  View details for DOI 10.1038/srep02137

  View details for Web of Science ID 000321287400008

  View details for PubMedID 23823870

  View details for PubMedCentralID PMC3705588

• Chd5 requires PHD-mediated histone 3 binding for tumor suppression. Cell reports Paul, S., Kuo, A., Schalch, T., Vogel, H., Joshua-Tor, L., McCombie, W. R., Gozani, O., Hammell, M., Mills, A. A. 2013; 3 (1): 92-102

  Abstract

  Chromodomain Helicase DNA binding protein 5 (CHD5) is a tumor suppressor mapping to 1p36, a genomic region that is frequently deleted in human cancer. Although CHD5 belongs to the CHD family of chromatin-remodeling proteins, whether its tumor-suppressive role involves an interaction with chromatin is unknown. Here we report that Chd5 binds the unmodified N terminus of H3 through its tandem plant homeodomains (PHDs). Genome-wide chromatin immunoprecipitation studies reveal preferential binding of Chd5 to loci lacking the active mark H3K4me3 and also identify Chd5 targets implicated in cancer. Chd5 mutations that abrogate H3 binding are unable to inhibit proliferation or transcriptionally modulate target genes, which leads to tumorigenesis in vivo. Unlike wild-type Chd5, Chd5-PHD mutants are unable to induce differentiation or efficiently suppress the growth of human neuroblastoma in vivo. Our work defines Chd5 as an N-terminally unmodified H3-binding protein and provides functional evidence that this interaction orchestrates chromatin-mediated transcriptional programs critical for tumor suppression.

  View details for DOI 10.1016/j.celrep.2012.12.009

  View details for PubMedID 23318260

  View details for PubMedCentralID PMC3575599

• Nuclear phosphatidylinositol-5-phosphate regulates ING2 stability at discrete chromatin targets in response to DNA damage. Scientific reports Bua, D. J., Martin, G. M., Binda, O., Gozani, O. 2013; 3: 2137-?

  Abstract

  ING2 (inhibitor of growth family member 2) is a component of a chromatin-regulatory complex that represses gene expression and is implicated in cellular processes that promote tumor suppression. However, few direct genomic targets of ING2 have been identified and the mechanism(s) by which ING2 selectively regulates genes remains unknown. Here we provide evidence that direct association of ING2 with the nuclear phosphoinositide phosphatidylinositol-5-phosphate (PtdIns(5)P) regulates a subset of ING2 targets in response to DNA damage. At these target genes, the binding event between ING2 and PtdIns(5)P is required for ING2 promoter occupancy and ING2-associated gene repression. Moreover, depletion of PtdIns(5)P attenuates ING2-mediated regulation of these targets in the presence of DNA damage. Taken together, these findings support a model in which PtdIns(5)P functions as a sub-nuclear trafficking factor that stabilizes ING2 at discrete genomic sites.

  View details for DOI 10.1038/srep02137

  View details for PubMedID 23823870

  View details for PubMedCentralID PMC3705588

• Phf19 links methylated Lys36 of histone H3 to regulation of Polycomb activity NATURE STRUCTURAL & MOLECULAR BIOLOGY Ballare, C., Lange, M., Lapinaite, A., Martin, G. M., Morey, L., Pascual, G., Liefke, R., Simon, B., Shi, Y., Gozani, O., Carlomagno, T., Aznar Benitah, S., Di Croce, L. 2012; 19 (12): 1257-?

  Abstract

  Polycomb-group proteins are transcriptional repressors with essential roles in embryonic development. Polycomb repressive complex 2 (PRC2) contains the methyltransferase activity for Lys27. However, the role of other histone modifications in regulating PRC2 activity is just beginning to be understood. Here we show that direct recognition of methylated histone H3 Lys36 (H3K36me), a mark associated with activation, by the PRC2 subunit Phf19 is required for the full enzymatic activity of the PRC2 complex. Using NMR spectroscopy, we provide structural evidence for this interaction. Furthermore, we show that Phf19 binds to a subset of PRC2 targets in mouse embryonic stem cells and that this is required for their repression and for H3K27me3 deposition. These findings show that the interaction of Phf19 with H3K36me2 and H3K36me3 is essential for PRC2 complex activity and for proper regulation of gene repression in embryonic stem cells.

  View details for DOI 10.1038/nsmb.2434

  View details for Web of Science ID 000311991700012

  View details for PubMedID 23104054

• Everybody's welcome: The big tent approach to epigenetic drug discovery. Drug discovery today. Therapeutic strategies Green, E. M., Gozani, O. 2012; 9 (2-3): e75-e81

  Abstract

  The rapid expansion of epigenetics research is fueled by the increasing understanding that epigenetic processes are critical to regulating cellular development and dysfunction of epigenetic programs is responsible for a diverse set of human pathologies, including cancer, autoimmune and neurodegenerative diseases. The expansive set of components contributing to epigenetic disease mechanisms and the often reversible nature of epigenetic lesions provide prime opportunities for the development of novel therapeutic strategies. Here, we provide an overview of epigenetics and its relationship to disease, discuss current epigenetics-based therapies and suggest new avenues for the identification of therapies targeting deregulated epigenetic programs in disease.

  View details for PubMedID 23505394

• On silico peptide microarrays for high-resolution mapping of antibody epitopes and diverse protein-protein interactions NATURE MEDICINE Price, J. V., Tangsombatvisit, S., Xu, G., Yu, J., Levy, D., Baechler, E. C., Gozani, O., Varma, M., Utz, P. J., Liu, C. L. 2012; 18 (9): 1434-?

  Abstract

  We developed a new, silicon-based peptide array for a broad range of biological applications, including potential development as a real-time point-of-care platform. We used photolithography on silicon wafers to synthesize microarrays (Intel arrays) that contained every possible overlapping peptide within a linear protein sequence covering the N-terminal tail of human histone H2B. These arrays also included peptides with acetylated and methylated lysine residues, reflecting post-translational modifications of H2B. We defined minimum binding epitopes for commercial antibodies recognizing the modified and unmodified H2B peptides. We further found that this platform is suitable for the highly sensitive characterization of methyltransferases and kinase substrates. The Intel arrays also revealed specific H2B epitopes that are recognized by autoantibodies in individuals with systemic lupus erythematosus who have elevated disease severity. By combining emerging nonfluorescence-based detection methods with an underlying integrated circuit, we are now poised to create a truly transformative proteomics platform with applications in bioscience, drug development and clinical diagnostics.

  View details for DOI 10.1038/nm.2913

  View details for Web of Science ID 000308472300042

  View details for PubMedID 22902875

  View details for PubMedCentralID PMC3491111

• SIRT7 links H3K18 deacetylation to maintenance of oncogenic transformation NATURE Barber, M. F., Michishita-Kioi, E., Xi, Y., Tasselli, L., Kioi, M., Moqtaderi, Z., Tennen, R. I., Paredes, S., Young, N. L., Chen, K., Struhl, K., Garcia, B. A., Gozani, O., Li, W., Chua, K. F. 2012; 487 (7405): 114-?

  Abstract

  Sirtuin proteins regulate diverse cellular pathways that influence genomic stability, metabolism and ageing. SIRT7 is a mammalian sirtuin whose biochemical activity, molecular targets and physiological functions have been unclear. Here we show that SIRT7 is an NAD(+)-dependent H3K18Ac (acetylated lysine 18 of histone H3) deacetylase that stabilizes the transformed state of cancer cells. Genome-wide binding studies reveal that SIRT7 binds to promoters of a specific set of gene targets, where it deacetylates H3K18Ac and promotes transcriptional repression. The spectrum of SIRT7 target genes is defined in part by its interaction with the cancer-associated E26 transformed specific (ETS) transcription factor ELK4, and comprises numerous genes with links to tumour suppression. Notably, selective hypoacetylation of H3K18Ac has been linked to oncogenic transformation, and in patients is associated with aggressive tumour phenotypes and poor prognosis. We find that deacetylation of H3K18Ac by SIRT7 is necessary for maintaining essential features of human cancer cells, including anchorage-independent growth and escape from contact inhibition. Moreover, SIRT7 is necessary for a global hypoacetylation of H3K18Ac associated with cellular transformation by the viral oncoprotein E1A. Finally, SIRT7 depletion markedly reduces the tumorigenicity of human cancer cell xenografts in mice. Together, our work establishes SIRT7 as a highly selective H3K18Ac deacetylase and demonstrates a pivotal role for SIRT7 in chromatin regulation, cellular transformation programs and tumour formation in vivo.

  View details for DOI 10.1038/nature11043

  View details for Web of Science ID 000305982900061

  View details for PubMedID 22722849

  View details for PubMedCentralID PMC3412143

• New marks on the block Set5 methylates H4 lysines 5, 8 and 12 NUCLEUS-AUSTIN Green, E. M., Morrison, A. J., Gozani, O. 2012; 3 (4): 335-339

  Abstract

  The methylation of lysine residues in the N-terminal tails of histones is a highly conserved mechanism that regulates critical functions of chromatin, such as the control of gene expression. Using a biochemical approach, we recently identified new methylation marks on the histone H4 tail in budding yeast at lysines 5, 8 and 12, catalyzed by the previously-uncharacterized enzyme Set5. Genetic studies revealed that Set5 functions in cellular processes that also rely on the global chromatin modifying complexes COMPASS and NuA4, which methylate H3 lysine 4 and acetylate H4 lysines 5, 8 and 12, respectively. The identification of new methylation events on the H4 tail raises many intriguing questions regarding their function and their interaction with known histone modifications. Here, we analyze the insights gained about the new enzyme Set5 and the implications for new functionality added to the H4 tail.

  View details for DOI 10.4161/nucl.20695

  View details for Web of Science ID 000315929000009

  View details for PubMedID 22688645

• Methylation by Set9 modulates FoxO3 stability and transcriptional activity AGING-US Calnan, D. R., Webb, A. E., White, J. L., Stowe, T. R., Goswami, T., Shi, X., Espejo, A., Bedford, M. T., Gozani, O., Gygi, S. P., Brunet, A. 2012; 4 (7): 462-479

  Abstract

  The FoxO family of transcription factors plays an important role in longevity and tumor suppression by regulating the expression of a wide range of target genes. FoxO3 has recently been found to be associated with extreme longevity in humans and to regulate the homeostasis of adult stem cell pools in mammals, which may contribute to longevity. The activity of FoxO3 is controlled by a variety of post-translational modifications that have been proposed to form a 'code' affecting FoxO3 subcellular localization, DNA binding ability, protein-protein interactions and protein stability. Lysine methylation is a crucial post-translational modification on histones that regulates chromatin accessibility and is a key part of the 'histone code'. However, whether lysine methylation plays a role in modulating FoxO3 activity has never been examined. Here we show that the methyltransferase Set9 directly methylates FoxO3 in vitro and in cells. Using a combination of tandem mass spectrometry and methyl-specific antibodies, we find that Set9 methylates FoxO3 at a single residue, lysine 271, a site previously known to be deacetylated by Sirt1. Methylation of FoxO3 by Set9 decreases FoxO3 protein stability, while moderately increasing FoxO3 transcriptional activity. The modulation of FoxO3 stability and activity by methylation may be critical for fine-tuning cellular responses to stress stimuli, which may in turn affect FoxO3's ability to promote tumor suppression and longevity.

  View details for Web of Science ID 000307474000004

  View details for PubMedID 22820736

• Smyd3 catalyzes a novel methylation mark and regulates cancer cell proliferation VanAller, G. S., Reynoird, N., Barbash, O., Huddleston, M. J., Le, B., Annan, R. S., Garcia, B. A., Tummino, P. J., Gozani, O., Kruger, R. G. AMER ASSOC CANCER RESEARCH. 2012

  View details for DOI 10.1158/1538-7445.AM2012-3145

  View details for Web of Science ID 000209701605297

• Smyd3 regulates cancer cell phenotypes and catalyzes histone H4 lysine 5 methylation EPIGENETICS Van Aller, G. S., Reynoird, N., Barbash, O., Huddleston, M., Liu, S., Zmoos, A., McDevitt, P., Sinnamon, R., Le, B., Mas, G., Annan, R., Sage, J., Garcia, B. A., Tummino, P. J., Gozani, O., Kruger, R. G. 2012; 7 (4): 340-343

  Abstract

  Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease.

  View details for Web of Science ID 000302493300004

  View details for PubMedID 22419068

  View details for PubMedCentralID PMC3368817

• Lysine Methylation and Regulation of Gene Expression Programs Gozani, O. FEDERATION AMER SOC EXP BIOL. 2012

  View details for Web of Science ID 000310711300283

• Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies ARTHRITIS RESEARCH & THERAPY Liu, C. L., Tangsombatvisit, S., Rosenberg, J. M., Mandelbaum, G., Gillespie, E. C., Gozani, O. P., Alizadeh, A. A., Utz, P. J. 2012; 14 (1)

  Abstract

  Autoreactivity to histones is a pervasive feature of several human autoimmune disorders, including systemic lupus erythematosus (SLE). Specific post-translational modifications (PTMs) of histones within neutrophil extracellular traps (NETs) may potentially drive the process by which tolerance to these chromatin-associated proteins is broken. We hypothesized that NETs and their unique histone PTMs might be capable of inducing autoantibodies that target histones.We developed a novel and efficient method for the in vitro production, visualization, and broad profiling of histone-PTMs of human and murine NETs. We also immunized Balb/c mice with murine NETs and profiled their sera on autoantigen and histone peptide microarrays for evidence of autoantibody production to their immunogen.We confirmed specificity toward acetyl-modified histone H2B as well as to other histone PTMs in sera from patients with SLE known to have autoreactivity against histones. We observed enrichment for distinctive histone marks of transcriptionally silent DNA during NETosis triggered by diverse stimuli. However, NETs derived from human and murine sources did not harbor many of the PTMs toward which autoreactivity was observed in patients with SLE or in MRL/lpr mice. Further, while murine NETs were weak autoantigens in vivo, there was only partial overlap in the immunoglobulin G (IgG) and IgM autoantibody profiles induced by vaccination of mice with NETs and those seen in patients with SLE.Isolated in vivo exposure to NETs is insufficient to break tolerance and may involve additional factors that have yet to be identified.

  View details for DOI 10.1186/ar3707

  View details for Web of Science ID 000304698800039

  View details for PubMedID 22300536

  View details for PubMedCentralID PMC3392818

• Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies (vol 14, R25, 2012) ARTHRITIS RESEARCH & THERAPY Liu, C. L., Tangsombatvisit, S., Rosenberg, J. M., Mandelbaum, G., Gillespie, E. C., Gozani, O. P., Alizadeh, A. A., Utz, P. J. 2012; 14 (4)

  View details for DOI 10.1186/ar3933

  View details for Web of Science ID 000314974600049

• Structure-activity relationships of methyl-lysine reader antagonists MEDCHEMCOMM Herold, J. M., James, L. I., Korboukh, V. K., Gao, C., Coil, K. E., Bua, D. J., Norris, J. L., Kireev, D. B., Brown, P. J., Jin, J., Janzen, W. P., Gozani, O., Frye, S. V. 2012; 3 (1): 45-51

  View details for DOI 10.1039/c1md00195g

  View details for Web of Science ID 000299975300007

• Correction: Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies. Arthritis research & therapy Liu, C. L., Tangsombatvisit, S., Rosenberg, J. M., Mandelbaum, G., Gillespie, E. C., Gozani, O. P., Alizadeh, A. A., Utz, P. J. 2012; 14 (4): 403-?

  View details for DOI 10.1186/ar3933

  View details for PubMedID 22894771

  View details for PubMedCentralID PMC3580574

• A proteomic approach for the identification of novel lysine methyltransferase substrates EPIGENETICS & CHROMATIN Levy, D., Liu, C. L., Yang, Z., Newman, A. M., Alizadeh, A. A., Utz, P. J., Gozani, O. 2011; 4

  Abstract

  Signaling via protein lysine methylation has been proposed to play a central role in the regulation of many physiologic and pathologic programs. In contrast to other post-translational modifications such as phosphorylation, proteome-wide approaches to investigate lysine methylation networks do not exist.In the current study, we used the ProtoArray® platform, containing over 9,500 human proteins, and developed and optimized a system for proteome-wide identification of novel methylation events catalyzed by the protein lysine methyltransferase (PKMT) SETD6. This enzyme had previously been shown to methylate the transcription factor RelA, but it was not known whether SETD6 had other substrates. By using two independent detection approaches, we identified novel candidate substrates for SETD6, and verified that all targets tested in vitro and in cells were genuine substrates.We describe a novel proteome-wide methodology for the identification of new PKMT substrates. This technological advance may lead to a better understanding of the enzymatic activity and substrate specificity of the large number (more than 50) PKMTs present in the human proteome, most of which are uncharacterized.

  View details for DOI 10.1186/1756-8935-4-19

  View details for PubMedID 22024134

• Hypoxia-induced methylation of a pontin chromatin remodeling factor PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lee, J. S., Kim, Y., Bhin, J., Shin, H. R., Nam, H. J., Lee, S. H., Yoon, J., Binda, O., Gozani, O., Hwang, D., Baek, S. H. 2011; 108 (33): 13510-13515

  Abstract

  Pontin is a chromatin remodeling factor that possesses both ATPase and DNA helicase activities. Although Pontin is frequently overexpressed in human cancers of various types and implicated in oncogenic functions, the upstream signaling network leading to the regulation of Pontin that in turn affects transcription of downstream target genes has not been extensively studied. Here, we identify Pontin is methylated by G9a/GLP methyltransferases in hypoxic condition and potentiates HIF-1α-mediated activation by increasing the recruitment of p300 coactivator to a subset of HIF-1α target promoters. Intriguingly, Pontin methylation results in the increased invasive and migratory properties by activating downstream target gene, Ets1. In contrast, inhibition of Pontin methylation results in the suppression of tumorigenic and metastatic properties. Together, our data provide new approaches by targeting Pontin methylation and its downstream targets for the development of therapeutic agents for human cancers.

  View details for DOI 10.1073/pnas.1106106108

  View details for Web of Science ID 000293895100036

  View details for PubMedID 21825155

  View details for PubMedCentralID PMC3158161

• CUL4B: Trash Talking at Chromatin MOLECULAR CELL Green, E. M., Gozani, O. 2011; 43 (3): 321-323

  Abstract

  In this issue, Nakagawa and Xiong (2011) reveal a mechanism targeting WDR5 for proteolysis dependent on the X-linked mental retardation gene, CUL4B. This provides a link between the stability of a chromatin factor and gene expression implicated in neurological pathogenesis.

  View details for DOI 10.1016/j.molce1.2011.07.015

  View details for Web of Science ID 000293680900002

  View details for PubMedID 21816341

  View details for PubMedCentralID PMC3151490

• Structural basis of SETD6-mediated regulation of the NF-kB network via methyl-lysine signaling NUCLEIC ACIDS RESEARCH Chang, Y., Levy, D., Horton, J. R., Peng, J., Zhang, X., Gozani, O., Cheng, X. 2011; 39 (15): 6380-6389

  Abstract

  SET domain containing 6 (SETD6) monomethylates the RelA subunit of nuclear factor kappa B (NF-κB). The ankyrin repeats of G9a-like protein (GLP) recognizes RelA monomethylated at Lys310. Adjacent to Lys310 is Ser311, a known phosphorylation site of RelA. Ser311 phosphorylation inhibits Lys310 methylation by SETD6 as well as binding of Lys310me1 by GLP. The structure of SETD6 in complex with RelA peptide containing the methylation site, in the presence of S-adenosyl-L-methionine, reveals a V-like protein structure and suggests a model for NF-κB binding to SETD6. In addition, structural modeling of the GLP ankyrin repeats bound to Lys310me1 peptide provides insight into the molecular basis for inhibition of Lys310me1 binding by Ser311 phosphorylation. Together, these findings provide a structural explanation for a key cellular signaling pathway centered on RelA Lys310 methylation, which is generated by SETD6 and recognized by GLP, and incorporate a methylation-phosphorylation switch of adjacent lysine and serine residues. Finally, SETD6 is structurally similar to the Rubisco large subunit methyltransferase. Given the restriction of Rubisco to plant species, this particular appearance of the protein lysine methyltransferase has been evolutionarily well conserved.

  View details for DOI 10.1093/nar/gkr256

  View details for Web of Science ID 000294555800013

  View details for PubMedID 21515635

  View details for PubMedCentralID PMC3159447

• Regulation of p53 function by lysine methylation EPIGENOMICS West, L. E., Gozani, O. 2011; 3 (3): 361-369

  Abstract

  The reversible and dynamic methylation of proteins on lysine residues can greatly increase the signaling potential of the modified factor. In addition to histones, several other nuclear factors such as the tumor suppressor and transcription factor p53 undergo lysine methylation, suggesting that this modification may be a common mechanism for modulating protein–protein interactions and key cellular signaling pathways. This article focuses on how lysine methylation events on the C-terminal tail of p53 are generated, sensed and transduced to modulate p53 functions.

  View details for DOI 10.2217/EPI.11.21

  View details for Web of Science ID 000293246400014

  View details for PubMedID 21826189

  View details for PubMedCentralID PMC3151012

• A Chemical Method for Labeling Lysine Methyltransferase Substrates CHEMBIOCHEM Binda, O., Boyce, M., Rush, J. S., Palaniappan, K. K., Bertozzi, C. R., Gozani, O. 2011; 12 (2): 330-334

  Abstract

  Several protein lysine methyltransferases (PKMTs) modify histones to regulate chromatin-dependent cellular processes, such as transcription, DNA replication and DNA damage repair. PKMTs are likely to have many additional substrates in addition to histones, but relatively few nonhistone substrates have been characterized, and the substrate specificity for many PKMTs has yet to be defined. Thus, new unbiased methods are needed to find PKMT substrates. Here, we describe a chemical biology approach for unbiased, proteome-wide identification of novel PKMT substrates. Our strategy makes use of an alkyne-bearing S-adenosylmethionine (SAM) analogue, which is accepted by the PKMT, SETDB1, as a cofactor, resulting in the enzymatic attachment of a terminal alkyne to its substrate. Such labeled proteins can then be treated with azide-functionalized probes to ligate affinity handles or fluorophores to the PKMT substrates. As a proof-of-concept, we have used SETDB1 to transfer the alkyne moiety from the SAM analogue onto a recombinant histone H3 substrate. We anticipate that this chemical method will find broad use in epigenetics to enable unbiased searches for new PKMT substrates by using recombinant enzymes and unnatural SAM cofactors to label and purify many substrates simultaneously from complex organelle or cell extracts.

  View details for DOI 10.1002/cbic.201000433

  View details for Web of Science ID 000286433800014

  View details for PubMedID 21243721

  View details for PubMedCentralID PMC3056122

• TRIM24 links a non-canonical histone signature to breast cancer NATURE Tsai, W., Wang, Z., Yiu, T. T., Akdemir, K. C., Xia, W., Winter, S., Tsai, C., Shi, X., Schwarzer, D., Plunkett, W., Aronow, B., Gozani, O., Fischle, W., Hung, M., Patel, D. J., Barton, M. C. 2010; 468 (7326): 927-U320

  Abstract

  Recognition of modified histone species by distinct structural domains within 'reader' proteins plays a critical role in the regulation of gene expression. Readers that simultaneously recognize histones with multiple marks allow transduction of complex chromatin modification patterns into specific biological outcomes. Here we report that chromatin regulator tripartite motif-containing 24 (TRIM24) functions in humans as a reader of dual histone marks by means of tandem plant homeodomain (PHD) and bromodomain (Bromo) regions. The three-dimensional structure of the PHD-Bromo region of TRIM24 revealed a single functional unit for combinatorial recognition of unmodified H3K4 (that is, histone H3 unmodified at lysine 4, H3K4me0) and acetylated H3K23 (histone H3 acetylated at lysine 23, H3K23ac) within the same histone tail. TRIM24 binds chromatin and oestrogen receptor to activate oestrogen-dependent genes associated with cellular proliferation and tumour development. Aberrant expression of TRIM24 negatively correlates with survival of breast cancer patients. The PHD-Bromo of TRIM24 provides a structural rationale for chromatin activation through a non-canonical histone signature, establishing a new route by which chromatin readers may influence cancer pathogenesis.

  View details for DOI 10.1038/nature09542

  View details for Web of Science ID 000285344600039

  View details for PubMedID 21164480

  View details for PubMedCentralID PMC3058826

• The MBT Repeats of L3MBTL1 Link SET8-mediated p53 Methylation at Lysine 382 to Target Gene Repression JOURNAL OF BIOLOGICAL CHEMISTRY West, L. E., Roy, S., Lachmi-Weiner, K., Hayashi, R., Shi, X., Appella, E., Kutateladze, T. G., Gozani, O. 2010; 285 (48): 37725-37732

  Abstract

  The p53 tumor suppressor protein is regulated by multiple post-translational modifications, including lysine methylation. We previously found that monomethylation of p53 at lysine 382 (p53K382me1) by the protein lysine methyltransferase (PKMT) SET8/PR-Set7 represses p53 transactivation of target genes. However, the molecular mechanism linking p53K382 monomethylation to repression is not known. Here we show in biochemical and crystallographic studies the preferential recognition of p53K382me1 by the triple malignant brain tumor (MBT) repeats of the chromatin compaction factor L3MBTL1. We demonstrate that SET8-mediated methylation of p53 at Lys-382 promotes the interaction between L3MBTL1 and p53 in cells, and the chromatin occupancy of L3MBTL1 at p53 target promoters. In the absence of DNA damage, L3MBTL1 interacts with p53K382me1 and p53-target genes are repressed, whereas depletion of L3MBTL1 results in a p53-dependent increase in p21 and PUMA transcript levels. Activation of p53 by DNA damage is coupled to a decrease in p53K382me1 levels, abrogation of the L3MBTL1-p53 interaction, and disassociation of L3MBTL1 from p53-target promoters. Together, we identify L3MBTL1 as the second known methyl-p53 effector protein, and provide a molecular explanation for the mechanism by which p53K382me1 is transduced to regulate p53 activity.

  View details for DOI 10.1074/jbc.M110.139527

  View details for Web of Science ID 000284424000064

  View details for PubMedID 20870725

  View details for PubMedCentralID PMC2988377

• Methylation of the Retinoblastoma Tumor Suppressor by SMYD2 JOURNAL OF BIOLOGICAL CHEMISTRY Saddic, L. A., West, L. E., Aslanian, A., Yates, J. R., Rubin, S. M., Gozani, O., Sage, J. 2010; 285 (48): 37733-37740

  Abstract

  The retinoblastoma tumor suppressor (RB) is a central cell cycle regulator and tumor suppressor. RB cellular functions are known to be regulated by a diversity of post-translational modifications such as phosphorylation and acetylation, raising the possibility that RB may also be methylated in cells. Here we demonstrate that RB can be methylated by SMYD2 at lysine 860, a highly conserved and novel site of modification. This methylation event occurs in vitro and in cells, and it is regulated during cell cycle progression, cellular differentiation, and in response to DNA damage. Furthermore, we show that RB monomethylation at lysine 860 provides a direct binding site for the methyl-binding domain of the transcriptional repressor L3MBTL1. These results support the idea that a code of post-translational modifications exists for RB and helps guide its functions in mammalian cells.

  View details for DOI 10.1074/jbc.M110.137612

  View details for Web of Science ID 000284424000065

  View details for PubMedID 20870719

  View details for PubMedCentralID PMC2988378

• Trimethylation of histone H3 lysine 4 impairs methylation of histone H3 lysine 9 Regulation of lysine methyltransferases by physical interaction with their substrates EPIGENETICS Binda, O., Leroy, G., Bua, D. J., Garcia, B. A., Gozani, O., Richard, S. 2010; 5 (8): 767-775

  Abstract

  Chromatin is broadly compartmentalized in two defined states: euchromatin and heterochromatin. Generally, euchromatin is trimethylated on histone H3 lysine 4 (H3K4(me3)) while heterochromatin contains the H3K9(me3) marks. The H3K9(me3) modification is added by lysine methyltransferases (KMTs) such as SETDB1. Herein, we show that SETDB1 interacts with its substrate H3, but only in the absence of the euchromatic mark H3K4(me3). In addition, we show that SETDB1 fails to methylate substrates containing the H3K4(me3) mark. Likewise, the functionally related H3K9 KMTs G9A, GLP, and SUV39H1 also fail to bind and to methylate H3K4(me3) substrates. Accordingly, we provide in vivo evidence that H3K9(me2)-enriched histones are devoid of H3K4(me2/3) and that histones depleted of H3K4(me2/3) have elevated H3K9(me2/3). The correlation between the loss of interaction of these KMTs with H3K4 (me3) and concomitant methylation impairment leads to the postulate that, at least these four KMTs, require stable interaction with their respective substrates for optimal activity. Thus, novel substrates could be discovered via the identification of KMT interacting proteins. Indeed, we find that SETDB1 binds to and methylates a novel substrate, the inhibitor of growth protein ING2, while SUV39H1 binds to and methylates the heterochromatin protein HP1α. Thus, our observations suggest a mechanism of post-translational regulation of lysine methylation and propose a potential mechanism for the segregation of the biologically opposing marks, H3K4(me3) and H3K9(me3). Furthermore, the correlation between H3-KMTs interaction and substrate methylation highlights that the identification of novel KMT substrates may be facilitated by the identification of interaction partners.

  View details for DOI 10.4161/epi.5.8.13278

  View details for Web of Science ID 000284263500013

  View details for PubMedID 21124070

  View details for PubMedCentralID PMC3052887

• Decoding Chromatin Goes High Tech CELL Levy, D., Gozani, O. 2010; 142 (6): 844-846

  Abstract

  Identifying proteins that recognize histone methylation is critical for understanding chromatin function. Vermeulen et al. (2010) now describe a cutting-edge strategy to identify and characterize several nuclear proteins and complexes that recognize five major histone trimethyl marks.

  View details for DOI 10.1016/j.cell.2010.08.032

  View details for Web of Science ID 000281855000007

  View details for PubMedID 20850007

• Members of the H3K4 trimethylation complex regulate lifespan in a germline-dependent manner in C. elegans NATURE Greer, E. L., Maures, T. J., Hauswirth, A. G., Green, E. M., Leeman, D. S., Maro, G. S., Han, S., Banko, M. R., Gozani, O., Brunet, A. 2010; 466 (7304): 383-U137

  Abstract

  The plasticity of ageing suggests that longevity may be controlled epigenetically by specific alterations in chromatin state. The link between chromatin and ageing has mostly focused on histone deacetylation by the Sir2 family, but less is known about the role of other histone modifications in longevity. Histone methylation has a crucial role in development and in maintaining stem cell pluripotency in mammals. Regulators of histone methylation have been associated with ageing in worms and flies, but characterization of their role and mechanism of action has been limited. Here we identify the ASH-2 trithorax complex, which trimethylates histone H3 at lysine 4 (H3K4), as a regulator of lifespan in Caenorhabditis elegans in a directed RNA interference (RNAi) screen in fertile worms. Deficiencies in members of the ASH-2 complex-ASH-2 itself, WDR-5 and the H3K4 methyltransferase SET-2-extend worm lifespan. Conversely, the H3K4 demethylase RBR-2 is required for normal lifespan, consistent with the idea that an excess of H3K4 trimethylation-a mark associated with active chromatin-is detrimental for longevity. Lifespan extension induced by ASH-2 complex deficiency requires the presence of an intact adult germline and the continuous production of mature eggs. ASH-2 and RBR-2 act in the germline, at least in part, to regulate lifespan and to control a set of genes involved in lifespan determination. These results indicate that the longevity of the soma is regulated by an H3K4 methyltransferase/demethylase complex acting in the C. elegans germline.

  View details for DOI 10.1038/nature09195

  View details for Web of Science ID 000279867100052

  View details for PubMedID 20555324

  View details for PubMedCentralID PMC3075006

• Binding of the MLL PHD3 Finger to Histone H3K4me3 Is Required for MLL-Dependent Gene Transcription JOURNAL OF MOLECULAR BIOLOGY Chang, P., Hom, R. A., Musselman, C. A., Zhu, L., Kuo, A., Gozani, O., Kutateladze, T. G., Cleary, M. L. 2010; 400 (2): 137-144

  Abstract

  The MLL (mixed-lineage leukemia) proto-oncogene encodes a histone methyltransferase that creates the methylated histone H3K4 epigenetic marks, commonly associated with actively transcribed genes. In addition to its canonical histone methyltransferase SET domain, the MLL protein contains three plant homeodomain (PHD) fingers that are well conserved between species but whose potential roles and requirements for MLL function are unknown. Here, we demonstrate that the third PHD domain of MLL (PHD3) binds histone H3 trimethylated at lysine 4 (H3K4me3) with high affinity and specificity and H3K4me2 with 8-fold lower affinity. Biochemical and structural analyses using NMR and fluorescence spectroscopy identified key amino acids essential for the interaction with H3K4me3. Site-directed mutations of the residues involved in recognition of H3K4me3 compromised in vitro H3K4me3 binding but not in vivo localization of full-length MLL to chromatin sites in target promoters of MEIS1 and HOXA genes. Whereas intact PHD3 finger was necessary for MLL occupancy at these promoters, H3K4me3 binding was critical for MLL transcriptional activity. These results demonstrate that MLL occupancy and target gene activation can be functionally separated. Furthermore, these findings reveal that MLL not only "writes" the H3K4me3 mark but also binds the mark, and this binding is required for the transcriptional maintenance functions of MLL.

  View details for DOI 10.1016/j.jmb.2010.05.005

  View details for Web of Science ID 000279786900003

  View details for PubMedID 20452361

  View details for PubMedCentralID PMC2886590

• Molecular Mechanism of MLL PHD3 and RNA Recognition by the Cyp33 RRM Domain JOURNAL OF MOLECULAR BIOLOGY Hom, R. A., Chang, P., Roy, S., Musselman, C. A., Glass, K. C., Selezneva, A. I., Gozani, O., Ismagilov, R. F., Cleary, M. L., Kutateladze, T. G. 2010; 400 (2): 145-154

  Abstract

  The nuclear protein cyclophilin 33 (Cyp33) is a peptidyl-prolyl cis-trans isomerase that catalyzes cis-trans isomerization of the peptide bond preceding a proline and promotes folding and conformational changes in folded and unfolded proteins. The N-terminal RNA-recognition motif (RRM) domain of Cyp33 has been found to associate with the third plant homeodomain (PHD3) finger of the mixed lineage leukemia (MLL) proto-oncoprotein and a poly(A) RNA sequence. Here, we report a 1.9 A resolution crystal structure of the RRM domain of Cyp33 and describe the molecular mechanism of PHD3 and RNA recognition. The Cyp33 RRM domain folds into a five-stranded antiparallel beta-sheet and two alpha-helices. The RRM domain, but not the catalytic module of Cyp33, binds strongly to PHD3, exhibiting a 2 muM affinity as measured by isothermal titration calorimetry. NMR chemical shift perturbation (CSP) analysis and dynamics data reveal that the beta strands and the beta2-beta3 loop of the RRM domain are involved in the interaction with PHD3. Mutations in the PHD3-binding site or deletions in the beta2-beta3 loop lead to a significantly reduced affinity or abrogation of the interaction. The RNA-binding pocket of the Cyp33 RRM domain, mapped on the basis of NMR CSP and mutagenesis, partially overlaps with the PHD3-binding site, and RNA association is abolished in the presence of MLL PHD3. Full-length Cyp33 acts as a negative regulator of MLL-induced transcription and reduces the expression levels of MLL target genes MEIS1 and HOXA9. Together, these in vitro and in vivo data provide insight into the multiple functions of Cyp33 RRM and suggest a Cyp33-dependent mechanism for regulating the transcriptional activity of MLL.

  View details for DOI 10.1016/j.jmb.2010.04.067

  View details for Web of Science ID 000279786900004

  View details for PubMedID 20460131

  View details for PubMedCentralID PMC3204800

• Negative Regulation of Hypoxic Responses via Induced Reptin Methylation MOLECULAR CELL Lee, J. S., Kim, Y., Kim, I. S., Kim, B., Choi, H. J., Lee, J. M., Shin, H. R., Kim, J. H., Kim, J., Seo, S., Lee, H., Binda, O., Gozani, O., Semenza, G. L., Kim, M., Il Kim, K., Hwang, D., Baek, S. H. 2010; 39 (1): 71-85

  Abstract

  Lysine methylation within histones is crucial for transcriptional regulation and thus links chromatin states to biological outcomes. Although recent studies have extended lysine methylation to nonhistone proteins, underlying molecular mechanisms such as the upstream signaling cascade that induces lysine methylation and downstream target genes modulated by this modification have not been elucidated. Here, we show that Reptin, a chromatin-remodeling factor, is methylated at lysine 67 in hypoxic conditions by the methyltransferase G9a. Methylated Reptin binds to the promoters of a subset of hypoxia-responsive genes and negatively regulates transcription of these genes to modulate cellular responses to hypoxia.

  View details for DOI 10.1016/j.molcel.2010.06.008

  View details for Web of Science ID 000280139200009

  View details for PubMedID 20603076

  View details for PubMedCentralID PMC4651011

• Structural Insight into p53 Recognition by the 53BP1 Tandem Tudor Domain JOURNAL OF MOLECULAR BIOLOGY Roy, S., Musselman, C. A., Kachirskaia, I., Hayashi, R., Glass, K. C., Nix, J. C., Gozani, O., Appella, E., Kutateladze, T. G. 2010; 398 (4): 489-496

  Abstract

  The tumor suppressor p53 and the DNA repair factor 53BP1 (p53 binding protein 1) regulate gene transcription and responses to genotoxic stresses. Upon DNA damage, p53 undergoes dimethylation at Lys382 (p53K382me2), and this posttranslational modification is recognized by 53BP1. The molecular mechanism of nonhistone methyl-lysine mark recognition remains unknown. Here we report a 1. 6-A-resolution crystal structure of the tandem Tudor domain of human 53BP1 bound to a p53K382me2 peptide. In the complex, dimethylated Lys382 is restrained by a set of hydrophobic and cation-pi interactions in a cage formed by four aromatic residues and an aspartate of 53BP1. The signature HKKme2 motif of p53, which defines specificity, is identified through a combination of NMR resonance perturbations, mutagenesis, measurements of binding affinities and docking simulations, and analysis of the crystal structures of 53BP1 bound to p53 peptides containing other dimethyl-lysine marks, p53K370me2 (p53 dimethylated at Lys370) and p53K372me2 (p53 dimethylated at Lys372). Binding of the 53BP1 Tudor domain to p53K382me2 may facilitate p53 accumulation at DNA damage sites and promote DNA repair as suggested by chromatin immunoprecipitation and DNA repair assays. Together, our data detail the molecular mechanism of p53-53BP1 association and provide the basis for deciphering the role of this interaction in the regulation of p53 and 53BP1 functions.

  View details for DOI 10.1016/j.jmb.2010.03.024

  View details for Web of Science ID 000277895500003

  View details for PubMedID 20307547

  View details for PubMedCentralID PMC2865857

• A Functional Link between the Histone Demethylase PHF8 and the Transcription Factor ZNF711 in X-Linked Mental Retardation MOLECULAR CELL Kleine-Kohlbrecher, D., Christensen, J., Vandamme, J., Abarrategui, I., Bak, M., Tommerup, N., Shi, X., Gozani, O., Rappsilber, J., Salcini, A. E., Helin, K. 2010; 38 (2): 165-178

  Abstract

  X-linked mental retardation (XLMR) is an inherited disorder that mostly affects males and is caused by mutations in genes located on the X chromosome. Here, we show that the XLMR protein PHF8 and a C. elegans homolog F29B9.2 catalyze demethylation of di- and monomethylated lysine 9 of histone H3 (H3K9me2/me1). The PHD domain of PHF8 binds to H3K4me3 and colocalizes with H3K4me3 at transcription initiation sites. Furthermore, PHF8 interacts with another XMLR protein, ZNF711, which binds to a subset of PHF8 target genes, including the XLMR gene JARID1C. Of interest, the C. elegans PHF8 homolog is highly expressed in neurons, and mutant animals show impaired locomotion. Taken together, our results functionally link the XLMR gene PHF8 to two other XLMR genes, ZNF711 and JARID1C, indicating that MR genes may be functionally linked in pathways, causing the complex phenotypes observed in patients developing MR.

  View details for DOI 10.1016/j.molcel.2010.03.002

  View details for Web of Science ID 000277598100005

  View details for PubMedID 20346720

  View details for PubMedCentralID PMC2989439

• The Target of the NSD Family of Histone Lysine Methyltransferases Depends on the Nature of the Substrate JOURNAL OF BIOLOGICAL CHEMISTRY Li, Y., Trojer, P., Xu, C., Cheung, P., Kuo, A., Drury, W. J., Qiao, Q., Neubert, T. A., Xu, R., Gozani, O., Reinberg, D. 2009; 284 (49): 34283-34295

  Abstract

  The NSD (nuclear receptor SET domain-containing) family of histone lysine methyltransferases is a critical participant in chromatin integrity as evidenced by the number of human diseases associated with the aberrant expression of its family members. Yet, the specific targets of these enzymes are not clear, with marked discrepancies being reported in the literature. We demonstrate that NSD2 can exhibit disparate target preferences based on the nature of the substrate provided. The NSD2 complex purified from human cells and recombinant NSD2 both exhibit specific targeting of histone H3 lysine 36 (H3K36) when provided with nucleosome substrates, but histone H4 lysine 44 is the primary target in the case of octamer substrates, irrespective of the histones being native or recombinant. This disparity is negated when NSD2 is presented with octamer targets in conjunction with short single- or double-stranded DNA. Although the octamers cannot form nucleosomes, the target is nonetheless nucleosome-specific as is the product, dimethylated H3K36. This study clarifies in part the previous discrepancies reported with respect to NSD targets. We propose that DNA acts as an allosteric effector of NSD2 such that H3K36 becomes the preferred target.

  View details for DOI 10.1074/jbc.M109.034462

  View details for Web of Science ID 000272165200059

  View details for PubMedID 19808676

  View details for PubMedCentralID PMC2797197

• Epigenome Microarray Platform for Proteome-Wide Dissection of Chromatin-Signaling Networks PLOS ONE Bua, D. J., Kuo, A. J., Cheung, P., Liu, C. L., Migliori, V., Espejo, A., Casadio, F., Bassi, C., Amati, B., Bedford, M. T., Guccione, E., Gozani, O. 2009; 4 (8)

  Abstract

  Knowledge of protein domains that function as the biological effectors for diverse post-translational modifications of histones is critical for understanding how nuclear and epigenetic programs are established. Indeed, mutations of chromatin effector domains found within several proteins are associated with multiple human pathologies, including cancer and immunodeficiency syndromes. To date, relatively few effector domains have been identified in comparison to the number of modifications present on histone and non-histone proteins. Here we describe the generation and application of human modified peptide microarrays as a platform for high-throughput discovery of chromatin effectors and for epitope-specificity analysis of antibodies commonly utilized in chromatin research. Screening with a library containing a majority of the Royal Family domains present in the human proteome led to the discovery of TDRD7, JMJ2C, and MPP8 as three new modified histone-binding proteins. Thus, we propose that peptide microarray methodologies are a powerful new tool for elucidating molecular interactions at chromatin.

  View details for DOI 10.1371/journal.pone.0006789

  View details for Web of Science ID 000269335000031

  View details for PubMedID 19956676

  View details for PubMedCentralID PMC2777412

• Cell cycle-dependent deacetylation of telomeric histone H3 lysine K56 by human SIRT6 CELL CYCLE Michishita, E., McCord, R. A., Boxer, L. D., Barber, M. F., Hong, T., Gozani, O., Chua, K. F. 2009; 8 (16): 2664-2666

  View details for Web of Science ID 000268983900036

  View details for PubMedID 19625767

• The structural insight into the biological role of PHD fingers Kutateladze, T. G., Champagne, K. S., Pena, P. V., Musselman, C. A., Hung, T., Saksouk, N., Cote, J., Gozani, O. FEDERATION AMER SOC EXP BIOL. 2009

  View details for Web of Science ID 000208621501262

• NMR assignments and histone specificity of the ING2 PHD finger MAGNETIC RESONANCE IN CHEMISTRY Pena, P. V., Musselman, C. A., Kuo, A. J., Gozani, O., Kutateladze, T. G. 2009; 47 (4): 352-358

  Abstract

  The ING2 plant homeodomain (PHD) finger is recruited to the nucleosome through specific binding to histone H3 trimethylated at lysine 4 (H3K4me3). Here, we describe backbone and side chain assignments of the ING2 PHD finger, analyze its binding to the unmodified and modified histone and p53 peptides, and map the histone H3 and H3K4me3 binding sites based on chemical shift perturbation analysis.

  View details for DOI 10.1002/mrc.2390

  View details for Web of Science ID 000264514200011

  View details for PubMedID 19184981

  View details for PubMedCentralID PMC2758779

• HBO1 HAT Complexes Target Chromatin throughout Gene Coding Regions via Multiple PHD Finger Interactions with Histone H3 Tail MOLECULAR CELL Saksouk, N., Avvakumov, N., Champagne, K. S., Hung, T., Doyon, Y., Cayrou, C., Paquet, E., Ullah, M., Landry, A., Cote, V., Yang, X., Gozani, O., Kutateladze, T. G., Cote, J. 2009; 33 (2): 257-265

  Abstract

  The HBO1 HAT protein is the major source of histone H4 acetylation in vivo and has been shown to play critical roles in gene regulation and DNA replication. A distinctive characteristic of HBO1 HAT complexes is the presence of three PHD finger domains in two different subunits: tumor suppressor proteins ING4/5 and JADE1/2/3. Biochemical and functional analyses indicate that these domains interact with histone H3 N-terminal tail region, but with a different specificity toward its methylation status. Their combinatorial action is essential in regulating chromatin binding and substrate specificity of HBO1 complexes, as well as cell growth. Importantly, localization analyses on the human genome indicate that HBO1 complexes are enriched throughout the coding regions of genes, supporting a role in transcription elongation. These results underline the importance and versatility of PHD finger domains in regulating chromatin association and histone modification crosstalk within a single protein complex.

  View details for DOI 10.1016/j.molcel.2009.01.007

  View details for Web of Science ID 000263204500016

  View details for PubMedID 19187766

  View details for PubMedCentralID PMC2677731

• SIRT6 stabilizes DNA-dependent protein kinase at chromatin for DNA double-strand break repair AGING-US McCord, R. A., Michishita, E., Hong, T., Berber, E., Boxer, L. D., Kusumoto, R., Guan, S., Shi, X., Gozani, O., Burlingame, A. L., Bohr, V. A., Chua, K. F. 2009; 1 (1): 109-121

  Abstract

  The Sir2 chromatin regulatory factor links maintenance of genomic stability to life span extension in yeast. The mammalian Sir2 family member SIRT6 has been proposed to have analogous functions, because SIRT6-deficiency leads to shortened life span and an aging-like degenerative phenotype in mice, and SIRT6 knockout cells exhibit genomic instability and DNA damage hypersensitivity. However, the molecular mechanisms underlying these defects are not fully understood. Here, we show that SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB) repair factor DNA-PK (DNA-dependent protein kinase) and promotes DNA DSB repair. In response to DSBs, SIRT6 associates dynamically with chromatin and is necessary for an acute decrease in global cellular acetylation levels on histone H3 Lysine 9. Moreover, SIRT6 is required for mobilization of the DNA-PK catalytic subunit (DNA-PKcs) to chromatin in response to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced site-specific DSB. Abrogation of these SIRT6 activities leads to impaired resolution of DSBs. Together, these findings elucidate a mechanism whereby regulation of dynamic interaction of a DNA repair factor with chromatin impacts on the efficiency of repair, and establish a link between chromatin regulation, DNA repair, and a mammalian Sir2 factor.

  View details for Web of Science ID 000276347200013

  View details for PubMedID 20157594

• Role for 53BP1 Tudor Domain Recognition of p53 Dimethylated at Lysine 382 in DNA Damage Signaling JOURNAL OF BIOLOGICAL CHEMISTRY Kachirskaia, I., Shi, X., Yamaguchi, H., Tanoue, K., Wen, H., Wang, E. W., Appella, E., Gozani, O. 2008; 283 (50): 34660-34666

  Abstract

  Modification of histone proteins by lysine methylation is a principal chromatin regulatory mechanism (Shi, Y., and Whetstine, J. R. (2007) Mol. Cell 25, 1-14). Recently, lysine methylation has been shown also to play a role in regulating non-histone proteins, including the tumor suppressor protein p53 (Huang, J., and Berger, S. L. (2008) Curr. Opin. Genet. Dev. 18, 152-158). Here, we identify a novel p53 species that is dimethylated at lysine 382 (p53K382me2) and show that the tandem Tudor domain of the DNA damage response mediator 53BP1 acts as an "effector" for this mark. We demonstrate that the 53BP1 tandem Tudor domain recognizes p53K382me2 with a selectivity relative to several other protein lysine methylation sites and saturation states. p53K382me2 levels increase with DNA damage, and recognition of this modification by 53BP1 facilitates an interaction between p53 and 53BP1. The generation of p53K382me2 promotes the accumulation of p53 protein that occurs upon DNA damage, and this increase in p53 levels requires 53BP1. Taken together, our study identifies a novel p53 modification, demonstrates a new effector function for the 53BP1 tandem Tudor domain, and provides insight into how DNA damage signals are transduced to stabilize p53.

  View details for DOI 10.1074/jbc.M806020200

  View details for Web of Science ID 000261469100021

  View details for PubMedID 18840612

  View details for PubMedCentralID PMC2596381

• Aire employs a histone-binding module to mediate immunological tolerance, linking chromatin regulation with organ-specific autoimmunity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Koh, A. S., Kuo, A. J., Park, S. Y., Cheung, P., Abramson, J., Bua, D., Carney, D., Shoelson, S. E., Gozani, O., Kingston, R. E., Benoist, C., Mathis, D. 2008; 105 (41): 15878-15883

  Abstract

  Aire induces ectopic expression of peripheral tissue antigens (PTAs) in thymic medullary epithelial cells, which promotes immunological tolerance. Beginning with a broad screen of histone peptides, we demonstrate that the mechanism by which this single factor controls the transcription of thousands of genes involves recognition of the amino-terminal tail of histone H3, but not of other histones, by one of Aire's plant homeodomain (PHD) fingers. Certain posttranslational modifications of H3 tails, notably dimethylation or trimethylation at H3K4, abrogated binding by Aire, whereas others were tolerated. Similar PHD finger-H3 tail-binding properties were recently reported for BRAF-histone deacetylase complex 80 and DNA methyltransferase 3L; sequence alignment, molecular modeling, and biochemical analyses showed these factors and Aire to have structure-function relationships in common. In addition, certain PHD1 mutations underlying the polyendocrine disorder autoimmune polyendocrinopathy-candidiases-ectodermaldystrophy compromised Aire recognition of H3. In vitro binding assays demonstrated direct physical interaction between Aire and nucleosomes, which was in part buttressed by its affinity to DNA. In vivo Aire interactions with chromosomal regions depleted of H3K4me3 were dependent on its H3 tail-binding activity, and this binding was necessary but not sufficient for the up-regulation of genes encoding PTAs. Thus, Aire's activity as a histone-binding module mediates the thymic display of PTAs that promotes self-tolerance and prevents organ-specific autoimmunity.

  View details for DOI 10.1073/pnas.0808470105

  View details for Web of Science ID 000260240900044

  View details for PubMedID 18840680

  View details for PubMedCentralID PMC2572939

• Histone H3K4me3 binding is required for the DNA repair and apoptotic activities of ING1 tumor suppressor JOURNAL OF MOLECULAR BIOLOGY Pena, P. V., Hom, R. A., Hung, T., Lin, H., Kuo, A. J., Wong, R. P., Subach, O. M., Champagne, K. S., Zhao, R., Verkhusha, V. V., Li, G., Gozani, O., Kutateladze, T. G. 2008; 380 (2): 303-312

  Abstract

  Inhibitor of growth 1 (ING1) is implicated in oncogenesis, DNA damage repair, and apoptosis. Mutations within the ING1 gene and altered expression levels of ING1 are found in multiple human cancers. Here, we show that both DNA repair and apoptotic activities of ING1 require the interaction of the C-terminal plant homeodomain (PHD) finger with histone H3 trimethylated at Lys4 (H3K4me3). The ING1 PHD finger recognizes methylated H3K4 but not other histone modifications as revealed by the peptide microarrays. The molecular mechanism of the histone recognition is elucidated based on a 2.1 A-resolution crystal structure of the PHD-H3K4me3 complex. The K4me3 occupies a deep hydrophobic pocket formed by the conserved Y212 and W235 residues that make cation-pi contacts with the trimethylammonium group. Both aromatic residues are essential in the H3K4me3 recognition, as substitution of these residues with Ala disrupts the interaction. Unlike the wild-type ING1, the W235A mutant, overexpressed in the stable clones of melanoma cells or in HT1080 cells, was unable to stimulate DNA repair after UV irradiation or promote DNA-damage-induced apoptosis, indicating that H3K4me3 binding is necessary for these biological functions of ING1. Furthermore, N216S, V218I, and G221V mutations, found in human malignancies, impair the ability of ING1 to associate with H3K4me3 or to induce nucleotide repair and cell death, linking the tumorigenic activity of ING1 with epigenetic regulation. Together, our findings reveal the critical role of the H3K4me3 interaction in mediating cellular responses to genotoxic stresses and offer new insight into the molecular mechanism underlying the tumor suppressive activity of ING1.

  View details for DOI 10.1016/j.jmb.2008.04.061

  View details for Web of Science ID 000257630000004

  View details for PubMedID 18533182

• RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination Matthews, A. G., Kuo, A. J., Ramon-Maiques, S., Han, S., Champagne, K. S., Kutateladze, T. G., Yang, W., Gozani, O., Oettinger, M. A. FEDERATION AMER SOC EXP BIOL. 2008

  View details for Web of Science ID 000208467802229

• Structural characterization of histone-binding PHD fingers Kutateladze, T. G., Champagne, K., Pena, P., Hom, R., Musselman, C., Hung, T., Gozani, O. FEDERATION AMER SOC EXP BIOL. 2008

  View details for Web of Science ID 000208467808339

• SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin NATURE Michishita, E., McCord, R. A., Berber, E., Kioi, M., Padilla-Nash, H., Damian, M., Cheung, P., Kusumoto, R., Kawahara, T. L., Barrett, J. C., Chang, H. Y., Bohr, V. A., Ried, T., Gozani, O., Chua, K. F. 2008; 452 (7186): 492-U16

  Abstract

  The Sir2 deacetylase regulates chromatin silencing and lifespan in Saccharomyces cerevisiae. In mice, deficiency for the Sir2 family member SIRT6 leads to a shortened lifespan and a premature ageing-like phenotype. However, the molecular mechanisms of SIRT6 function are unclear. SIRT6 is a chromatin-associated protein, but no enzymatic activity of SIRT6 at chromatin has yet been detected, and the identity of physiological SIRT6 substrates is unknown. Here we show that the human SIRT6 protein is an NAD+-dependent, histone H3 lysine 9 (H3K9) deacetylase that modulates telomeric chromatin. SIRT6 associates specifically with telomeres, and SIRT6 depletion leads to telomere dysfunction with end-to-end chromosomal fusions and premature cellular senescence. Moreover, SIRT6-depleted cells exhibit abnormal telomere structures that resemble defects observed in Werner syndrome, a premature ageing disorder. At telomeric chromatin, SIRT6 deacetylates H3K9 and is required for the stable association of WRN, the factor that is mutated in Werner syndrome. We propose that SIRT6 contributes to the propagation of a specialized chromatin state at mammalian telomeres, which in turn is required for proper telomere metabolism and function. Our findings constitute the first identification of a physiological enzymatic activity of SIRT6, and link chromatin regulation by SIRT6 to telomere maintenance and a human premature ageing syndrome.

  View details for DOI 10.1038/nature06736

  View details for PubMedID 18337721

• The plant homeodomain finger of RAG2 recognizes histone H3 methylated at both lysine-4 and arginine-2 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ramon-Maiques, S., Kuo, A. J., Carney, D., Matthews, A. G., Oettinger, M. A., Gozani, O., Yang, W. 2007; 104 (48): 18993-18998

  Abstract

  Recombination activating gene (RAG) 1 and RAG2 together catalyze V(D)J gene rearrangement in lymphocytes as the first step in the assembly and maturation of antigen receptors. RAG2 contains a plant homeodomain (PHD) near its C terminus (RAG2-PHD) that recognizes histone H3 methylated at lysine 4 (H3K4me) and influences V(D)J recombination. We report here crystal structures of RAG2-PHD alone and complexed with five modified H3 peptides. Two aspects of RAG2-PHD are unique. First, in the absence of the modified peptide, a peptide N-terminal to RAG2-PHD occupies the substrate-binding site, which may reflect an autoregulatory mechanism. Second, in contrast to other H3K4me3-binding PHD domains, RAG2-PHD substitutes a carboxylate that interacts with arginine 2 (R2) with a Tyr, resulting in binding to H3K4me3 that is enhanced rather than inhibited by dimethylation of R2. Five residues involved in histone H3 recognition were found mutated in severe combined immunodeficiency (SCID) patients. Disruption of the RAG2-PHD structure appears to lead to the absence of T and B lymphocytes, whereas failure to bind H3K4me3 is linked to Omenn Syndrome. This work provides a molecular basis for chromatin-dependent gene recombination and presents a single protein domain that simultaneously recognizes two distinct histone modifications, revealing added complexity in the read-out of combinatorial histone modifications.

  View details for DOI 10.1073/pnas.0709170104

  View details for Web of Science ID 000251498700024

  View details for PubMedID 18025461

  View details for PubMedCentralID PMC2141896

• Recognition of unmethylated histone H3 lysine 4 links BHC80 to LSD1-mediated gene repression NATURE Lan, F., Collins, R. E., De Cegli, R., Alpatov, R., Horton, J. R., Shi, X., Gozani, O., Cheng, X., Shi, Y. 2007; 448 (7154): 718-U14

  Abstract

  Histone methylation is crucial for regulating chromatin structure, gene transcription and the epigenetic state of the cell. LSD1 is a lysine-specific histone demethylase that represses transcription by demethylating histone H3 on lysine 4 (ref. 1). The LSD1 complex contains a number of proteins, all of which have been assigned roles in events upstream of LSD1-mediated demethylation apart from BHC80 (also known as PHF21A), a plant homeodomain (PHD) finger-containing protein. Here we report that, in contrast to the PHD fingers of the bromodomain PHD finger transcription factor (BPTF) and inhibitor of growth family 2 (ING2), which bind methylated H3K4 (H3K4me3), the PHD finger of BHC80 binds unmethylated H3K4 (H3K4me0), and this interaction is specifically abrogated by methylation of H3K4. The crystal structure of the PHD finger of BHC80 bound to an unmodified H3 peptide has revealed the structural basis of the recognition of H3K4me0. Knockdown of BHC80 by RNA inhibition results in the de-repression of LSD1 target genes, and this repression is restored by the reintroduction of wild-type BHC80 but not by a PHD-finger mutant that cannot bind H3. Chromatin immunoprecipitation showed that BHC80 and LSD1 depend reciprocally on one another to associate with chromatin. These findings couple the function of BHC80 to that of LSD1, and indicate that unmodified H3K4 is part of the 'histone code'. They further raise the possibility that the generation and recognition of the unmodified state on histone tails in general might be just as crucial as post-translational modifications of histone for chromatin and transcriptional regulation.

  View details for DOI 10.1038/nature06034

  View details for Web of Science ID 000248598000050

  View details for PubMedID 17687328

  View details for PubMedCentralID PMC2702779

• Stabilized phosphatidylinositol-5-phosphate analogues as ligands for the nuclear protein ING2: Chemistry, biology, and molecular modeling JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Huang, W., Zhang, H., Davrazou, F., Kutateladze, T. G., Shi, X., Gozani, O., Prestwich, G. D. 2007; 129 (20): 6498-6506

  Abstract

  The interaction of PtdIns(5)P with the tumor suppressor protein ING2 has been implicated in the regulation of chromatin modification. To enhance the stability of PtdIns(5)P for studies of the biological role in vivo, two phosphatase-resistant moieties were used to replace the labile 5-phosphate. The total asymmetric synthesis of the 5-methylenephosphonate (MP) and 5-phosphothionate (PT) analogues of PtdIns(5)P is described herein, and the resulting metabolically stabilized lipid analogues were evaluated in three ways. First, liposomes containing either the dioleoyl MP or PT analogues bound to recombinant ING2 similar to liposomes containing dipalmitoyl PtdIns(5)P, indicating that the replacement of the hydrolyzable 5-phosphate group does not compromise the binding. Second, the dioleoyl MP and PT PtdIns(5)P analogues were equivalent to dipalmitoyl PtdIns(5)P in augmenting cell death induced by a DNA double-strand break in HT1080 cells. Finally, molecular modeling and docking of the MP or PT analogues to the C-terminus PtdInsP-binding region of ING2 (consisting of a PHD finger and a polybasic region) revealed a number of complementary surface and electrostatic contacts between the lipids and ING2.

  View details for DOI 10.1021/ja070195b

  View details for Web of Science ID 000246535300040

  View details for PubMedID 17469822

  View details for PubMedCentralID PMC2553394

• Structures and function of PHD ringers of ING tumor suppressors Experimental Biology 2007 Annual Meeting Pena, P. V., Davrazou, F., Hom, R., Champagne, K., Shi, X., Walter, K. L., Verkhusha, V. V., Gozani, O., Zhao, R., Kutateladze, T. G. FEDERATION AMER SOC EXP BIOL. 2007: A283–A283

  View details for Web of Science ID 000245708502202

• RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination Matthews, A., Kuo, A., Ramon-Maiques, S., Han, S., Gallardo, M., Yang, W., Gozani, O., Oettinger, M. AMER ASSOC IMMUNOLOGISTS. 2007

  View details for Web of Science ID 000209758201064

• Proteome-wide analysis in Saccharomyces cerevisiae identifies several PHD fingers as novel direct and selective binding modules of histone H3 methylated at either lysine 4 or lysine 36 JOURNAL OF BIOLOGICAL CHEMISTRY Shi, X., Kachirskaia, I., Walter, K. L., Kuo, J. A., Lake, A., Davrazou, F., Chan, S. M., Martin, D. G., Fingerman, I. M., Briggs, S. D., Howe, L., Utz, P. J., Kutateladze, T. G., Lugovskoy, A. A., Bedford, M. T., Gozani, O. 2007; 282 (4): 2450-2455

  Abstract

  The PHD finger motif is a signature chromatin-associated motif that is found throughout eukaryotic proteomes. Here we have determined the histone methyl-lysine binding activity of the PHD fingers present within the Saccharomyces cerevisiae proteome. We provide evidence on the genomic scale that PHD fingers constitute a general class of effector modules for histone H3 trimethylated at lysine 4 (H3K4me3) and histone H3 trimethylated at lysine 36 (H3K36me3). Structural modeling of PHD fingers demonstrates a conserved mechanism for recognizing the trimethyl moiety and provides insight into the molecular basis of affinity for the different methyl-histone ligands. Together, our study suggests that a common function for PHD fingers is to transduce methyl-lysine events and sheds light on how a single histone modification can be linked to multiple biological outcomes.

  View details for DOI 10.1074/jbc.C600286200

  View details for Web of Science ID 000243593200036

  View details for PubMedID 17142463

  View details for PubMedCentralID PMC2735445

• The Yng1p plant homeodomain finger is a methyl-histone binding module that recognizes lysine 4-methylated histone H3 MOLECULAR AND CELLULAR BIOLOGY Martin, D. G., Baetz, K., Shi, X., Walter, K. L., MacDonald, V. E., Wlodarski, M. J., Gozani, O., Hieter, P., Howe, L. 2006; 26 (21): 7871-7879

  Abstract

  The ING (inhibitor of growth) protein family includes a group of homologous nuclear proteins that share a highly conserved plant homeodomain (PHD) finger domain at their carboxyl termini. Members of this family are found in multiprotein complexes that posttranslationally modify histones, suggesting that these proteins serve a general role in permitting various enzymatic activities to interact with nucleosomes. There are three members of the ING family in Saccharomyces cerevisiae: Yng1p, Yng2p, and Pho23p. Yng1p is a component of the NuA3 histone acetyltransferase complex and is required for the interaction of NuA3 with chromatin. To gain insight into the function of the ING proteins, we made use of a genetic strategy to identify genes required for the binding of Yng1p to histones. Using the toxicity of YNG1 overexpression as a tool, we showed that Yng1p interacts with the amino-terminal tail of histone H3 and that this interaction can be disrupted by loss of lysine 4 methylation within this tail. Additionally, we mapped the region of Yng1p required for overexpression of toxicity to the PHD finger, showed that this region capable of binding lysine 4-methylated histone H3 in vitro, and demonstrated that mutations of the PHD finger that abolish binding in vitro are no longer toxic in vivo. These results identify a novel function for the Yng1p PHD finger in promoting stabilization of the NuA3 complex at chromatin through recognition of histone H3 lysine 4 methylation.

  View details for DOI 10.1128/MCB.00573-06

  View details for Web of Science ID 000241462400005

  View details for PubMedID 16923967

  View details for PubMedCentralID PMC1636756

• Molecular mechanism of histone H3K4me3 recognition by plant homeodomain of ING2 NATURE Pena, P. V., Davrazou, F., Shi, X., Walter, K. L., Verkhusha, V. V., Gozani, O., Zhao, R., Kutateladze, T. G. 2006; 442 (7098): 100-103

  Abstract

  Covalent modifications of histone tails have a key role in regulating chromatin structure and controlling transcriptional activity. In eukaryotes, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with active chromatin and gene expression. We recently found that plant homeodomain (PHD) finger of tumour suppressor ING2 (inhibitor of growth 2) binds H3K4me3 and represents a new family of modules that target this epigenetic mark. The molecular mechanism of H3K4me3 recognition, however, remains unknown. Here we report a 2.0 A resolution structure of the mouse ING2 PHD finger in complex with a histone H3 peptide trimethylated at lysine 4. The H3K4me3 tail is bound in an extended conformation in a deep and extensive binding site consisting of elements that are conserved among the ING family of proteins. The trimethylammonium group of Lys 4 is recognized by the aromatic side chains of Y215 and W238 residues, whereas the intermolecular hydrogen-bonding and complementary surface interactions, involving Ala 1, Arg 2, Thr 3 and Thr 6 of the peptide, account for the PHD finger's high specificity and affinity. Substitution of the binding site residues disrupts H3K4me3 interaction in vitro and impairs the ability of ING2 to induce apoptosis in vivo. Strong binding of other ING and YNG PHD fingers suggests that the recognition of H3K4me3 histone code is a general feature of the ING/YNG proteins. Elucidation of the mechanisms underlying this novel function of PHD fingers provides a basis for deciphering the role of the ING family of tumour suppressors in chromatin regulation and signalling.

  View details for DOI 10.1038/nature04814

  View details for Web of Science ID 000238724500045

  View details for PubMedID 16728977

  View details for PubMedCentralID PMC3190580

• PtdIns(5)P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection EMBO JOURNAL Pendaries, C., Tronchere, H., Arbibe, L., Mounier, J., Gozani, O., Cantley, L., Fry, M. J., Gaits-Iacovoni, F., Sansonetti, P. J., Payrastre, B. 2006; 25 (5): 1024-1034

  Abstract

  The virulence factor IpgD, delivered into nonphagocytic cells by the type III secretion system of the pathogen Shigella flexneri, is a phosphoinositide 4-phosphatase generating phosphatidylinositol 5 monophosphate (PtdIns5P). We show that PtdIns5P is rapidly produced and concentrated at the entry foci of the bacteria, where it colocalises with phosphorylated Akt during the first steps of infection. Moreover, S. flexneri-induced phosphorylation of host cell Akt and its targets specifically requires IpgD. Ectopic expression of IpgD in various cell types, but not of its inactive mutant, or addition of short-chain penetrating PtdIns5P is sufficient to induce Akt phosphorylation. Conversely, sequestration of PtdIns5P or reduction of its level strongly decreases Akt phosphorylation in infected cells or in IpgD-expressing cells. Accordingly, IpgD and PtdIns5P production specifically activates a class IA PI 3-kinase via a mechanism involving tyrosine phosphorylations. Thus, S. flexneri parasitism is shedding light onto a new mechanism of PI 3-kinase/Akt activation via PtdIns5P production that plays an important role in host cell responses such as survival.

  View details for DOI 10.1038/sj.emboj.7601001

  View details for Web of Science ID 000236225400010

  View details for PubMedID 16482216

  View details for PubMedCentralID PMC1409730

• The fellowships of the INGs JOURNAL OF CELLULAR BIOCHEMISTRY Shi, X. B., Gozani, O. 2005; 96 (6): 1127-1136

  Abstract

  The inhibitor of growth (ING) family of proteins is an evolutionarily conserved family, with members present from yeast to humans. The mammalian ING proteins are candidate tumor suppressor proteins and accordingly can cooperate with p53 to arrest proliferation and induce apoptosis. ING proteins are also reported to function in the promotion of cellular senescence, the regulation of DNA damage responses and the inhibition of angiogenesis. At the molecular level, ING proteins are thought to function as chromatin regulatory molecules, acting as co-factors for distinct histone and factor acetyl-transferase (H/FAT) and deacetylase (HDAC) enzyme complexes. Further, ING proteins interact with a number of additional proteins involved in the regulation of critical nuclear processes, such as gene expression and DNA replication, and also function as nuclear phosphoinositide (PtdInsP) receptors. Despite the increasing number of known molecular interacting partners for ING proteins, the specific biochemical action of mammalian ING proteins and its relationship to tumor suppression remain elusive. In this Prospect, we summarize the present understanding of the binding partners and physiologic roles of ING proteins and propose a general molecular paradigm for how ING proteins might function to prevent cancer.

  View details for DOI 10.1002/jcb.20625

  View details for Web of Science ID 000233353000003

  View details for PubMedID 16167330

• A PHD finger motif in the C terminus of RAG2 modulates recombination activity JOURNAL OF BIOLOGICAL CHEMISTRY Elkin, S. K., Ivanov, D., Ewalt, M., Ferguson, C. G., Hyberts, S. G., Sun, Z. Y., Prestwich, G. D., Yuan, J. Y., Wagner, G., Oettinger, M. A., Gozani, O. P. 2005; 280 (31): 28701-28710

  Abstract

  The RAG1 and RAG2 proteins catalyze V(D)J recombination and are essential for generation of the diverse repertoire of antigen receptor genes and effective immune responses. RAG2 is composed of a "core" domain that is required for the recombination reaction and a C-terminal nonessential or "non-core" region. Recent evidence has emerged arguing that the non-core region plays a critical regulatory role in the recombination reaction, and mutations in this region have been identified in patients with immunodeficiencies. Here we present the first structural data for the RAG2 protein, using NMR spectroscopy to demonstrate that the C terminus of RAG2 contains a noncanonical PHD finger. All of the non-core mutations of RAG2 that are implicated in the development of immunodeficiencies are located within the PHD finger, at either zinc-coordinating residues or residues adjacent to an alpha-helix on the surface of the domain that participates in binding to the signaling molecules, phosphoinositides. Functional analysis of disease and phosphoinositide-binding mutations reveals novel intramolecular interactions within the non-core region and suggests that the PHD finger adopts two distinct states. We propose a model in which the equilibrium between these states modulates recombination activity. Together, these data identify the PHD finger as a novel and functionally important domain of RAG2.

  View details for DOI 10.1074/jbc.M504731200

  View details for Web of Science ID 000230857300061

  View details for PubMedID 15964836

• Modification of protein sub-nuclear localization by synthetic phosphoinositides: Evidence for nuclear phosphoinositide signaling mechanisms 45th International Symposium on Regulation of Enzyme Activity and Synthesis in Normal and Neoplastic Tissues Gozani, O., Field, S. J., Ferguson, C. G., Ewalt, M., Mahlke, C., Cantley, L. C., Prestwich, G. D., Yuan, J. Y. ELSEVIER SCIENCE BV. 2005: 171–185

  Abstract

  PtdInsPs are critical signaling molecules that regulate diverse cellular functions. One method to study PtdInsP biology involves using synthetic PtdInsP analogs to activate endogenous PtdInsP-mediated events in living cells. Such methodology has been successfully employed to explore the role of several PtdInsP-biological outcomes in the cytoplasm. However, this strategy has not previously been used to examine the function of PtdInsPs in the nucleus of live cells, primarily because there has not been a well-defined PtdInsP-binding protein to provide functional nuclear readouts. Here we have shown that synthetic PtdIns(5)P analogs access and function in the nucleus. We have found that these molecules modify the sub-nuclear localization of PHD finger-containing proteins in live cells and in real time. This work demonstrates that synthetic PtdInsPs and PtdInsP derivatives may be powerful tools for probing nuclear PtdInsP functions. Finally, our work supports a model that endogenous PtdInsPs regulate sub-nuclear localization and function of endogenous nuclear PtdInsP-binding proteins.

  View details for DOI 10.1016/j.advenzreg.2005.02.010

  View details for Web of Science ID 000235349900013

  View details for PubMedID 16199078

• The PHD finger of the chromatin-associated protein ING2 functions as a nuclear phosphoinositide receptor CELL Gozani, O., Karuman, P., Jones, D. R., Ivanov, D., Cha, J., Lugovskoy, A. A., Baird, C. L., Zhu, H., Field, S. J., Lessnick, S. L., Villasenor, J., Mehrotra, B., Chen, J., Rao, V. R., Brugge, J. S., Ferguson, C. G., Payrastre, B., Myszka, D. G., Cantley, L. C., Wagner, G., Divecha, N., Prestwich, G. D., Yuan, J. Y. 2003; 114 (1): 99-111

  Abstract

  Phosphoinositides (PtdInsPs) play critical roles in cytoplasmic signal transduction pathways. However, their functions in the nucleus are unclear, as specific nuclear receptors for PtdInsPs have not been identified. Here, we show that ING2, a candidate tumor suppressor protein, is a nuclear PtdInsP receptor. ING2 contains a plant homeodomain (PHD) finger, a motif common to many chromatin-regulatory proteins. We find that the PHD fingers of ING2 and other diverse nuclear proteins bind in vitro to PtdInsPs, including the rare PtdInsP species, phosphatidylinositol 5-phosphate (PtdIns(5)P). Further, we demonstrate that the ING2 PHD finger interacts with PtdIns(5)P in vivo and provide evidence that this interaction regulates the ability of ING2 to activate p53 and p53-dependent apoptotic pathways. Together, our data identify the PHD finger as a phosphoinositide binding module and a nuclear PtdInsP receptor, and suggest that PHD-phosphoinositide interactions directly regulate nuclear responses to DNA damage.

  View details for Web of Science ID 000184219900009

  View details for PubMedID 12859901

• Life and death in paradise. Nature cell biology Gozani, O., Boyce, M., Yoo, L., Karuman, P., Yuan, J. 2002; 4 (6): E159-62

  Abstract

  Over 500 researchers participated in a recent American Association for Cancer Research special conference, entitled "Apoptosis and Cancer: Basic Mechanisms and Therapeutic Opportunities in the Post-Genomic Era" (February 13-17, 2002) in sunny Hawaii (Hilton Waikoloa village, Kona, Hawaii). The meeting participants presented the most recent findings on the mechanisms regulating cell death in cancer. In the past decade, apoptosis research has undergone a quantum leap, metamorphosing from a descriptive, phenomenological discipline into a molecularly defined, highly complex signalling field. This transformation was highlighted in the conference's opening talk by meeting co-organizer, John Reed (The Burnham Institute, La Jolla, CA). Reed and colleagues used published protein functional information and bio-informatic mining of the available human genome databases to tabulate the number of human proteins predicted to be involved in regulating apoptosis. The list includes 11 catalytically active caspases, 26 CARD (caspase associated recruitment domain)-, 32 DD (death domain)-, 12 DED (death effector domain)-, 8 BIR (baculovirus inhibitor of apoptosis protein region)-, 24 BH (Bcl-2 homology)-, and 34 PAAD/PYD (pyrin/PAAD)-containing sequences.

  View details for PubMedID 12042835

• The Peutz-Jegher gene product LKB1 is a mediator of p53-dependent cell death MOLECULAR CELL Karuman, P., Gozani, O., Odze, R. D., Zhou, X. C., Zhu, H., Shaw, R., Brien, T. P., Bozzuto, C. D., Ooi, D., Cantley, L. C., Yuan, J. Y. 2001; 7 (6): 1307-1319

  Abstract

  Here, we investigate the mechanism and function of LKB1, a Ser/Thr kinase mutated in Peutz-Jegher syndrome (PJS). We demonstrate that LKB1 physically associates with p53 and regulates specific p53-dependent apoptosis pathways. LKB1 protein is present in both the cytoplasm and nucleus of living cells and translocates to mitochondria during apoptosis. In vivo, LKB1 is highly upregulated in pyknotic intestinal epithelial cells. In contrast, polyps arising in Peutz-Jegher patients are devoid of LKB1 staining and have reduced numbers of apoptotic cells. We propose that a deficiency in apoptosis is a key factor in the formation of multiple benign intestinal polyps in PJS patients, and possibly for the subsequent development of malignant tumors in these patients.

  View details for Web of Science ID 000169547400017

  View details for PubMedID 11430832

• Characterization of a protein complex containing spliceosomal proteins SAPS 49, 130, 145, and 155 MOLECULAR AND CELLULAR BIOLOGY Das, B. K., Xia, L., Palandjian, L., Gozani, O., Chyung, Y., Reed, R. 1999; 19 (10): 6796-6802

  Abstract

  SF3b is a U2 snRNP-associated protein complex essential for spliceosome assembly. Although evidence that SF3b contains the spliceosomal proteins SAPs 49, 130, 145, and 155 has accumulated, a protein-mediated association between all of these proteins has yet to be directly demonstrated. Here we report the isolation of a cDNA encoding SAP 130, which completes the cloning of the putative SF3b complex proteins. Using antibodies to SAP 130 and other putative SF3b components, we showed that SAPs 130, 145, and 155 are present in a protein complex in nuclear extracts and that these proteins associate with one another in purified U2 snRNP. Moreover, SAPs 155 and 130 interact with each other (directly or indirectly) within this complex, and SAPs 49 and 145 are known to interact directly with each other. Thus, together with prior work, our studies indicate that SAPs 49, 130, 145, and 155 are indeed components of SF3b. The Saccharomyces cerevisiae homologs of SAPs 49 and 145 are encoded by essential genes. We show here that the S. cerevisiae homologs of SAPs 130 and 155 (scSAP 130/RSE1 and scSAP 155, respectively) are also essential. Recently, the SF3b proteins were found in purified U12 snRNP, which functionally substitutes for U2 snRNP in the minor spliceosome. This high level of conservation, together with the prior observation that the SF3b proteins interact with pre-mRNA very close to the branch site, suggest that the SF3b complex plays a critical role near or at the spliceosome catalytic core.

  View details for Web of Science ID 000082660200032

  View details for PubMedID 10490618

• Cyclin E associates with components of the pre-mRNA splicing machinery in mammalian cells MOLECULAR AND CELLULAR BIOLOGY Seghezzi, W., Chua, K., Shanahan, F., Gozani, O., Reed, R., Lees, E. 1998; 18 (8): 4526-4536

  Abstract

  Cyclin E-cdk2 is a critical regulator of cell cycle progression from G1 into S phase in mammalian cells. Despite this important function little is known about the downstream targets of this cyclin-kinase complex. Here we have identified components of the pre-mRNA processing machinery as potential targets of cyclin E-cdk2. Cyclin E-specific antibodies coprecipitated a number of cyclin E-associated proteins from cell lysates, among which are the spliceosome-associated proteins, SAP 114, SAP 145, and SAP 155, as well as the snRNP core proteins B' and B. The three SAPs are all subunits of the essential splicing factor SF3, a component of U2 snRNP. Cyclin E antibodies also specifically immunoprecipitated U2 snRNA and the spliceosome from splicing extracts. We demonstrate that SAP 155 serves as a substrate for cyclin E-cdk2 in vitro and that its phosphorylation in the cyclin E complex can be inhibited by the cdk-specific inhibitor p21. SAP 155 contains numerous cdk consensus phosphorylation sites in its N terminus and is phosphorylated prior to catalytic step II of the splicing pathway, suggesting a potential role for cdk regulation. These findings provide evidence that pre-mRNA splicing may be linked to the cell cycle machinery in mammalian cells.

  View details for Web of Science ID 000074950000013

  View details for PubMedID 9671462

• A potential role for U2AF-SAP 155 interactions in recruiting U2 snRNP to the branch site MOLECULAR AND CELLULAR BIOLOGY Gozani, O., Potashkin, J., Reed, R. 1998; 18 (8): 4752-4760

  Abstract

  Base pairing between U2 snRNA and the branchpoint sequence (BPS) is essential for pre-mRNA splicing. Because the metazoan BPS is short and highly degenerate, this interaction alone is insufficient for specific binding of U2 snRNP. The splicing factor U2AF binds to the pyrimidine tract at the 3' splice site in the earliest spliceosomal complex, E, and is essential for U2 snRNP binding in the spliceosomal complex A. We show that the U2 snRNP protein SAP 155 UV cross-links to pre-mRNA on both sides of the BPS in the A complex. SAP 155's downstream cross-linking site is immediately adjacent to the U2AF binding site, and the two proteins interact directly in protein-protein interaction assays. Using UV cross-linking, together with functional analyses of pre-mRNAs containing duplicated BPSs, we show a direct correlation between BPS selection and UV cross-linking of SAP 155 on both sides of the BPS. Together, our data are consistent with a model in which U2AF binds to the pyrimidine tract in the E complex and then interacts with SAP 155 to recruit U2 snRNP to the BPS.

  View details for Web of Science ID 000074950000036

  View details for PubMedID 9671485

• Phosphorylation of spliceosomal protein SAP 155 coupled with splicing catalysis GENES & DEVELOPMENT Wang, C. Y., Chua, K., Seghezzi, W., Lees, E., Gozani, O., Reed, R. 1998; 12 (10): 1409-1414

  Abstract

  The U2 snRNP component SAP 155 contacts pre-mRNA on both sides of the branch site early in spliceosome assembly and is therefore positioned near or at the spliceosome catalytic center. We have isolated a cDNA encoding human SAP 155 and identified its highly related Saccharomyces cerevisiae homolog (50% identity). The carboxy-terminal two-thirds of SAP 155 shows the highest conservation and is remarkably similar to the regulatory subunit A of the phosphatase PP2A. Significantly, SAP 155 is phosphorylated concomitant with or just after catalytic step one, making this the first example of a protein modification tightly regulated with splicing catalysis.

  View details for Web of Science ID 000073793500003

  View details for PubMedID 9585501

• Identification of proteins that interact with exon sequences, splice sites, and the branchpoint sequence during each stage of spliceosome assembly MOLECULAR AND CELLULAR BIOLOGY Chiara, M. D., Gozani, O., Bennett, M., CHAMPIONARNAUD, P., Palandjian, L., Reed, R. 1996; 16 (7): 3317-3326

  Abstract

  We have carried out a systematic analysis of the proteins that interact with specific intron and exon sequences during each stage of mammalian spliceosome assembly. This was achieved by site-specifically labeling individual nucleotides within the 5' and 3' splice sites, the branchpoint sequence (BPS), or the exons with 32P and identifying UV-cross-linked proteins in the E, A, B, or C spliceosomal complex. Significantly, two members of the SR family of splicing factors, which are known to promote E-complex assembly, cross-link within exon sequences to a region approximately 25 nucleotides upstream from the 5' splice site. At the 5' splice site, cross-linking of the U5 small nuclear ribonucleoprotein particle protein, U5(200), was detected in both the B and C complexes. As observed in yeast cells, U5(200), also cross-links to intron/exon sequences at the 3' splice site in the C complex and may play a role in aligning the 5' and 3' exons for ligation. With label at the branch site, we detected three distinct proteins, designated BPS72,BpS70, and BPS56, which replace one another in the E, A, and C complexes. Another dynamic exchange was detected with pre-mRNA labeled at the AG dinucleotide of the 3' splice site. In this case, a protein, AG100,cross-links in the A complex and is replaced by another protein, AG75, in the C complex. The observation that these proteins are specifically associated with critical pre-mRNA sequence elements in functional complexes at different stages of spliceosome assembly implicates roles for these factors in key recognition events during the splicing pathway.

  View details for Web of Science ID A1996UT08600010

  View details for PubMedID 8668147

• Evidence that sequence-independent binding of highly conserved U2 snRNP proteins upstream of the branch site is required for assembly of spliceosomal complex A GENES & DEVELOPMENT Gozani, O., Feld, R., Reed, R. 1996; 10 (2): 233-243

  Abstract

  A critical step in the pre-mRNA splicing reaction is the stable binding of U2 snRNP to the branchpoint sequence (BPS) to form the A complex. The multimeric U2 snRNP protein complexes SF3a and SF3b are required for A complex assembly, but their specific roles in this process are not known. Saccharomyces cerevisiae homologs of all of the SF3a, but none of the SF3b, subunits have been identified. Here we report the isolation of a cDNA encoding the mammalian SF3b subunit SAP 145 and the identification of its probable yeast homolog (29% identity). This first indication that the homology between yeast and metazoan A complex proteins can be extended to SF3b adds strong new evidence that the mechanism of A complex assembly is highly conserved. To investigate this mechanism in the mammalian system we analyzed proteins that cross-link to 32P-site-specifically labeled pre-mRNA in the A complex. This analysis revealed that SAP 145, together with four other SF3a/SF3b subunits, UV cross-links to pre-mRNA in a 20-nucleotide region upstream of the BPS. Mutation of this region, which we have designated the anchoring site, has no apparent effect on U2 snRNP binding. In contrast, when a 2'O methyl oligonucleotide complementary to the anchoring site is added to the spliceosome assembly reaction, A complex assembly and cross-linking of the SF3a/SF3b subunits are blocked. These data indicate that sequence-independent binding of the highly conserved SF3a/SF3b subunits upstream of the branch site is essential for anchoring U2 snRNP to pre-mRNA.

  View details for Web of Science ID A1996TT48900010

  View details for PubMedID 8566756

• ACCUMULATION OF A NOVEL SPLICEOSOMAL COMPLEX ON PRE-MESSENGER-RNAS CONTAINING BRANCH SITE MUTATIONS MOLECULAR AND CELLULAR BIOLOGY CHAMPIONARNAUD, P., Gozani, O., Palandjian, L., Reed, R. 1995; 15 (10): 5750-5756

  Abstract

  Pre-mRNA assembles into spliceosomal complexes in the stepwise pathway E-->A-->B-->C. We show that mutations in the metazoan branchpoint sequence (BPS) have no apparent effect on E complex formation but block the assembly of the A complex and the UV cross-linking of U2 small nuclear ribonucleoprotein particle (snRNP) proteins. Unexpectedly, a novel complex, designated E*, assembles on pre-mRNAs containing BPS mutations. Unlike the E complex, the E* complex accumulates in the presence of ATP. U1 snRNP and U2AF, which are tightly bound to pre-mRNA in the E complex, are not tightly bound in the E* complex. Significantly, previous work showed that U1 snRNP and U2AF become destabilized from pre-mRNA after E complex assembly on normal pre-mRNAs. Thus, our data are consistent with a model in which there are two steps in the transition from the E complex to the A complex (E-->E*-->A). In the first step, U1 snRNP and U2AF are destabilized in an ATP-dependent, BPS-independent reaction. In the second step, the stable binding of U2 snRNP occurs in a BPS-dependent reaction.

  View details for Web of Science ID A1995RV77200059

  View details for PubMedID 7565727

• A NOVEL SET OF SPLICEOSOME-ASSOCIATED PROTEINS AND THE ESSENTIAL SPLICING FACTOR PSF BIND STABLY TO PRE-MESSENGER-RNA PRIOR TO CATALYTIC STEP-II OF THE SPLICING REACTION EMBO JOURNAL Gozani, O., PATTON, J. G., Reed, R. 1994; 13 (14): 3356-3367

  Abstract

  We have isolated and determined the protein composition of the spliceosomal complex C. The pre-mRNA in this complex has undergone catalytic step I, but not step II, of the splicing reaction. We show that a novel set of 14 spliceosome-associated proteins (SAPs) and the essential splicing factor PSF are specifically associated with the C complex, implicating these proteins in catalytic step II. Significantly, immunodepletion and biochemical complementation studies demonstrate directly that PSF is essential for catalytic step II. Purified PSF is known to UV crosslink to pyrimidine tracts, and our data show that PSF UV crosslinks to pre-mRNA in purified C complex. Thus, PSF may replace the 3' splice site binding factor U2AF65 which is destabilized during spliceosome assembly. Finally, we show that SAPs 60 and 90, which are present in both the B and C complexes, are specifically associated with U4 and U6 snRNPs, and thus may have important roles in the functioning of these snRNPs during the splicing reaction.

  View details for Web of Science ID A1994NZ03600016

  View details for PubMedID 8045264