Honors & Awards
Career Development Fellow, Leukemia and Lymphoma Society (2018-2021)
Stanford Dean’s Fellowship, Stanford University (2018)
Fields Prize in Microbiology and Immunobiology (Top PhD Thesis), Harvard Medical School (2017)
HHMI International Predoctoral Fellowship, Howard Hughes Medical Institute (2015-2017)
Library Research Award (Best Thesis), University of Wisconsin-Madison (2011)
Trewartha Senior Thesis Grant, University of Wisconsin-Madison (2010)
National Science Scholarship, Agency of Science, Technology and Research (ASTAR) (2009-2011)
Doctor of Philosophy, Harvard University (2017)
The Epstein-Barr Virus Regulome in Lymphoblastoid Cells.
Cell host & microbe
2017; 22 (4): 561–73.e4
Epstein-Barr virus (EBV) transforms B cells to continuously proliferating lymphoblastoid cell lines (LCLs), which represent an experimental model for EBV-associated cancers. EBV nuclear antigens (EBNAs) and LMP1 are EBV transcriptional regulators that are essential for LCL establishment, proliferation, and survival. Starting with the 3D genome organization map of LCL, we constructed a comprehensive EBV regulome encompassing 1,992 viral/cellular genes and enhancers. Approximately 30% of genes essential for LCL growth were linked to EBV enhancers. Deleting EBNA2 sites significantly reduced their target gene expression. Additional EBV super-enhancer (ESE) targets included MCL1, IRF4, and EBF. MYC ESE looping to the transcriptional stat site of MYC was dependent on EBNAs. Deleting MYC ESEs greatly reduced MYC expression and LCL growth. EBNA3A/3C altered CDKN2A/B spatial organization to suppress senescence. EZH2 inhibition decreased the looping at the CDKN2A/B loci and reduced LCL growth. This study provides a comprehensive view of the spatial organization of chromatin during EBV-driven cellular transformation.
View details for DOI 10.1016/j.chom.2017.09.001
View details for PubMedID 29024646
Epstein-Barr Virus Oncoprotein Super-enhancers Control B Cell Growth
CELL HOST & MICROBE
2015; 17 (2): 205-216
Super-enhancers are clusters of gene-regulatory sites bound by multiple transcription factors that govern cell transcription, development, phenotype, and oncogenesis. By examining Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs), we identified four EBV oncoproteins and five EBV-activated NF-κB subunits co-occupying ∼1,800 enhancer sites. Of these, 187 had markedly higher and broader histone H3K27ac signals, characteristic of super-enhancers, and were designated "EBV super-enhancers." EBV super-enhancer-associated genes included the MYC and BCL2 oncogenes, which enable LCL proliferation and survival. EBV super-enhancers were enriched for B cell transcription factor motifs and had high co-occupancy of STAT5 and NFAT transcription factors (TFs). EBV super-enhancer-associated genes were more highly expressed than other LCL genes. Disrupting EBV super-enhancers by the bromodomain inhibitor JQ1 or conditionally inactivating an EBV oncoprotein or NF-κB decreased MYC or BCL2 expression and arrested LCL growth. These findings provide insight into mechanisms of EBV-induced lymphoproliferation and identify potential therapeutic interventions.
View details for DOI 10.1016/j.chom.2014.12.013
View details for Web of Science ID 000349761700010
View details for PubMedID 25639793
View details for PubMedCentralID PMC4539236
Epstein-Barr Virus Nuclear Antigen 3C binds to BATF/IRF4 or SPI1/IRF4 composite sites and recruits Sin3A to repress CDKN2A
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2014; 111 (1): 421-426
Epstein-Barr virus nuclear antigen 3C (EBNA3C) repression of CDKN2A p14(ARF) and p16(INK4A) is essential for immortal human B-lymphoblastoid cell line (LCL) growth. EBNA3C ChIP-sequencing identified >13,000 EBNA3C sites in LCL DNA. Most EBNA3C sites were associated with active transcription; 64% were strong H3K4me1- and H3K27ac-marked enhancers and 16% were active promoters marked by H3K4me3 and H3K9ac. Using ENCODE LCL transcription factor ChIP-sequencing data, EBNA3C sites coincided (±250 bp) with RUNX3 (64%), BATF (55%), ATF2 (51%), IRF4 (41%), MEF2A (35%), PAX5 (34%), SPI1 (29%), BCL11a (28%), SP1 (26%), TCF12 (23%), NF-κB (23%), POU2F2 (23%), and RBPJ (16%). EBNA3C sites separated into five distinct clusters: (i) Sin3A, (ii) EBNA2/RBPJ, (iii) SPI1, and (iv) strong or (v) weak BATF/IRF4. EBNA3C signals were positively affected by RUNX3, BATF/IRF4 (AICE) and SPI1/IRF4 (EICE) cooccupancy. Gene set enrichment analyses correlated EBNA3C/Sin3A promoter sites with transcription down-regulation (P < 1.6 × 10(-4)). EBNA3C signals were strongest at BATF/IRF4 and SPI1/IRF4 composite sites. EBNA3C bound strongly to the p14(ARF) promoter through SPI1/IRF4/BATF/RUNX3, establishing RBPJ-, Sin3A-, and REST-mediated repression. EBNA3C immune precipitated with Sin3A and conditional EBNA3C inactivation significantly decreased Sin3A binding at the p14(ARF) promoter (P < 0.05). These data support a model in which EBNA3C binds strongly to BATF/IRF4/SPI1/RUNX3 sites to enhance transcription and recruits RBPJ/Sin3A- and REST/NRSF-repressive complexes to repress p14(ARF) and p16(INK4A) expression.
View details for DOI 10.1073/pnas.1321704111
View details for Web of Science ID 000329350700100
View details for PubMedID 24344258
View details for PubMedCentralID PMC3890834
Epstein-Barr virus subverts mevalonate and fatty acid pathways to promote infected B-cell proliferation and survival.
2019; 15 (9): e1008030
Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with multiple human malignancies. EBV drives B-cell proliferation, which contributes to the pathogenesis of multiple lymphomas. Yet, knowledge of how EBV subverts host biosynthetic pathways to transform resting lymphocytes into activated lymphoblasts remains incomplete. Using a temporal proteomic dataset of EBV primary human B-cell infection, we identified that cholesterol and fatty acid biosynthetic pathways were amongst the most highly EBV induced. Epstein-Barr nuclear antigen 2 (EBNA2), sterol response element binding protein (SREBP) and MYC each had important roles in cholesterol and fatty acid pathway induction. Unexpectedly, HMG-CoA reductase inhibitor chemical epistasis experiments revealed that mevalonate pathway production of geranylgeranyl pyrophosphate (GGPP), rather than cholesterol, was necessary for EBV-driven B-cell outgrowth, perhaps because EBV upregulated the low-density lipoprotein receptor in newly infected cells for cholesterol uptake. Chemical and CRISPR genetic analyses highlighted downstream GGPP roles in EBV-infected cell small G protein Rab activation. Rab13 was highly EBV-induced in an EBNA3-dependent manner and served as a chaperone critical for latent membrane protein (LMP) 1 and 2A trafficking and target gene activation in newly infected and in lymphoblastoid B-cells. Collectively, these studies identify highlight multiple potential therapeutic targets for prevention of EBV-transformed B-cell growth and survival.
View details for DOI 10.1371/journal.ppat.1008030
View details for PubMedID 31518366
- Genome-wide CRISPR-based gene knockout screens reveal cellular factors and pathways essential for nasopharyngeal carcinoma JOURNAL OF BIOLOGICAL CHEMISTRY 2019; 294 (25): 9734–45
Genome-wide CRISPR-based gene knockout screens reveal cellular factors and pathways essential for nasopharyngeal carcinoma.
The Journal of biological chemistry
Early diagnosis of nasopharyngeal carcinoma (NPC) is difficult because of a lack of specific symptoms. Many patients have advanced disease at diagnosis, and these patients poorly respond to treatment. New treatments are therefore needed to improve outcomes of NPC. To better understand the molecular pathogenesis of NPC, here we used NPC cell line in a genome-wide CRISPR-based knockout screen to identify the cellular factors and pathways associated with NPC (i.e. dependency factors). This screen identified the MYST histone acetyl transferase complex; NF-kB signaling, purine synthesis, and linear ubiquitination pathways; and MDM2 proto-oncogene (MDM2) as NPC dependency factors/pathways. Using cell growth, cDNA rescue, and inhibitor assays, we found that perturbation of these pathways greatly reduces the growth of NPC cell lines, but does not affect growth of SV40-immortalized normal nasopharyngeal epithelial cells. These results suggest that targeting these pathways/proteins may hold promise for achieving better treatment of patients with NPC.
View details for PubMedID 31073033
RNA-seq analyses of gene expression during Epstein-Barr virus infection of primary B lymphocytes.
Journal of virology
Epstein-Barr virus (EBV) infection of human primary resting B lymphocytes (RBLs) leads to establishment of lymphoblastoid cell lines (LCLs) that can grow indefinitely in vitro. EBV transforms RBLs through expression of viral latency genes and these genes alter host transcription programs. To globally measure the transcriptome changes during EBV transformation, primary human resting B lymphocytes (RBLs) were infected with B95.8 EBV for 0, 2, 4, 7, 14, 21, and 28 days, and polyA plus RNAs were analyzed by RNA-seq. ANOVA analyses found 3669 protein coding genes that were differentially expressed (FDR<0.01). 94% of LCL genes that are essential for LCL growth and survival were differentially expressed. Pathway analyses identified significant enrichment of pathways involved in cell proliferation, DNA repair, metabolism, and anti-viral responses. RNA-seq also identified lncRNAs differentially expressed during EBV infection. CRISPRi and CRISPRa found that CYTOR and NORAD lncRNAs were important for LCL growth. During EBV infection, type III EBV latency genes were expressed rapidly after infection. Immediately after LCL establishment, EBV lytic genes were also expressed in LCLs and ∼4% of the LCLs express gp350. ChIP-seq and POLR2A ChIA-PET data linked EBV enhancers to 90% of EBV-regulated genes. Many genes were linked to enhancers occupied by multiple EBNAs or NF-kB subunits. Incorporating these assays, we generated a comprehensive EBV regulome in LCLs.IMPORTANCEEBV immortalization of RBL is a useful model system to study EBV oncogenesis. By incorporating RNA-seq, ChIP-seq, ChIA-PET, and genome-wide CRISPR screen, we identified key pathways that EBV usurps to enable B cell growth and transformation. Multiple layers of regulation could be achieved by co-operations between multiple EBV transcription factors binding to the same enhancers. EBV manipulated the expression of most cell genes essential for LCL growth and survival. In addition to proteins, lncRNAs regulated by EBV also contributed to LCL growth and survival. The data presented in this paper not only allowed us to further define the molecular pathogenesis of EBV, but also serve as a useful resource to the EBV research community.
View details for DOI 10.1128/JVI.00226-19
View details for PubMedID 31019051
TAF family proteins and MEF2C are essential for Epstein-Barr virus MYC super-enhancer activity.
Journal of virology
Super-enhancers (SEs) are clusters of enhancers marked by extraordinary high and broad chromatin immune-precipitation followed by deep sequencing (ChIP-seq) signals for H3K27ac or other transcription factors (TFs). SEs play pivotal roles in development and oncogenesis. Epstein-Barr virus (EBV) super-enhancers (ESEs) are co-occupied by all essential EBV oncogenes and EBV-activated NF-kB subunits. Perturbation of ESEs stops lymphoblastoid cell line (LCL) growth. To further characterize ESEs and identify proteins critical for ESEs function, MYC ESEs were cloned upstream of a GFP reporter. Reporters driven by MYC ESEs 525kb and 428kb upstream of MYC (525ESE and 428ESE) had very high activities in LCLs but not in EBV-negative BJAB cells. EBNA2 activated MYC ESE driven Luciferase reporters. CRISPRi targeting 525ESE significantly decreased MYC expression. Genome-wide CRISPR screens identified factors essential for ESE activity. TAF family proteins including TAF8, TAF11, and TAF3 were essential for the activity of the integrated 525ESE driven reporter in LCLs. TAF8 and TAF11 knockout significantly decreased 525ESE activity and MYC transcription. MEF2C was also identified to be essential for 525ESE activity. Depletion of MEF2C decreased 525ESE reporter activity, MYC expression, and LCL growth. MEF2C cDNA resistant to CRIPSR cutting rescued MEF2C knockout, restored 525ESE reporter activity and MYC expression. MEF2C depletion decreased IRF4, EBNA2, and SPI1 binding to 525ESE in LCLs. MEF2C depletion also affected the expression of other ESE target genes including ETS1 and BCL2. These data indicated that in addition to EBNA2, TAF family members and MEF2C are essential for ESE activity, MYC expression, and LCL growth.ImportanceSEs play critical roles in cancer development. Since SEs assemble much bigger protein complexes on enhancers than typical enhancers (TEs), they are more sensitive than TEs to perturbations. Understanding the protein composition of SEs that are linked to key oncogenes may identify novel therapeutic targets. A genome-wide CRISPR screen specifically identified proteins essential for MYC ESE activity but not SV40 enhancer. These proteins were not only essential for the reporter activity[underln],[/underln] but were also important for MYC expression and LCL growth. Targeting these proteins may lead to new therapies for EBV-associated cancers.
View details for DOI 10.1128/JVI.00513-19
View details for PubMedID 31167905
CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation.
2019; 28 (5): 1307–22.e8
CD40 has major roles in B cell development, activation, and germinal center responses. CD40 hypoactivity causes immunodeficiency whereas its overexpression causes autoimmunity and lymphomagenesis. To systematically identify B cell autonomous CD40 regulators, we use CRISPR/Cas9 genome-scale screens in Daudi B cells stimulated by multimeric CD40 ligand. These highlight known CD40 pathway components and reveal multiple additional mechanisms regulating CD40. The nuclear ubiquitin ligase FBXO11 supports CD40 expression by targeting repressors CTBP1 and BCL6. FBXO11 knockout decreases primary B cell CD40 abundance and impairs class-switch recombination, suggesting that frequent lymphoma monoallelic FBXO11 mutations may balance BCL6 increase with CD40 loss. At the mRNA level, CELF1 controls exon splicing critical for CD40 activity, while the N6-adenosine methyltransferase WTAP negatively regulates CD40 mRNA abundance. At the protein level, ESCRT negatively regulates activated CD40 levels while the negative feedback phosphatase DUSP10 limits downstream MAPK responses. These results serve as a resource for future studies and highlight potential therapeutic targets.
View details for DOI 10.1016/j.celrep.2019.06.079
View details for PubMedID 31365872
CRISPR/Cas9-Mediated Genome Editing in Epstein-Barr Virus-Transformed Lymphoblastoid B-Cell Lines.
Current protocols in molecular biology
2018; 121: 31.12.1–31.12.23
Epstein-Barr virus (EBV) efficiently transforms primary human B cells into immortalized lymphoblastoid cell lines (LCLs), which are extensively used in human genetic, immunological and virological studies. LCLs provide unlimited sources of DNA for genetic investigation, but can be difficult to manipulate, for instance because low retroviral or lentiviral transduction frequencies hinder experiments that require co-expression of multiple components. This unit details Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 engineering for robust LCL genome editing. We describe the generation and delivery of single-guide RNAs (sgRNAs), or dual-targeting sgRNAs, via lentiviral transduction of LCLs that stably express Cas9 protein. CRISPR/Cas9 editing allows LCL loss-of-function studies, including knock-out of protein-coding genes or deletion of DNA regulatory elements, and can be adapted for large-scale screening approaches. Low transfection efficiencies are a second barrier to performing CRISPR editing in LCLs, which are not typically lipid-transfectable. To circumvent this barrier, we provide an optimized protocol for LCL nucleofection of Cas9/sgRNA ribonucleoprotein complexes (RNPs) as an alternative route to achieve genome editing in LCLs. These editing approaches can also be employed in other B-cell lines, including Burkitt lymphoma and diffuse large B-cell lymphoma cells, and are highly reproducible. © 2018 by John Wiley & Sons, Inc.
View details for PubMedID 29337376
Modulating Gene Expression in Epstein-Barr Virus (EBV)-Positive B Cell Lines with CRISPRa and CRISPRi.
Current protocols in molecular biology
2018; 121: 31.13.1–31.13.18
Epstein-Barr virus (EBV) transforms small resting primary B cells into large lymphoblastoid cells which are able to grow and survive in vitro indefinitely. These cells represent a model for oncogenesis. In this unit, variants of conventional clustered regularly interspaced short palindromic repeats (CRISPR), namely the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) methods, are discussed in the context of gene regulation at genomic DNA promoter and enhancer elements. Lymphoblastoid B cell lines (LCLs) stably expressing nuclease-deficient Cas9 (dCas9)-VP64 (Cas9 associated with CRISPRa) or dCas9-KRAB (Cas9 associated with CRISPRi) are transduced with lentivirus that encodes a single guide RNA (sgRNA) that targets a specific gene locus. The ribonucleoprotein complex formed by the dCas9 molecule and its cognate sgRNA enables sequence-specific binding at a promoter or enhancer of interest to affect the expression of genes regulated by the targeted promoter or enhancer. © 2018 by John Wiley & Sons, Inc.
View details for PubMedID 29337370
View details for PubMedCentralID PMC5774230
CRISPR/Cas9 Screens Reveal Epstein-Barr Virus-Transformed B Cell Host Dependency Factors
CELL HOST & MICROBE
2017; 21 (5): 580-?
Epstein-Barr virus (EBV) causes endemic Burkitt lymphoma (BL) and immunosuppression-related lymphomas. These B cell malignancies arise by distinct transformation pathways and have divergent viral and host expression programs. To identify host dependency factors resulting from these EBV+, B cell-transformed cell states, we performed parallel genome-wide CRISPR/Cas9 loss-of-function screens in BL and lymphoblastoid cell lines (LCLs). These highlighted 57 BL and 87 LCL genes uniquely important for their growth and survival. LCL hits were enriched for EBV-induced genes, including viral super-enhancer targets. Our systematic approach uncovered key mechanisms by which EBV oncoproteins activate the PI3K/AKT pathway and evade tumor suppressor responses. LMP1-induced cFLIP was found to be critical for LCL defense against TNFα-mediated programmed cell death, whereas EBV-induced BATF/IRF4 were critical for BIM suppression and MYC induction in LCLs. Finally, EBV super-enhancer-targeted IRF2 protected LCLs against Blimp1-mediated tumor suppression. Our results identify viral transformation-driven synthetic lethal targets for therapeutic intervention.
View details for DOI 10.1016/j.chom.2017.04.005
View details for Web of Science ID 000400892500009
View details for PubMedID 28494239
Epstein-Barr Virus LMP1 Mediated Oncogenicity.
Journal of virology
Epstein-Barr virus Latent Membrane Protein 1 (LMP1) is expressed in multiple human malignancies, including nasopharynegeal carcinoma, Hodgkin and immunosuppression-associated lymphomas. LMP1 mimics CD40 signaling to activate multiple growth and survival pathways, in particular NF-κB. LMP1 has critical roles in EBV-driven B-cell transformation, and its expression causes fatal lymphoproliferative disease in immunosuppressed mice. Here, we review recent developments in studies of LMP1 signaling, LMP1-induced host dependency factors, mouse models of LMP1 lymphomagenesis and anti-LMP1 immunotherapy approaches.
View details for DOI 10.1128/JVI.01718-16
View details for PubMedID 28835489
Epstein-Barr virus nuclear antigen 3A partially coincides with EBNA3C genome-wide and is tethered to DNA through BATF complexes
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2015; 112 (2): 554-559
Epstein-Barr Virus (EBV) conversion of B-lymphocytes to Lymphoblastoid Cell Lines (LCLs) requires four EBV nuclear antigen (EBNA) oncoproteins: EBNA2, EBNALP, EBNA3A, and EBNA3C. EBNA2 and EBNALP associate with EBV and cell enhancers, up-regulate the EBNA promoter, MYC, and EBV Latent infection Membrane Proteins (LMPs), which up-regulate BCL2 to protect EBV-infected B-cells from MYC proliferation-induced cell death. LCL proliferation induces p16(INK4A) and p14(ARF)-mediated cell senescence. EBNA3A and EBNA3C jointly suppress p16(INK4A) and p14(ARF), enabling continuous cell proliferation. Analyses of the EBNA3A human genome-wide ChIP-seq landscape revealed 37% of 10,000 EBNA3A sites to be at strong enhancers; 28% to be at weak enhancers; 4.4% to be at active promoters; and 6.9% to be at weak and poised promoters. EBNA3A colocalized with BATF-IRF4, ETS-IRF4, RUNX3, and other B-cell Transcription Factors (TFs). EBNA3A sites clustered into seven unique groups, with differing B-cell TFs and epigenetic marks. EBNA3A coincidence with BATF-IRF4 or RUNX3 was associated with stronger EBNA3A ChIP-Seq signals. EBNA3A was at MYC, CDKN2A/B, CCND2, CXCL9/10, and BCL2, together with RUNX3, BATF, IRF4, and SPI1. ChIP-re-ChIP revealed complexes of EBNA3A on DNA with BATF. These data strongly support a model in which EBNA3A is tethered to DNA through a BATF-containing protein complexes to enable continuous cell proliferation.
View details for DOI 10.1073/pnas.1422580112
View details for Web of Science ID 000347732300069
View details for PubMedID 25540416
The NF-kappa B Genomic Landscape in Lymphoblastoid B Cells
2014; 8 (5): 1595-1606
The nuclear factor κB (NF-κΒ) subunits RelA, RelB, cRel, p50, and p52 are each critical for B cell development and function. To systematically characterize their responses to canonical and noncanonical NF-κB pathway activity, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) analysis in lymphoblastoid B cell lines (LCLs). We found a complex NF-κB-binding landscape, which did not readily reflect the two NF-κB pathway paradigms. Instead, 10 subunit-binding patterns were observed at promoters and 11 at enhancers. Nearly one-third of NF-κB-binding sites lacked κB motifs and were instead enriched for alternative motifs. The oncogenic forkhead box protein FOXM1 co-occupied nearly half of NF-κB-binding sites and was identified in protein complexes with NF-κB on DNA. FOXM1 knockdown decreased NF-κB target gene expression and ultimately induced apoptosis, highlighting FOXM1 as a synthetic lethal target in B cell malignancy. These studies provide a resource for understanding mechanisms that underlie NF-κB nuclear activity and highlight opportunities for selective NF-κB blockade.
View details for DOI 10.1016/j.celrep.2014.07.037
View details for Web of Science ID 000341574800033
View details for PubMedID 25159142
View details for PubMedCentralID PMC4163118
Epstein-Barr virus nuclear antigen leader protein localizes to promoters and enhancers with cell transcription factors and EBNA2
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (46): 18537-18542
Epstein-Barr virus (EBV) nuclear antigens EBNALP (LP) and EBNA2 (E2) are coexpressed in EBV-infected B lymphocytes and are critical for lymphoblastoid cell line outgrowth. LP removes NCOR and RBPJ repressive complexes from promoters, enhancers, and matrix-associated deacetylase bodies, whereas E2 activates transcription from distal enhancers. LP ChIP-seq analyses identified 19,224 LP sites of which ~50% were ± 2 kb of a transcriptional start site. LP sites were enriched for B-cell transcription factors (TFs), YY1, SP1, PAX5, BATF, IRF4, ETS1, RAD21, PU.1, CTCF, RBPJ, ZNF143, SMC3, NFκB, TBLR, and EBF. E2 sites were also highly enriched for LP-associated cell TFs and were more highly occupied by RBPJ and EBF. LP sites were highly marked by H3K4me3, H3K27ac, H2Az, H3K9ac, RNAPII, and P300, indicative of activated transcription. LP sites were 29% colocalized with E2 (LP/E2). LP/E2 sites were more similar to LP than to E2 sites in associated cell TFs, RNAPII, P300, and histone H3K4me3, H3K9ac, H3K27ac, and H2Az occupancy, and were more highly transcribed than LP or E2 sites. Gene affected by CTCF and LP cooccupancy were more highly expressed than genes affected by CTCF alone. LP was at myc enhancers and promoters and of MYC regulated ccnd2, 23 med complex components, and MYC regulated cell survival genes, igf2r and bcl2. These data implicate LP and associated TFs and DNA looping factors CTCF, RAD21, SMC3, and YY1/INO80 chromatin-remodeling complexes in repressor depletion and gene activation necessary for lymphoblastoid cell line growth and survival.
View details for DOI 10.1073/pnas.1317608110
View details for Web of Science ID 000326830900057
View details for PubMedID 24167291
A Divalent Ion Is Crucial in the Structure and Dominant-Negative Function of ID Proteins, a Class of Helix-Loop-Helix Transcription Regulators
2012; 7 (10)
Inhibitors of DNA binding and differentiation (ID) proteins, a dominant-negative group of helix-loop-helix (HLH) transcription regulators, are well-characterized key players in cellular fate determination during development in mammals as well as Drosophila. Although not oncogenes themselves, their upregulation by various oncogenic proteins (such as Ras, Myc) and their inhibitory effects on cell cycle proteins (such as pRb) hint at their possible roles in tumorigenesis. Furthermore, their potency as inhibitors of cellular differentiation, through their heterodimerization with subsequent inactivation of the ubiquitous E proteins, suggest possible novel roles in engineering induced pluripotent stem cells (iPSCs). We present the high-resolution 2.1Å crystal structure of ID2 (HLH domain), coupled with novel biochemical insights in the presence of a divalent ion, possibly calcium (Ca2+), in the loop of ID proteins, which appear to be crucial for the structure and activity of ID proteins. These new insights will pave the way for new rational drug designs, in addition to current synthetic peptide options, against this potent player in tumorigenesis as well as more efficient ways for stem cells reprogramming.
View details for DOI 10.1371/journal.pone.0048591
View details for Web of Science ID 000310705600051
View details for PubMedID 23119064
Structural analysis and dimerization profile of the SCAN domain of the pluripotency factor Zfp206
NUCLEIC ACIDS RESEARCH
2012; 40 (17): 8721-8732
Zfp206 (also named as Zscan10) belongs to the subfamily of C(2)H(2) zinc finger transcription factors, which is characterized by the N-terminal SCAN domain. The SCAN domain mediates self-association and association between the members of SCAN family transcription factors, but the structural basis and selectivity determinants for complex formation is unknown. Zfp206 is important for maintaining the pluripotency of embryonic stem cells presumably by combinatorial assembly of itself or other SCAN family members on enhancer regions. To gain insights into the folding topology and selectivity determinants for SCAN dimerization, we solved the 1.85 Å crystal structure of the SCAN domain of Zfp206. In vitro binding studies using a panel of 20 SCAN proteins indicate that the SCAN domain Zfp206 can selectively associate with other members of SCAN family transcription factors. Deletion mutations showed that the N-terminal helix 1 is critical for heterodimerization. Double mutations and multiple mutations based on the Zfp206SCAN-Zfp110SCAN model suggested that domain swapped topology is a possible preference for Zfp206SCAN-Zfp110SCAN heterodimer. Together, we demonstrate that the Zfp206SCAN constitutes a protein module that enables C(2)H(2) transcription factor dimerization in a highly selective manner using a domain-swapped interface architecture and identify novel partners for Zfp206 during embryonal development.
View details for DOI 10.1093/nar/gks611
View details for Web of Science ID 000309464300053
View details for PubMedID 22735705