Academic Appointments


  • Basic Life Science Research Associate, Bioengineering

All Publications


  • NuSeT: A deep learning tool for reliably separating and analyzing crowded cells PLoS Computational Biology Yang, L., et al 2020
  • A fluorogenic array for temporally unlimited single-molecule tracking NATURE CHEMICAL BIOLOGY Ghosh, R. P., Franklin, J., Draper, W. E., Shi, Q., Betran, B., Spakowitz, A. J., Liphardt, J. T. 2019; 15 (4): 401-+
  • A Mutation in Histone H2B Represents a New Class of Oncogenic Driver. Cancer discovery Bennett, R. L., Bele, A. n., Small, E. C., Will, C. M., Nabet, B. n., Oyer, J. A., Huang, X. n., Ghosh, R. P., Grzybowski, A. T., Yu, T. n., Zhang, Q. n., Riva, A. n., Lele, T. P., Schatz, G. C., Kelleher, N. L., Ruthenburg, A. J., Liphardt, J. n., Licht, J. D. 2019

    Abstract

    By examination of the cancer genomics database we identified a new set of mutations in core histones that frequently recur in cancer patient samples and are predicted to disrupt nucleosome stability. In support of this idea, we characterized a glutamate to lysine mutation of histone H2B at amino acid 76 (H2B-E76K) found particularly in bladder and head and neck cancer that disrupts the interaction between H2B and H4. Although H2B-E76K forms dimers with H2A, it does not form stable histone octamers with H3 and H4 in vitro and when reconstituted with DNA forms unstable nucleosomes with increased sensitivity to nuclease. Expression of the equivalent H2B mutant in yeast restricted growth at high temperature and led to defective nucleosome-mediated gene repression. Significantly, H2B-E76K expression in the normal mammary epithelial cell line MCF10A increased cellular proliferation, cooperated with PIK3CA to promote colony formation, caused a significant drift in gene expression and fundamental changes in chromatin accessibility particularly at gene regulatory elements. Taken together, these data demonstrate that mutations in the globular domains of core histones may give rise to an oncogenic program due to nucleosome dysfunction and deregulation of gene expression.

    View details for DOI 10.1158/2159-8290.CD-19-0393

    View details for PubMedID 31337617

  • Satb1 integrates DNA binding site geometry and torsional stress to differentially target nucleosome-dense regions. Nature communications Ghosh, R. P., Shi, Q. n., Yang, L. n., Reddick, M. P., Nikitina, T. n., Zhurkin, V. B., Fordyce, P. n., Stasevich, T. J., Chang, H. Y., Greenleaf, W. J., Liphardt, J. T. 2019; 10 (1): 3221

    Abstract

    The Satb1 genome organizer regulates multiple cellular and developmental processes. It is not yet clear how Satb1 selects different sets of targets throughout the genome. Here we have used live-cell single molecule imaging and deep sequencing to assess determinants of Satb1 binding-site selectivity. We have found that Satb1 preferentially targets nucleosome-dense regions and can directly bind consensus motifs within nucleosomes. Some genomic regions harbor multiple, regularly spaced Satb1 binding motifs (typical separation ~1 turn of the DNA helix) characterized by highly cooperative binding. The Satb1 homeodomain is dispensable for high affinity binding but is essential for specificity. Finally, we find that Satb1-DNA interactions are mechanosensitive. Increasing negative torsional stress in DNA enhances Satb1 binding and Satb1 stabilizes base unpairing regions against melting by molecular machines. The ability of Satb1 to control diverse biological programs may reflect its ability to combinatorially use multiple site selection criteria.

    View details for DOI 10.1038/s41467-019-11118-8

    View details for PubMedID 31324780

  • ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing. Nature methods Chen, X., Shen, Y., Draper, W., Buenrostro, J. D., Litzenburger, U., Cho, S. W., Satpathy, A. T., Carter, A. C., Ghosh, R. P., East-Seletsky, A., Doudna, J. A., Greenleaf, W. J., Liphardt, J. T., Chang, H. Y. 2016

    Abstract

    Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion, termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility, and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control.

    View details for DOI 10.1038/nmeth.4031

    View details for PubMedID 27749837

  • Rapid disorganization of mechanically interacting systems of mammary acini PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shi, Q., Ghosh, R. P., Engelke, H., Rycroft, C. H., Cassereau, L., Sethian, J. A., Weaver, V. M., Liphardt, J. T. 2014; 111 (2): 658-663

    Abstract

    Cells and multicellular structures can mechanically align and concentrate fibers in their ECM environment and can sense and respond to mechanical cues by differentiating, branching, or disorganizing. Here we show that mammary acini with compromised structural integrity can interconnect by forming long collagen lines. These collagen lines then coordinate and accelerate transition to an invasive phenotype. Interacting acini begin to disorganize within 12.5 ± 4.7 h in a spatially coordinated manner, whereas acini that do not interact mechanically with other acini disorganize more slowly (in 21.8 ± 4.1 h) and to a lesser extent (P < 0.0001). When the directed mechanical connections between acini were cut with a laser, the acini reverted to a slowly disorganizing phenotype. When acini were fully mechanically isolated from other acini and also from the bulk gel by box-cuts with a side length <900 μm, transition to an invasive phenotype was blocked in 20 of 20 experiments, regardless of waiting time. Thus, pairs or groups of mammary acini can interact mechanically over long distances through the collagen matrix, and these directed mechanical interactions facilitate transition to an invasive phenotype.

    View details for DOI 10.1073/pnas.1311312110

    View details for Web of Science ID 000329614500032

    View details for PubMedID 24379367

    View details for PubMedCentralID PMC3896145

  • Binding of the Rett Syndrome Protein, MeCP2, to Methylated and Unmethylated DNA and Chromatin IUBMB LIFE Hansen, J. C., Ghosh, R. P., Woodcock, C. L. 2010; 62 (10): 732–38

    Abstract

    Methylated CpG Binding Protein 2 (MeCP2) is a nuclear protein named for its ability to selectively recognize methylated DNA. Much attention has been focused on understanding MeCP2 structure and function in the context of its role in Rett syndrome, a severe neurodevelopmental disorder that afflicts one in 10,000-15,000 girls. Early studies suggested a connection between DNA methylation, MeCP2, and establishment of a repressive chromatin structure at specific gene promoters. However, it is now recognized that MeCP2 can both activate and repress specific genes depending on the context. Likewise, in the cell, MeCP2 is bound to unmethylated DNA and chromatin in addition to methylated DNA. Thus, to understand the molecular basis of MeCP2 functionality, it is necessary to unravel the complex interrelationships between MeCP2 binding to unmethylated and methylated regions of the genome. MeCP2 is unusual and interesting in that it is an intrinsically disordered protein, that is, much of its primary sequence fails to fold into secondary structure and yet is functional. The unique structure of MeCP2 is the subject of the first section of this article. We then discuss recent investigations of the in vitro binding of MeCP2 to unmethylated and methylated DNA, and the potential ramifications of this work for in vivo function. We close by focusing on mechanistic studies indicating that the binding of MeCP2 to chromatin results in compaction into local (secondary) and global (tertiary) higher order structures. MeCP2 also competes with histone H1 for nucleosomal binding sites. The recent finding that MeCP2 is found at near stoichiometric levels with nucleosomes in neuronal cells underscores the multiple modes of engagement of MeCP2 with the genome, which include the cooperative tracking of methylation density.

    View details for DOI 10.1002/iub.386

    View details for Web of Science ID 000284218600004

    View details for PubMedID 21031501

    View details for PubMedCentralID PMC3096928

  • MeCP2 Binds Cooperatively to Its Substrate and Competes with Histone H1 for Chromatin Binding Sites MOLECULAR AND CELLULAR BIOLOGY Ghosh, R. P., Horowitz-Scherer, R. A., Nikitina, T., Shlyakhtenko, L. S., Woodcock, C. L. 2010; 30 (19): 4656–70

    Abstract

    Sporadic mutations in the hMeCP2 gene, coding for a protein that preferentially binds symmetrically methylated CpGs, result in the severe neurological disorder Rett syndrome (RTT). In the present work, employing a wide range of experimental approaches, we shed new light on the many levels of MeCP2 interaction with DNA and chromatin. We show that strong methylation-independent as well as methylation-dependent binding by MeCP2 is influenced by DNA length. Although MeCP2 is strictly monomeric in solution, its binding to DNA is cooperative, with dimeric binding strongly correlated with methylation density, and strengthened by nearby A/T repeats. Dimeric binding is abolished in the F155S and R294X severe RTT mutants. MeCP2 also binds chromatin in vitro, resulting in compaction-related changes in nucleosome architecture that resemble the classical zigzag motif induced by histone H1 and considered important for 30-nm-fiber formation. In vivo chromatin binding kinetics and in vitro steady-state nucleosome binding of both MeCP2 and H1 provide strong evidence for competition between MeCP2 and H1 for common binding sites. This suggests that chromatin binding by MeCP2 and H1 in vivo should be viewed in the context of competitive multifactorial regulation.

    View details for DOI 10.1128/MCB.00379-10

    View details for Web of Science ID 000281721400009

    View details for PubMedID 20679481

    View details for PubMedCentralID PMC2950531

  • Unique Physical Properties and Interactions of the Domains of Methylated DNA Binding Protein 2 BIOCHEMISTRY Ghosh, R. P., Nikitina, T., Horowitz-Scherer, R. A., Gierasch, L. M., Uversky, V. N., Hite, K., Hansen, J. C., Woodcock, C. L. 2010; 49 (20): 4395–4410

    Abstract

    Methylated DNA binding protein 2 (MeCP2) is a methyl CpG binding protein whose key role is the recognition of epigenetic information encoded in DNA methylation patterns. Mutation or misregulation of MeCP2 function leads to Rett syndrome as well as a variety of other autism spectrum disorders. Here, we have analyzed in detail the properties of six individually expressed human MeCP2 domains spanning the entire protein with emphasis on their interactions with each other, with DNA, and with nucleosomal arrays. Each domain contributes uniquely to the structure and function of the full-length protein. MeCP2 is approximately 60% unstructured, with nine interspersed alpha-molecular recognition features (alpha-MoRFs), which are polypeptide segments predicted to acquire secondary structure upon forming complexes with binding partners. Large increases in secondary structure content are induced in some of the isolated MeCP2 domains and in the full-length protein by binding to DNA. Interactions between some MeCP2 domains in cis and trans seen in our assays likely contribute to the structure and function of the intact protein. We also show that MeCP2 has two functional halves. The N-terminal portion contains the methylated DNA binding domain (MBD) and two highly disordered flanking domains that modulate MBD-mediated DNA binding. One of these flanking domains is also capable of autonomous DNA binding. In contrast, the C-terminal portion of the protein that harbors at least two independent DNA binding domains and a chromatin-specific binding domain is largely responsible for mediating nucleosomal array compaction and oligomerization. These findings led to new mechanistic and biochemical insights regarding the conformational modulations of this intrinsically disordered protein, and its context-dependent in vivo roles.

    View details for DOI 10.1021/bi9019753

    View details for Web of Science ID 000277649200011

    View details for PubMedID 20405910

    View details for PubMedCentralID PMC2872689

  • Chromatin Higher-order Structure and Dynamics COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Woodcock, C. L., Ghosh, R. P. 2010; 2 (5): a000596

    Abstract

    The primary role of the nucleus as an information storage, retrieval, and replication site requires the physical organization and compaction of meters of DNA. Although it has been clear for many years that nucleosomes constitute the first level of chromatin compaction, this contributes a relatively small fraction of the condensation needed to fit the typical genome into an interphase nucleus or set of metaphase chromosomes, indicating that there are additional "higher order" levels of chromatin condensation. Identifying these levels, their interrelationships, and the principles that govern their occurrence has been a challenging and much discussed problem. In this article, we focus on recent experimental advances and the emerging evidence indicating that structural plasticity and chromatin dynamics play dominant roles in genome organization. We also discuss novel approaches likely to yield important insights in the near future, and suggest research areas that merit further study.

    View details for DOI 10.1101/cshperspect.a000596

    View details for Web of Science ID 000279882700003

    View details for PubMedID 20452954

    View details for PubMedCentralID PMC2857170

  • Rett syndrome-causing mutations in human MeCP2 result in diverse structural changes that impact folding and DNA interactions JOURNAL OF BIOLOGICAL CHEMISTRY Ghosh, R. P., Horowitz-Scherer, R. A., Nikitina, T., Gierasch, L. M., Woodcock, C. L. 2008; 283 (29): 20523–34

    Abstract

    Most cases of Rett syndrome (RTT) are caused by mutations in the methylated DNA-binding protein, MeCP2. Here, we have shown that frequent RTT-causing missense mutations (R106W, R133C, F155S, T158M) located in the methylated DNA-binding domain (MBD) of MeCP2 have profound and diverse effects on its structure, stability, and DNA-binding properties. Fluorescence spectroscopy, which reports on the single tryptophan in the MBD, indicated that this residue is strongly protected from the aqueous environment in the wild type but is more exposed in the R133C and F155S mutations. In the mutant proteins R133C, F155S, and T158M, the thermal stability of the domain was strongly reduced. Thermal stability of the wild-type protein was increased in the presence of unmethylated DNA and was further enhanced by DNA methylation. DNA-induced thermal stability was also seen, but to a lesser extent, in each of the mutant proteins. Circular dichroism (CD) of the MBD revealed differences in the secondary structure of the four mutants. Upon binding to methylated DNA, the wild type showed a subtle but reproducible increase in alpha-helical structure, whereas the F155S and R106W did not acquire secondary structure with DNA. Each of the mutant proteins studied is unique in terms of the properties of the MBD and the structural changes induced by DNA binding. For each mutation, we examined the extent to which the magnitude of these differences correlated with the severity of RTT patient symptoms.

    View details for DOI 10.1074/jbc.M803021200

    View details for Web of Science ID 000257565300065

    View details for PubMedID 18499664

    View details for PubMedCentralID PMC2459279

  • MeCP2-chromatin interactions include the formation of chromatosome-like structures and are altered in mutations causing Rett syndrome JOURNAL OF BIOLOGICAL CHEMISTRY Nikitina, T., Ghosh, R. P., Horowitz-Scherer, R. A., Hansen, J. C., Grigoryev, S. A., Woodcock, C. L. 2007; 282 (38): 28237–45

    Abstract

    hMeCP2 (human methylated DNA-binding protein 2), mutations of which cause most cases of Rett syndrome (RTT), is involved in the transmission of repressive epigenetic signals encoded by DNA methylation. The present work focuses on the modifications of chromatin architecture induced by MeCP2 and the effects of RTT-causing mutants. hMeCP2 binds to nucleosomes close to the linker DNA entry-exit site and protects approximately 11 bp of linker DNA from micrococcal nuclease. MeCP2 mutants differ in this property; the R106W mutant gives very little extra protection beyond the approximately 146-bp nucleosome core, whereas the large C-terminal truncation R294X reveals wild type behavior. Gel mobility assays show that linker DNA is essential for proper MeCP2 binding to nucleosomes, and electron microscopy visualization shows that the protein induces distinct conformational changes in the linker DNA. When bound to nucleosomes, MeCP2 is in close proximity to histone H3, which exits the nucleosome core close to the proposed MeCP2-binding site. These findings firmly establish nucleosomal linker DNA as a crucial binding partner of MeCP2 and show that different RTT-causing mutations of MeCP2 are correspondingly defective in different aspects of the interactions that alter chromatin architecture.

    View details for DOI 10.1074/jbc.M704304200

    View details for Web of Science ID 000249455600073

    View details for PubMedID 17660293

  • Multiple modes of interaction between the methylated DNA binding protein MeCP2 and chromatin MOLECULAR AND CELLULAR BIOLOGY Nikitina, T., Shi, X., Ghosh, R. P., Horowitz-Scherer, R. A., Hansen, J. C., Woodcock, C. L. 2007; 27 (3): 864–77

    Abstract

    Mutations of the methylated DNA binding protein MeCP2, a multifunctional protein that is thought to transmit epigenetic information encoded as methylated CpG dinucleotides to the transcriptional machinery, give rise to the debilitating neurodevelopmental disease Rett syndrome (RTT). In this in vitro study, the methylation-dependent and -independent interactions of wild-type and mutant human MeCP2 with defined DNA and chromatin substrates were investigated. A combination of electrophoretic mobility shift assays and visualization by electron microscopy made it possible to understand the different conformational changes underlying the gel shifts. MeCP2 is shown to have, in addition to its well-established methylated DNA binding domain, a methylation-independent DNA binding site (or sites) in the first 294 residues, while the C-terminal portion of MeCP2 (residues 295 to 486) contains one or more essential chromatin interaction regions. All of the RTT-inducing mutants tested were quantitatively bound to chromatin under our conditions, but those that tend to be associated with the more severe RTT symptoms failed to induce the extensive compaction observed with wild-type MeCP2. Two modes of MeCP2-driven compaction were observed, one promoting nucleosome clustering and the other forming DNA-MeCP2-DNA complexes. MeCP2 binding to DNA and chromatin involves a number of different molecular interactions, some of which result in compaction and oligomerization. The multifunctional roles of MeCP2 may be reflected in these different interactions.

    View details for DOI 10.1128/MCB.01593-06

    View details for Web of Science ID 000243946000007

    View details for PubMedID 17101771

    View details for PubMedCentralID PMC1800686