Kenneth S. Zaret, Ph.D.

Research Interest

Ken’s laboratory discovered “pioneer factors” that bind to silent chromatin, endow the competence for cell differentiation, and promote cellular reprogramming. Recently, his lab found broad chromatin domains that can resist pioneer factor binding and serve as impediments to cellular reprogramming; these domains appear to help commit cells to particular fates. Finally, his lab has unveiled how inductive signaling in the embryo leads to chromatin modifications that affect cell fate choices, thereby identifying specific enzymatic targets for small molecules to modulate cell fate control.

Zhaolan (Joe) Zhou, Ph.D.

Mechanisms of Gene Regulation. Pre-mRNA splicing is a fundamental step in eukaryotic gene expression. It is conducted by the spliceosome, a large biological machine that was poorly characterized when I started my thesis research. I successfully developed an affinity purification approach and isolated the first functional spliceosome assembled in vitro. In collaboration with other colleagues, we obtained an electron microscopic view of the spliceosome and identified 145 distinct spliceosomal proteins, many of which have known roles in gene transcription, mRNA export and translation. We also discovered that Aly, an mRNA export factor, is specifically recruited to spliced mRNA by the splicing reaction and promotes efficient mRNA export. We further demonstrated the recruitment of Aly is mediated by direct protein-protein interactions with the splicing factor, UAP56. These studies provided mechanistic insight into the well-known phenomenon that intron-containing genes are often expressed more highly in mammalian cells than their cDNA counterparts, and offered the first biochemical evidence that multiple steps of gene expression, such as gene transcription, splicing and export, are functionally coupled.

Zhou Z and Reed R* (1998). Human homologs of yeast prp16 and prp17 reveal conservation of the mechanism for catalytic step II of pre-mRNA splicing. EMBO J, 17(7): 2095-106. PMID9524131.
Zhou Z#, Luo MJ#, Straesser K, Katahira J, Hurt E and Reed R* (2000). The protein Aly links pre-messenger RNA splicing to nuclear export in metazoans. Nature, 407: 401-405. PMID11014198.
Zhou Z, Licklider LJ, Gygi SP and Reed R* (2002). Comprehensive proteomic analysis of the human spliceosome. Nature, 419: 182-185. PMID12226669.
Zhou Z, Sim J, Griffith J and Reed R* (2002). Purification and electron microscopic visualization of functional human spliceosomes. Proc Natl Acad Sci USA, 99: 12203-12207. PMID12215496.

Molecular basis of Rett syndrome (RTT). RTT is a debilitating neurodevelopmental disorder caused by mutations in the gene encoding methyl-CpG binding protein 2 (MeCP2). One characteristic feature of RTT is the regression of learned motor and language skills after 6-18 months of normal development. The onset of the disease coincides with synaptic maturation driven by sensory experience in humans. As a postdoctoral fellow, I started to pursue the role of MeCP2 in neuronal activity-dependent gene regulation and investigate the possibility that defective experience-dependent synaptic maturation may underlie the pathogenesis of RTT. I found that MeCP2 is selectively phosphorylated in the brain in a neuronal activity-dependent manner and this event mediates dendritic morphogenesis and spine maturation. Together with other colleagues, we found that activity-dependent phosphorylation of MeCP2 is indispensable for synapse development and function in animal models of Rett syndrome. I went on and led a research team demonstrating that MeCP2 regulates gene expression and neuronal development in a brain region, neuronal cell type and age-specific manner, paving the way to investigate the pathogenesis of Rett syndrome in cell types of interest.

Zhou Z#, Hong E#, Cohen S, Zhao W, Ho SY, Chen W, Savner E, Hu L, Steen J, Weitz C and Greenberg ME* (2006). Brain-specific phosphorylation of MeCP2 regulates activity-dependent Bdnf transcription, dendritic growth, and spine maturation. Neuron, 52: 255–269. PMID17046689.
Cohen S, Gabel HW, Hutchinson AN, Sadacca LA, Ebert DA, Harmin DA, Greenberg RS, Verdine VK, Zhou Z, Wetsel WC, West AE and Greenberg ME* (2011). Genome-wide activity-dependent MeCP2 phosphorylation regulates nervous system development and function. Neuron, 72: 72-85. PMID21982370.
Wang IT#, Reyes AR#, and Zhou Z* (2013). Neuronal morphology in MeCP2 mouse models is intrinsically variable and depends on age, cell type, and Mecp2 mutation. Neurobiology of Disease, 58C: 3-12. PMID23659895.
Zhao YT#, Goffin D#, Johnson BS# and Zhou Z* (2013). Loss of MeCP2 function is associated with distinct gene expression changes in the striatum. Neurobiology of Disease, 59C: 257-266. PMID23948639.

Pathophysiology of Rett syndrome (RTT). Major advances in RTT research have greatly benefited from studies of knockout, conditional knockout, and conditional rescue mouse models of MeCP2. However, nearly one-third of RTT mutations are missense mutations in the methyl-CpG binding domain (MBD) of MeCP2. To specifically address the contribution of these missense mutations to RTT etiology and provide the research community with clinically relevant mouse models, I led a research team that developed the first knockin mouse model faithfully recapitulating an RTT-associated MeCP2 T158A mutation, and subsequently expanded this to other common mutations such as T158M and R106W. We were the first to demonstrate that missense mutations in the MBD impair MeCP2 binding to methylated DNA in vivo and concomitantly reduce MeCP2 protein stability. Through transgenic studies, we demonstrated that elevating MeCP2 T158M mutant expression improves MeCP2 binding to DNA and significantly ameliorates RTT-like phenotypes, encouraging therapeutic avenues that target MeCP2 protein stability or MeCP2 expression as treatment for RTT. Through studies of these unique mouse models, my lab also found that MeCP2 modulates gene expression in a cellular compartment-dependent and cell type-specific manner. By overcoming X-linked cellular heterogeneity in mosaic female models of RTT, we were the first to uncover cell and non-cell autonomous gene expression changes related to RTT etiology. Our findings have enhanced the understanding of RTT pathogenesis and pointed the field to a direction of examining MeCP2 function in cellular context of clinical interest.

Goffin D, Allen M, Amorim M, Zhang L, Wang I-TJ, Reyes A-RS, Mercado-Berton A, Ong C, Cohen S, Hu L, Blendy JA, Carlson G,Siegel S, Greenberg ME and Zhou Z* (2012). Rett Syndrome mutation MeCP2 T158A mutation disrupts DNA binding, protein stability and ERP responses. Nature Neuroscience, 15: 274-283. PMID22119903.
GoffinD, Brodkin ES, BlendyJA, SiegelSJ and ZhouZ* (2014). Cellular origins of auditory event-related potential deficits in Rett syndrome. Nature Neuroscience, 17(6): 804-806. PMID24777420.
Lamonica JM, Kwon DY, Goffin D, Fenik P, Johnson BS, Cui Y, Guo H, Veasey S and Zhou Z* (2017). Elevating expression of MeCP2 T158M rescues DNA binding and Rett syndrome-like phenotypes. Journal of Clinical Investigation, 127 (5): 1889-1904. PMID28394263.
Johnson BS#, Zhao Y#, Fasolino M#, Lamonica JM, Kim YJ, Georgakilas G, Wood KH, Bu D, Cui Y, Goffin D, Vahedi G, Kim TH and ZhouZ* (2017). Biotin tagging of MeCP2 reveals contextual insights into the Rett syndrome transcriptome. Nature Medicine, 23(10): 1203-1214. PMID28920956.

Pathophysiology of CDKL5 deficiency disorder (CDD). While I was studying MeCP2 phosphorylation and trying to identify its up-stream kinase, several human genetic studies linked mutations in the X-linked gene encoding cyclin-dependent kinase-like 5 (CDKL5) to atypical RTT, a variant with early-onset epileptic features. In vitro biochemistry and cell culture studies supported an interaction between CDKL5 and MeCP2. However, experimental evidence remained contentious. I decided to take a genetic approach and investigate CDKL5 function in vivo, and therefore led a research team in the development and characterization of the first knockout mouse model of CDKL5. My lab found that CDKL5 dysfunction disrupts many key signal transduction pathways and long-range neural circuit communication, leading to CDD-like phenotypes in mice. We then carried out conditional knockout studies to dissect the cellular origin of these phenotypes and uncovered crucial roles for CDKL5 in glutamatergic neurons for learning and memory and in GABAergic neurons for social interaction and repetitive behaviors. We have now developed conditional rescue mouse lines to assess the reversibility of CDD-related phenotypes and knockin mouse models bearing CDD patient mutations to support preclinical studies.

Wang IT, Allen M, GoffinD, ZhuX, Fairless AH, Brodkin ES, SiegelSJ, MarshED, BlendyJA, and ZhouZ* (2012). Loss of CDKL5 disrupts kinome profile and ERP response leading to autistic-like phenotypes in mice. Proc Natl Acad Sci USA.109: 21516-21521. PMID23236174.
Tang S, Wang I-T, Yue C, Takano H, Terzic B, Pance K, Lee JY, Cui Y, Coulter DA* and Zhou Z* (*co-corresponding) (2017). Loss of CDKL5 in glutamatergic neurons disrupts hippocampal microcircuitry and leads to memory impairment in mice. Journal of Neuroscience, 37(31): 7420-7437. PMID28674172.
Tang S#, Terzic B#, Wang I-T, Sarmieto N, Sizov K, Cui Y, Takano H, Marsh ED, Zhou Z* and Coulter DA* (*co-corresponding) (2019): Altered NMDAR Signaling Underlies Autistic-like Features in Mouse Models of CDKL5 Deficiency Disorder. Nature Communications, 10: 2655. doi: 10.1038/s41467-019-10689-w. PMID:31201320.
Mulcahey PJ#, Tang S#, Takano H#, White A, Davila Portillo DR, Kane OM, Marsh ED, Zhou Z, Coulter DA (2020): Aged heterozygous Cdkl5 mutant mice exhibit spontaneous epileptic spasms. Experimental Neurology 332: 113388. PMID:32585155.

Epigenetic basis of autism and major depressive disorder (MDD). The genetic underpinnings of neuropsychiatric disorders are highly complex, involving multifaceted interactions between risk genes and the environment. It is known that environmental factors such as adverse early life events confer significantly greater susceptibility to psychiatric conditions later in life, yet the epigenetic mechanisms by which environmental factors interact with genetic programs in the nervous system remain poorly understood. Sponsored by the Biobehavioral Research Awards for Innovative New Scientists (BRAINS) from NIMH, I led a research team that conceived a novel, Cre-dependent biotinylation strategy and developed a series of genetically modified mice that allow for genome-wide profiling of DNA methylation, histone modifications and RNA expression from cell types of interest, thus overcoming the extensive cellular heterogeneity of the brain. My lab found that long genes implicated in autism harbor broad enhancer-like chromatin domains and causally link chromatin genes to autism etiology. My lab has also developed a computational pipeline to identify an integrated epigenetic code in target cell types and in response to environmental stimuli. Furthermore, to investigate the causal role of stress-induced epigenetic changes to behavioral maladaptation, my research team adopted a modified CRISPR/Cas9 strategy to alter DNA methylation and histone acetylation at loci of interest. Having established a chronic unpredictable stress (CUS) paradigm to induce the expression of MDD-like phenotypes in our genetically modified mice, we are currently in the process of employing genomic and genome-editing expertise to interrogate the epigenetic mechanisms underlying the pathogenesis of MDD.

Wood KH, Johnson BS, Welsh SA, Lee JY, Cui Y, Krizman E, Brodkin ES, Blendy JA, Robinson MB, Bartolomei MS and Zhou Z* (2016): Tagging of Methyl-CpG-binding Domain Proteins Reveals Different Spatiotemporal Expression and Supports Distinct Functions. Epigenomics 4: 455-473. PMID27066839.
Kwon DY, Zhao YT, Lamonica JM and Zhou Z* (2017). Locus-specific histone deacetylation using a synthetic CRISPR-Cas9-based HDAC. Nature Communications, 8:15315. Doi:10.1038/ncomms15315. PMID28497787.
Zhao YT, Kwon DY, Johnson BS, Fasolino M, Lamonica JM, Kim YJ, Zhao BS, He C, Vahedi G, Kim TH and Zhou Z* (2018). Long genes linked to autism harbor broad enhancer-like chromatin domains. Genome Research, 28:933-942. PMID: 29848492.
Kwon DY, Hu P, Zhao Y-T, Beagan JA, Nofziger JH, Cui Y, Xu B, Zaitseva D, Phillips-Cremins JE, Blendy JA, Wu H, Zhou Z* (2020): Neuronal YY1 in the prefrontal cortex regulates transcriptionaland behavioral responses to chronic stress. BioRxiv, doi:

Research Interest

A fundamental question in Genetics and Neuroscience is how the brain executes genetic programs while maintaining the ability to adapt to the environment. The underlying molecular mechanisms are not well understood, but epigenetic regulation, mediated by DNA methylation and chromatin organization, provides an intricate platform bridging genetics and the environment, and allows for the integration of intrinsic and environmental signals into the genome and subsequent translation of the genome into stable yet adaptive functions in the brain. Impaired epigenetic regulation has been implicated in many neurodevelopmental and neuropsychiatric disorders.

The Zhou laboratory is interested in understanding the epigenetic mechanisms that integrate environmental factors with genetic code to govern brain development and function, elucidating the pathophysiology of specific neurodevelopmental disorders with known genetic causes such as Rett syndrome (RTT) and CDKL5 deficiency disorder (CDD), and illuminating the pathogenesis of selective neuropsychiatric disorders with complex genetic traits such as autism and major depressive disorder (MDD). We use a variety of cutting-edge genomic technologies, together with cellular and physiological assays in genetically modified mice, to pursue our interests. We aim to ultimately translate our findings into therapeutic development to improve the treatment for neurodevelopmental and neuropsychiatric disorders.

Irfan A. Asangani, Ph.D.

Research Interest

Cancer cells display an altered landscape of chromatin leading to broad changes in the gene expression. In addition, genes involved in chromatin remodeling and epigenetic regulation are frequently and specifically mutated in a wide variety of cancers including prostate cancer. While known to serve important roles in the control of gene expression and development, these largely unexpected mutation findings have illuminated newly recognized mechanisms central to the genesis of cancer. Gaining insight into the mechanism of chromatin regulation in cancer will offer the potential to reveal novel approaches and targets for effective therapeutic intervention.
Our laboratory employs a multidisciplinary approach to study these molecular epigenetic events associated with cancer towards the overarching goal of translating this knowledge into clinical tools by developing novel diagnostic, prognostic and therapeutic strategies. Additionally, we investigate the mechanisms of resistance to targeted therapies and develop novel combinatorial approaches that act on compensatory/new pathways in resistant tumors. Our basic strategy is to develop and deploy rational polytherapy upfront that suppresses the survival and emergence of resistant tumor cells.

Ben E. Black, Ph.D.

Centromere structural biochemistry. The work in the Black Lab in this area is focused on understanding the physical nature of the epigenetic information generated by the incorporation of the histone H3 variant, CENP-A into chromatin. How do the DNA and proteins work together to form a chromatin domain that is distinguished from the rest of the chromosome as the site to build a mitotic kinetochore and as the site for persistent centromere maintenance through cell divisions? The Black Lab’s crystal structure of CENP-A, described in Sekulic et al., 2010 was the first of this protein from any species, in any context, and represents a landmark study in the centromere field. More recently the Black Lab devised a ChIP-seq-based strategy to probe centromeric chromatin architecture at very high-resolution with a study (Hasson et al., 2013) that resolved a longstanding conflict regarding the nature of human centromeric nucleosomes. The Black Lab also combined a battery of biophysical approaches alongside cell-based functional assays to identify CENP-C as an essential collaborator in maintaining centromere identity in Falk et al., 2015. In the course of these studies, the Black Lab found that CENP-C surprisingly alters the shape and the dynamics of the CENP-A nucleosome when it binds, revealing a novel mode of regulation that nucleosome-binding proteins can bring to bear on chromatin.

  • Allu, P.K., Dawicki-McKenna, J.M., Van Eeuwen, T., Slavin, M., Braitbard, M., Xu, C., Kalisman, N., Murakami, K., and E. Black*. 2019. Structure of the human core centromeric nucleosome complex. Curr. Biol., 29:2625-2639. (*corresponding author) [PMCID: PMC6702948]
  • Guo, L.Y., P.K. Allu, L. Zandarashvili, K.L. McKinley, N. Sekulic, J.M. Dawicki-McKenna, D. Fachinetti, G.A. Logsdon, R.M. Jamiolkowski, D.W. Cleveland, I.M. Cheeseman, and B.E. Black*. 2017. Centromeres are maintained by fastening CENP-A to DNA and directing an arginine anchor-dependent nucleosome structural transition. Nat. Commun., 8:15775. (*corresponding author) [PMCID: PMC5472775]
  • Falk, S.J., L.Y. Guo, N. Sekulic, E.M. Smoak, T. Mani, G.A. Logsdon, K. Gupta, L.E.T. Jansen, G.D. Van Duyne, S.A. Vinogradov, M.A. Lampson, and B.E. Black*. 2015. CENP-C reshapes and stabilizes CENP-A nucleosomes at the centromere. Science, 348:699-703. (*corresponding author; contributed equally) [PMCID: in progress]
  • Hasson, D. †, T. Panchenko, K.J. Salimian, M.U. Salman, N. Sekulic, A. Alonso, P.E. Warburton, and B.E.  Black*. 2013. The octamer is the major form of CENP-A nucleosomes at human centromeres. Nat. Struct. Mol. Biol., 20:687-695. (*corresponding author; contributed equally and listed in alphabetical order) [PMCID: PMC3760417]
  • Sekulic, N., E.A. Bassett, D.J. Rogers, and B.E. Black*. 2010. The structure of (CENP-A—H4)2 reveals physical features that mark centromeres. Nature, 467:347-351. (*corresponding author) [PMCID: PMC2946842]

Centromere chromatin assembly. Given the importance of CENP-A in defining the properties of centromeric nucleosomes, one key question in chromatin biology and epigenetics is that of how histone variants (including CENP-A) are delivered to – and incorporated into – the correct nucleosomes at appropriate locations, and how they are ‘sorted’ from each other by so-called histone chaperones. Starting with the discovery of the cis-acting element within CENP-A that targets it to centromeres (which he called the CENP-A targeting domain, CATD), Dr. Black has contributed highly to the understanding of these processes. In a paper (Bassett et al., 2012), the Black Lab identified the precise mode of recognition of CENP-A by HJURP using a very effective combination of cell-based functional assays, conventional biochemistry, and high-resolution biophysical approaches. Using these data, the Black Lab formulated a new model for centromere assembly in which HJURP stabilizes the histone fold domains of both CENP-A and its partner histone H4 for a substantial portion of the cell cycle prior to mediating chromatin assembly at the centromere. The Black Lab has also used a new approach to establish a new functional centromere at an ectopic locus to understand the relationship between the elements that direct new CENP-A chromatin assembly and the first steps in centromere establishment.

  • Logsdon, G.L., C.W. Gambogi, M.A. Liskovykh, E.J. Barrey, V. Larionov, K.H. Miga, P. Heun, and E. Black*. 2019. Human artificial chromosomes that bypass centromeric DNA. Cell, 178:624-639. (*corresponding author) [PMCID: PMC6657561]
  • Logsdon, G.L., E. Barrey, E.A. Bassett, J.E. DeNizio, L.Y. Guo, T. Panchenko, J.M. Dawicki-McKenna, P. Heun, and B.E. Black*. 2015. Both tails and the centromere targeting domain of CENP-A are required for centromere establishment. J. Cell Biol., 208:521-531. (*corresponding author) [PMCID: PMC4347640]
  • Bassett, E.A., J. DeNizio, M.C. Barnhart-Dailey, T. Panchenko, N. Sekulic, D.J. Rogers, D.R. Foltz, and B.E. Black*. 2012. HJURP uses distinct CENP-A surfaces to recognize and to stabilize CENP-A/ histone H4 for centromere assembly. Dev. Cell, 22: 749-762. (*corresponding author) [PMCID: PMC3353549]
  • Black, B.E.*, and D.W. Cleveland*. 2011. Epigenetic centromere propagation and the nature of CENP-A nucleosomes. Cell, 144:471-479. (*corresponding authors) [PMCID: PMC3061232]
  • Foltz, D.R., L.E.T. Jansen, A.O. Bailey, J.R. Yates III, E.A. Bassett, S. Wood, B.E. Black, and D.W. Cleveland. 2009. Centromere specific assembly of CENP-A nucleosomes is mediated by HJURP. Cell, 137:472-484. [PMCID: PMC2747366]

Aurora B-mediated mitotic error correction. While studying patient-derived cells harboring neocentromeres, the Black Lab made the observation that the Aurora B kinase is highly enriched at chromosomes that have spindle attachment errors. This appears to be quite a fundamental observation. Black and colleagues found this to be a common feature of healthy, diploid cells, but one that is absent from the aneuploid, tumor-derived cells typically used for mammalian mitosis research. Further investigation revealed dynamic modulation of Aurora B levels at each centromere in a chromosome autonomous fashion that greatly expands the dynamic range of this kinase in phosphorylating kinetochore substrates. It appears that this feedback leads to highly efficient mitotic error correction; a discovery that greatly impacts the understanding of Aurora B function.

  • Zaystev, A.V., D. Sagura-Peña, M. Godzi, A. Calderon, E.R. Ballister, R. Stamatov, A.M. Mayo, L. Peterson, B.E. Black, F.L. Ataullakhanov, M.A. Lampson, E.L. Grishchuk. 2016. Bistability of a coupled Aurora B kinase-phosphatase system in cell division. Elife, 5:e10644. [PMCID: PMC4798973]
  • Wood, T. Panchenko, M.A. Lampson*, and B.E. Black*. 2011. Feedback control in sensing chromosome biorientation by the Aurora B kinase. Curr. Biol., 21:1158-1165. (*corresponding authors) [PMCID: PMC3156581]
  • Bassett, E.A., S. Wood, K.J. Salimian, S. Ajith, D.R. Foltz, and B.E. Black*. 2010. Epigenetic centromere specification directs Aurora B accumulation but is insufficient to efficiently correct mitotic errors. J. Cell Biol., 190:177-185. (*corresponding author) [PMCID: PMC2930274]

Hydrogen/deuterium exchange-mass spectrometry (HXMS) with chromatin proteins. The Black Lab has emerged as the world leader in applying HXMS to chromatin-associated proteins. This powerful approach probes structure and dynamics in solution, and is a strong complement to more conventional structural biology techniques. The Black Lab has used it successfully to gain insight into a diverse set of chromatin assembly complexes and natively unstructured nucleosomal DNA binding proteins, gaining insight into complexes that have been recalcitrant to other standard approaches (e.g. crystallography and NMR). Along the way the Black Lab has advanced HXMS technology and dispelled the earlier misconceptions that the approach is low-resolution (it is not, and the Black Lab has achieved near amino acid resolution of HX behavior on several proteins) and merely a probe of what happens on the surfaces of proteins (it is not, and the Black Lab has gained important insight into the core of individual proteins and proteins within large multi-subunit complexes).

  • Zandarashvili, L., M.F. Langelier, U.K. Velagapudi, M.A. Hancock, J.D. Steffen, R. Billur, Z.M. Hannan, A.J. Wicks, D.B. Krastev, S.J. Pettitt, C.J. Lord, T.T. Talele, J.M. Pascal*, and E. Black*. 2020. Structural basis for allosteric PARP-1 retention on DNA breaks. Science, 368:eaax6367. (*corresponding authors; contributed equally) [PMCID: in progress]
  • Langelier, M.F., L. Zandarashvili, P.M. Aguiar, B.E. Black*, and J.M. Pascal*. 2018. NAD+ analog reveals PARP-1 substrate-blocking mechanism and allosteric communication from catalytic center to DNA-binding domains. Nat. Commun., 9:844. (*corresponding authors) [PMCID: PMC5829251]
  • Dawicki-McKenna, J.M., M.F. Langelier, J.E. DeNizio, A.A. Riccio, C.D. Cao, K.R. Karch, M. McCauley, J.D. Steffen, B.E. Black*, and J.M. Pascal*. 2015. PARP-1 activation requires local unfolding of an autoinhibitory domain. Mol. Cell, 60:755-768. (*corresponding author; contributed equally) [PMCID: PMC4712911]
  • DeNizio, J., S.J. Elsässer, and B.E. Black*. 2014. DAXX co-folds with H3.3/H4 using high local stability conferred by the H3.3 variant recognition residues. Nucleic Acids Res., 42:4318-4331. (*corresponding author) [PMCID: PMC3985662]
  • Black, B.E., D.R. Foltz, S. Chakravarthy, K. Luger, V.L. Woods Jr., and D.W. Cleveland. 2004. Structural determinants for generating centromeric chromatin. Nature 430:578-582.

Centromere inheritance through the mammalian germline. This is a relatively recent research direction in my group, and we are already making headway into understanding how the epigenetic centromere mark represented by nucleosomes containing CENP-A is successfully transmitted through the male and female germlines. Both male and female germlines present major challenges to the centromere, and we are using mouse as a model system to understand how this faithfully occurs.

  • Lampson, M.A.*, and B.E. Black*. 2018. Cellular and molecular mechanisms of centromere drive. Cold Spring Harb. Symp. Quant. Biol., online ahead of print. (*corresponding authors)
  • Das, A., E.M. Smoak, R. Linares-Saldana, M.A. Lampson*, and B.E. Black*. 2017. Centromere inheritance through the germline. Chromosoma, 126:595-604. (*corresponding authors) [PMCID: PMC5693723]
  • Iwata-Otsubo, A. †, J.M. Dawicki-McKenna, T. Akera, S.J. Falk, L. Chmátal, K. Yang, B.A. Sullivan, R.M. Schultz, M.A. Lampson*, and B.E. Black*. 2017. Expanded satellite repeats amplify a discrete CENP-A nucleosome assembly site on chromosomes that drive in female meiosis. Curr. Biol., 27:2365-2373. (*corresponding authors; contributed equally) [PMCID: PMC5567862]
  • Smoak, E.M., P. Stein, R.M. Schultz, M.A. Lampson*, and B.E. Black*. 2016. Long-term retention of CENP-A nucleosomes in mammalian oocytes underpins transgenerational inheritance of centromere identity. Curr. Biol., 26:1110-1116. (*corresponding authors) [PMCID: PMC4846481]

Research Interest

The Black Lab is answering the most pressing questions in chromosome biology, such as:

  • How does genetic inheritance actually work?
  • How was epigenetic information transmitted to us from our parents?
  • Can building new artificial chromosomes help us understand how natural chromosomes work?
  • How are the key enzymes protecting the integrity of our genome specifically and potently activated by potential catastrophes like DNA breaks or chromosome misattachment to the mitotic spindle?

Roberto Bonasio, Ph.D.

Research Interest

The Bonasio laboratory investigates the molecular mechanisms of epigenetic memory with a focus on the role of noncoding RNAs. These processes are key to a number of biological phenomena, including embryonic development, cancer, stem cell pluripotency, and brain function. We approach these fundamental biological questions from both a mechanistic and systems-level perspective. We combine biochemistry and molecular biology with bioinformatics and genomics in conventional systems, such as mammalian cells, and nonconventional model organisms, such as ants, which offer new, unexplored avenues to study epigenetics.

Roger Greenberg, M.D., Ph.D.

BRCA1 dependent DNA damage recognition and repair. Seminal studies connecting the breast and ovarian tumor suppressor protein BRCA1 to DNA repair arose from observations that BRCA1 was present in large nuclear foci at DNA double-strand breaks (DSBs). The molecular events underlying BRCA1 foci formation were predicted to be important to its roles in genome integrity and tumor suppression given that the most common clinical BRCA1 missense mutations abrogated foci localization. We provided the first insights into the molecular nature of BRCA1 DSB recognition events by reporting that BRCA1 is targeted to ubiquitin chains that arise at DSB chromatin (Sobhian et al. Science 2007). Our findings revealed that BRCA1 interacts with a 5-membered ubiquitin binding protein complex, which selectively interacts with lysine63-linked (K63-Ub) ubiquitin chains. The 5-member RAP80 complex contains a deubiquitinating enzyme that specifically hydrolyzes K63-Ub and a novel gene on chromosome 19 that we named MERIT40 (Mediator of RAP80 Interactions and Targeting 40 kd) (Shao et al Genes Dev 2009). This work provided the first evidence that nondegradative ubiquitin chains are a recognition signal for the assembly of DNA repair protein complexes at damaged chromatin, becoming a paradigm for DNA damage recognition. Our subsequent studies provided insights into the importance of ubiquitin signaling to BRCA1 dependent DNA repair and tumor suppression (see references b-e and contribution 4).

Sobhian B, Shao G, Lilli DR, Culhane AC, Moreau L, Xia B, Livingston DM* and Greenberg RA*. RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites.Science 316(5828): 1198- 202, 2007 (PMC2706583) * co-corresponding authorship.
Jiang Q, Paramasivam M, Aressy B, Wu J, Bellani M, Tong W, Seidman MM, Greenberg RA. MERIT40 cooperates with BRCA2 to resolve DNA inter-strand crosslinks.Genes & Development 2015 Sept [Epub ahead of print]
Tang J, Cho NW, Cui G, Manion EM, Shanbhag NM, Botuyan MV, Mer G, Greenberg RA. TIP60 limits 53BP1 accumulation at DNA double-strand breaks to promote BRCA1-dependent homologous recombination.Nat Struct Mol Biol 20:317-25 2013. (PMC3594358)
Shao G, Patterson-Fortin J, Messick TE, Feng D, Shanbhag N, Wang Y, and Greenberg RA. MERIT 40 controls BRCA1-Rap80 complex integrity and recruitment to DNA double-strand breaks.Genes Dev. 23(6): 740-54, 2009 (PMC2661612)
Coleman KA, Greenberg RA. The BRCA1-RAP80 Complex Regulates DNA Repair Mechanism Utilization by Restricting End Resection.J Biol Chem 286(15): 13669-80. 2011 (PMC3075711).

ATM dependent DNA double-strand break silencing. A longstanding question had been how DNA double-strand break responses communicate with RNA Pol II transcriptional processes on contiguous stretches of chromatin. We developed the first system to study this process with the capacity to visualize DSB responses and nascent transcription in real time in human cells. This methodology consists of a reporter system in which we induce DSBs at lac operator repeats that are 4kb upstream of a transgene that harbors MS2 stem loops within its 3’-UTR, enabling real time visualization of nascent transcription by coexpression of a YFP-MS2 protein. Using this system and complementary approaches, we demonstrated an ATM dependent silencing of transcription that extended at least 4 kilobases from the site of DNA damage (Shanbhag et al. Cell 2010). This seminal study has resulted in a wide range of investigation into the biological significance and underlying mechanisms of ATM dependent DSB silencing. The work has implications for fundamental biological processes such as meiotic sex chromosome inactivation, viral latency, and human diseases such as Ataxia Telangiectasia.

Shanbhag NM, Rafalska-Metcalf IU, Balane-Bolivar C, Janicki SM, and Greenberg RA. ATM dependent chromatin changes silence transcription in cis to DNA double-strand breaks.Cell 141(6): 970-81. 2010.
Shanbhag NM, Greenberg RA. The dynamics of DNA damage repair and transcription. Methods in Molecular Biology 1042: 227-35, 2013.
Harding SM, Boiarsky J, and Greenberg RA. ATM dependent Silencing Links Nucleolar Chromatin Reorganization to DNA Damage Recognition.Cell Reports October 2015P.

Mechanisms responsible for ALT telomere mobility and recombination. Telomere length maintenance is a requisite feature of cellular immortalization and a hallmark of cancer. Approximately 85% of cancers rely on the re-expression of telomerase reverse transcriptase, while nearly 15% utilize a recombination-based mechanism known as alternative lengthening of telomeres (ALT). We developed a methodology for real time visualization of ALT (Cho et al. Cell 2014). This entails inducible expression of the FokI endonuclease fused to a telomere specific binding protein (mcherryTRF1-FokI). DSBs initiated rapid directional ALT telomere movement that extended for up to 4 μM, culminating in synapsis and homology dependent telomere synthesis. This unprecedented directional chromatin mobility was due to a specialized homology searching mechanism that is characterized by extensive single stranded DNA generation and homologous recombination between non-sister chromatids. Critical to this noncanonical form of homology search is the meiotic recombination complex Hop2-Mnd1, which is aberrantly reexpressed in ALT cells. These findings have implications for understanding large-scale chromatin dynamics, fundamental mechanisms of homology searches, and potential targets to selectively inhibit telomere maintenance in ALT positive cancers.

Cho NW, Dilley RL, Lampson MA, and Greenberg RA. Interchromosomal Homology Searches Drive Directional ALT Telomere Movement and Synapsis.Cell. 159: 108-121 2014 (PMC4177039). Highlighted in Cell 2014.

Discovery of new breast cancer susceptibility genes and biallelic BRCA1 mutations as a causing a new Fanconi Anemia Subtype (FANCS). Approximately 20% of familial breast cancer occurs as a consequence of germline heterozygous mutations in BRCA1 and BRCA2, suggesting the presence of additional genetic causes. We posited that several members of the RAP80 complex would be tumor suppressor genes based on their importance for BRCA1 dependent DNA repair. Indeed, we have reported germline deleterious mutations in RAP80 and Abraxas associated with familial breast cancer (refs c and d). Mutations within MERIT40 and BRCC36 were subsequently found by several other groups to confer cancer susceptibility. We have also identified biallelic mutations in BRCA1 as a cause of a new Fanconi Anemia subtype, and received HUGO approval to designate BRCA1 as FANCS. Biallelic mutations within BRCA1 were previously thought to be incompatible with viability in humans and genetic testing protocols had erroneously incorporated this assumption into recommended interpretations of genomic sequencing data. Our findings revealed that missense alleles within the BRCT regions were compatible with viability in humans when occurring in trans to another deleterious BRCA1 allele, and conferred multiple developmental anomalies consistent with Fanconi Anemia along with breast and ovarian cancer susceptibility. This discovery has altered genetic testing paradigms.

Sawyer SL, Tian L, Kähkönen M, Schwartzentruber J, Kircher M, University of Washington Centre for Mendelian Genomics, FORGE Canada Consortium, Majewski J, Dyment DA, Innes AM, Boycott KM, Moreau LA, Moilanen JS, Greenberg RA. Biallelic Mutations in BRCA1 Cause a New Fanconi Anemia Subtype.Cancer Discov 5(2):135-422014 2015.
Domchek SM*, Tang J, Jill Stopfer, Lilli DR, Tischkowitz M, Foulkes WD, Monteiro ANA, Messick TE, Powers J, Yonker A, Couch FJ, Goldgar D, Nathanson KL, Greenberg RA*:Biallelic deleterious BRCA1 mutations in a woman with early-onset ovarian cancer.Cancer Discovery 3: 399-405 2013 (PMC3625496) Notes: *co-corresponding authors. Highlighted in Cancer Discovery 2013
Solyom S, Aressy B, Pylkäs K, Patterson-Fortin J, Hartikainen JM, Kallioniemi A, Kauppila S, Nikkilä J, Kosma VM, Mannermaa A, Greenberg RA*, Winqvist R* Recurrent breast cancer predispositionassociated Abraxas mutation disrupts nuclear localization and DNA damage response functions of BRCA1.Science Trans Med 22;4(122):122ra23, 2012 (PMC in process).* co-corresponding authorship.
Nikkilä J, Coleman K, Morrissey D, Pylkäs K, Erkko H, Messick TE, Karppinen SM, Amelina A, Winqvist R*, and Greenberg RA*. Familial breast cancer screening reveals an alteration in the RAP80 UIM domain that impairs DNA damage response function. Oncogene. 28(16): 1843-52. 2009 (PMC2692655). * co-corresponding authorship

Deubiquitinating enzyme biochemistry and biological function in signal transduction. We have defined the biochemical, structural, and in vivo functional underpinnings of Zn2+ dependent (JAMM Domain) deubiquitinating enzymes, and their roles in DNA damage response and inflammatory cytokine signaling. Specifically, we have implicated BRCC36 in lysine63-linked ubiquitin specific DUB activity in the nucleus at DNA damage sites, and in the cytoplasm in stabilizing type I interferon receptor (Sobhian et al. Science 2007; Zheng et al. Cell Rep 2013). This body of work revealed that this class of DUBs is generally not active a single polypeptide, but requires interaction with MPN- domain proteins (Patterson-Fortin J. Biol Chem 2010). In collaboration with Frank Sicheri’s group at the University of Toronto, we have solved the crystal structure of active and inactive DUB complexes, uncovering the molecular basis behind JAMM domain DUB activity (Zeqiraj et al Molecular Cell 2015). This work makes possible the development of first in class JAMM domain DUB inhibitors based on our structural and biological insights.

Zeqiraj E, Tian L, Piggott CA, Pillon MC, Duffy NM, Ceccarelli DF, Keszei AF, Lorenzen K, Kurinov I, Orlicky S, Gish G, Heck AJR, Guarné A, Greenberg RA* and Sicheri F* Higher order assembly of BRCC36–KIAA0157 is required for DUB activity and biological function. Molecular Cell 2015, [ePub ahead of Print]. * co-corresponding authorship.
Zheng H, Gupta V, Patterson-Fortin J, Bhattacharya S, Katlinski, Wu J, Varghese B, Carbone CJ, Aressy B, Fuchs SY*, and Greenberg RA*. A novel BRISC-SHMT complex deubiquitinates IFNAR1 and regulates interferon responses. Cell Reports, Sept 26 2013. (PMC24075985). * co-corresponding authorship.
Patterson-Fortin J, Shao G, Bretscher H, Messick TE, Greenberg RA. Differential regulation of JAMM domain deubiquitinating enzyme activity within the RAP80 complex. J Biol Chem 285(40): 30971-81, 2010 (PMC2945588).
Sobhian B, Shao G, Lilli DR, Culhane AC, Moreau L, Xia B, Livingston DM* and Greenberg RA*. RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites. Science 316(5828): 1198- 202, 2007 (PMC2706583) * co-corresponding authorship.

Research Interest

The Greenberg lab is interested in understanding how chromatin responses to DNA damage impact genome integrity, cancer susceptibility, and response to anti-cancer therapy. Our basic findings have led to the identification of three new breast cancer susceptibility genes, a human syndrome associated with biallelic BRCA1 mutations, and insights into mechanisms by which chromatin responses affect response to targeted therapies.

Elizabeth Heller, Ph.D.

Proteomic characterization of inhibitory synapses. During doctoral training at The Rockefeller University under the mentorship of Dr. Nathaniel Heintz, I aimed to genetically tag and purify individual synapse types in the mammalian brain, in order to characterize their protein content using an innovative biochemical enrichment strategy coupled with high throughput proteomic analysis. In pursuit of this goal, I developed the first protocol for the specific biochemical isolation and characterization of the elusive inhibitory synapse.  We made a remarkable discovery, namely, that inhibitory synapses consist of structural proteins and ion channels, yet are completely lacking in the signaling molecules that comprise the major component of excitatory synapses.

Selimi F, Cristea IM, Heller E, Chait BT, Heintz N. Proteomic studies of a single CNS synapse type: the parallel fiber/purkinje cell synapse. PLoS Biol. 2009 Apr 14;7(4):e83. PubMed PMID: 19402746; PubMed Central PMCID: PMC2672601.
Heller EA, Zhang W, Selimi F, Earnheart JC, Ślimak MA, Santos-Torres J, Ibañez-Tallon I, Aoki C, Chait BT, Heintz N. The biochemical anatomy of cortical inhibitory synapses. PLoS One. 2012;7(6):e39572. PubMed PMID: 22768092; PubMed Central PMCID: PMC3387162.

Identification of critical period for sleep-consolidated spatial memory. During my undergraduate training I conducted an independent study under Dr. Ted Abel, aimed at elucidating the time course of sleep-induced memory formation in mice by examining memory deficits that result from sleep deprivation during discrete times following learning. We found that fear conditioning is blocked by sleep deprivation during a time period 5-10 hours post training, but unaffected by sleep deprivation for five hours immediately following training. This finding provided critical insights into the time-course of sleep-induced memory consolidation.

Graves LA, Heller EA, Pack AI, Abel T. Sleep deprivation selectively impairs memory consolidation for contextual fear conditioning. Learn Mem. 2003 May-Jun;10(3):168-76. PubMed PMID: 12773581; PubMed Central PMCID: PMC202307.

Locus-specific epigenetic editing for the study of addiction and depression. My postdoctoral research aimed to investigate the causal molecular mechanisms by which chromatin modifications contribute to reward-related pathology in the mammalian brain. There is a preponderance of compelling evidence implicating epigenetic modifications in the pathology of addiction and depression, in both human patients and animal models, yet previous studies have been unable to distinguish between the mere presence and the functional relevance of epigenetic modifications at relevant loci. To elucidate the molecular function of epigenetic regulation relevant to reward pathology, I have developed the use of engineered transcription factors to deliver histone modifications to a specific gene of interest in reward-related regions of the mammalian brain (major publications listed in Personal Statement).
Identification of cellular and molecular mechanisms underlying addiction and stress. In addition to pursuing my main postdoctoral research project, described above, I have also worked with others both inside and outside of the Nestler lab to investigate the molecular basis of drug addiction. For example, I have studied the role of serum- and glucocorticoid-inducible kinase 1 (SGK1) in regulating morphine and cocaine reward, and found that while its transcription and activity are upregulated in vivo by morphine and cocaine, exogenous SGK1 overexpression causes opposite behavioral responses to these two drugs. I have also contributed to several additional studies on the epigenetics of addiction, such as the role of nucleosome remodeling and the Sirtuin family of histone deacetylase.

Ferguson D, Koo JW, Feng J, Heller E, Rabkin J, Heshmati M, Renthal W, Neve R, Liu X, Shao N, Sartorelli V, Shen L, Nestler EJ. Essential role of SIRT1 signaling in the nucleus accumbens in cocaine and morphine action. J Neurosci. 2013 Oct 9;33(41):16088-98. PubMed PMID: 24107942; PubMed Central PMCID: PMC3792451.
Cates HM, Thibault M, Pfau M, Heller E, Eagle A, Gajewski P, Bagot R, Colangelo C, Abbott T, Rudenko G, Neve R, Nestler EJ, Robison AJ. Threonine 149 phosphorylation enhances ΔFosB transcriptional activity to control psychomotor responses to cocaine. J Neurosci. 2014 Aug 20;34(34):11461-9. PubMed PMID: 25143625; PubMed Central PMCID: PMC4138349.
Koo JW, Lobo MK, Chaudhury D, Labonté B, Friedman A, Heller E, Peña CJ, Han MH, Nestler EJ. Loss of BDNF signaling in D1R-expressing NAc neurons enhances morphine reward by reducing GABA inhibition. Neuropsychopharmacology. 2014 Oct;39(11):2646-53. PubMed PMID: 24853771; PubMed Central PMCID: PMC4207344.
Heller EA, Kaska S, Fallon B, Ferguson D, Kennedy PJ, Neve RL, Nestler EJ, Mazei-Robison MS. Morphine and cocaine increase serum- and glucocorticoid-inducible kinase 1 activity in the ventral tegmental area. J Neurochem. 2015 Jan;132(2):243-53. PubMed PMID: 25099208; PubMed Central PMCID: PMC4302038.

Research Interest

The Heller Lab studies the mechanisms by which remodeling of the epigenome leads to aberrant neuronal gene function and behavior.  To approach this problem, we directly manipulate histone and DNA modifications at specific genes in vivo, using viral delivery of epigenetic editing tools.  We focus on uncovering the mechanisms by which chromatin modifications interact with the transcriptional machinery following exposure to psychostimulants, such as drugs of abuse and stress. Because the behavioral disease traits of addiction and depression persist long after cessation of the harmful experience,  stable epigenetic remodeling is an attractive mechanism for such long-lasting effects and presents an intriguing target for therapeutic intervention.

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