Posts classified under: Program Members

Penn Epigenetics > Program Members

Irfan A. Asangani, Ph.D.

Research Interest

The Asangani Lab is affiliated with the Department of Cancer Biology at the Perelman School of Medicine, University of Pennsylvania, Philadelphia. Our lab investigates how epigenetic regulators, such as chromatin modifying enzymes and chromatin-associated proteins, cooperate with transcription factors to orchestrate transcriptional addiction in cancer cells. We aim to translate this knowledge into clinical tools by developing novel diagnostic, prognostic, and therapeutic strategies.

Ben E. Black, Ph.D.

Contributions to Science

1. Centromere structural biochemistry. The work in my 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? Our 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. We 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 addition, we reconstituted the core centromeric nucleosome complex (CCNC) that includes both CENP-C and CENP-N and defined their independent modes of binding and roles of each in maintaining centromere identity in Guo et al., 2017. In the course of these studies, we 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 B.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: PMC4610723]
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]
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.

2. Aurora B-mediated mitotic error correction. While studying patient-derived cells harboring neocentromeres, my team 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. We find 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 impact 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]
Salimian, K.J., E.R. Ballister, E.M. Smoak, S. 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]

3. 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 I called the CENP-A targeting domain, CATD), I have contributed highly to the understanding of these processes. My group 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, we 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. More recently we devised a ChIP-seq-based strategy to probe centromeric chromatin architecture at very high-resolution with a study (Hasson et al., 2013; 3b, below) that resolved a longstanding conflict regarding the nature of human centromeric nucleosomes assembled by HJURP. We’ve 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. These findings led to our landmark development of new types of human artificial chromosomes (HACs) that improve the technology and bring mammalian synthetic chromosomes one step closer to reality in Logsdon et al., 2019.

Logsdon, G.L., C.W. Gambogi, M.A. Liskovykh, E.J. Barrey, V. Larionov, K.H. Miga, P. Heun, and B.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]
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]
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]

4. Hydrogen/deuterium exchange-mass spectrometry (HXMS) with chromatin proteins. My group 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. We have 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 we have advanced HXMS technology and dispelled the earlier misconceptions that the approach is low-resolution (it is not, and we have 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 we have 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 B.E. Black*. 2020. Structural basis for allosteric PARP-1 retention on DNA breaks. Science, 368:eaax6367. (*corresponding authors; †contributed equally) [PMCID: PMC7347020]
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]
Hansen, J.C.*, B.B. Wexler, D.J. Rogers, K.C. Hite, T. Panchenko, S. Ajith, and B.E. Black*. 2011. DNA binding restricts the intrinsic conformational flexibility of Methyl CpG Binding Protein 2 (MeCP2). J. Biol. Chem., 286:18938-18948. (*corresponding authors) [PMCID: PMC3099709]

5. 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.

Das, A., A. Iwata-Otsubo, A. Destouni, J.M. Dawicki-McKenna, K.G. Boese, B.E. Black*, and M.A. Lampson*. 2022. Epigenetic, genetic and maternal effects enable stable centromere inheritance. Nat. Cell Biol., 24:748-756. (*corresponding authors) [PMCID: PMC9107508]
Lampson, M.A.*, and B.E. Black*. 2017. Cellular and molecular mechanisms of centromere drive. Cold Spring Harb. Symp. Quant. Biol., 82:249-257. (*corresponding authors) [PMCID: PMC6041145]
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.

Contributions to Science

1. Chromatin complexes and noncoding RNAs
The maintenance of epigenetic memory critically relies on proper targeting of complexes that modify chromatin structure. A key question is how are these complexes targeted to the proper genomic regions in different lineages. Using protein biochemistry and next generation sequencing, I discovered that multiple proteins of the Polycomb group, critical for epigenetic repression, bind to RNA and that their RNA binding activity contributes both to chromatin localization and to regulation of PRC2 activity at specific genes (Kaneko 2013). More recently, I have adapted my protein–RNA interaction mapping technique to identify additional regions of PRC2 that interact RNA and found the protein surface likely responsible for the RNA-mediated inhibition of its activity (Zhang 2019). Because we identified RNA-binding sites on both “flavors” of PRC2 (PRC2.1 and PRC2.2) we also investigated their function, discovering distinct contribution of these two complexes to the epigenetic changes that occur during neural differentiation (Petacovici 2021). We also discovered the RNA-binding activity of another chromatin modifier, TET2, and found that this enzyme regulates levels of small noncoding RNAs derived from tRNAs (He 2020). Our studies on Polyomb
Kaneko S, Son J, Shen SS, Reinberg D†, Bonasio R†. PRC2 binds active promoters and contacts nascent RNA in embryonic stem cells. Nature Structural and Molecular Biology 2013;20:1258–64. PMID: 24141703; PMCID: PMC3839660.
Zhang Q*, McKenzie NJ*, Warneford-Thomson R*, Gail EH, Flanigan SF, Owen BM, Lauman R, Levina V, Garcia BA, Schittenhelm RB, Bonasio R†, Davidovich C†. RNA exploits an exposed regulatory site to inhibit the enzymatic activity of PRC2. Nature Structural and Molecular Biology. 2019;26:297. PMID: 30833789.
He C†, Bozler J, Janssen KA, Wilusz JE, Garcia BA, Schorn AJ, Bonasio R†. TET2 chemically modifies tRNAs and regulates tRNA fragment levels. Nature Structural and Molecular Biology 2020; doi:10.1038/s41594-020-00526-w. PMID: 33230319.
Petracovici A, Bonasio R. Distinct PRC2 subunits regulate maintenance and establishment of Polycomb repression during differentiation. Molecular Cell 2021; doi:10.1016/j.molcel.2021.03.038. PMID: 33887196.

2. Epigenetics of social behavior in ants
My ultimate research goal is to understand how epigenetic processes of gene regulation impact complex organism-level phenomena such as brain function and behavior. To this end, since my postdoctoral work I have led efforts to develop ants as experimental organisms for neuroepigenetic research. Ant workers and queens exhibit dramatically different morphologies, lifespans, and behaviors, which are encoded by the same genome and, therefore, must be specified at the epigenetic level. With our high-quality assemblies of the ant genomes and annotation of coding and non-coding genes (Shields 2018), as well as the recent demonstration of feasibility of reverse genetics by genome editing, we have contributed to establish Harpegnathos saltator as a molecular model system for neuroepigenetic research. Our transcriptomic analyses combined with genetic and social manipulations revealed key roles for neuropeptides (Gospocic 2017) and steroid hormones (Gospocic 2021) in establishing and maintaining distinct neurotranscriptomes in the different castes. We also discovered that social reprogramming in Harpegnathos causes extensive cellular plasticity in the brain, including the expansion of a neuroprotective glia population (Sheng 2020), which has spurred my interest in a new direction for my research: aging and neurodegeneration.
Gospocic J, Shields EJ, Glastad KM, Lin Y, Penick CA, Yan H, Mikheyev AS, Linksvayer TA, Garcia BA, Berger SL, Liebig J, Reinberg D, Bonasio R. The neuropeptide corazonin controls social behavior and caste identity in ants. Cell 2017;170:748. PMID 28802044; PMCID: PMC5564227.
Shields EJ, Sheng L, Weiner AK, Garcia BA, Bonasio R. High-quality genome assemblies reveal long non-coding RNAs expressed in ant brains. Cell Reports 2018;23:3078. PMID 29874592; PMCID: PMC6023404.
Sheng L*, Shields EJ*, Gospocic J, Glastad KM, Ratchasanmuang P, Raj A, Little S, Bonasio R. Social reprogramming in ants induces longevity-associated glia remodeling. Sci Adv 2020;6: eaba9869. PMID 32875108; PMCID: PMC7438095.
Gospocic J*, Glastad KM*, Sheng L, Shields EJ, Berger SL‡, Bonasio R‡. Kr-h1 maintains distinct caste-specific neurotranscriptomes in response to socially regulated hormones. Cell 2021;184:5807. PMID: 34739833.

3. Technology development
Throughout my career I have been actively engaged in developing new technologies. Some of them became part of the studies described above and below, others were reported on their own. The first primary publications originating from my own lab were a novel method to detect protein–RNA interactions based on their proximity and not their affinity (Beck 2014) and a mass spectrometry screen to map the RNA-binding sites of hundreds of known and novel RNA-binding proteins in the nucleus of embryonic stem cells (He 2016). My lab was also an early adopter of high-throughput single-cell RNA sequencing techniques (e.g. Drop-seq) and helped other laboratories on campus to obtain access to this technology (Nicetto 2019). Most recently, we developed a new technique to map R-loops (Yan 2019).
Beck D*, Narendra V*, Drury WJ 3rd, Casey R, Jansen PW, Yuan ZF, Garcia BA, Vermeulen M, Bonasio R. In vivo proximity labeling for the detection of protein–protein and protein–RNA interactions. Journal of Proteome Research 2014;13:6135. PMID: 25311790; PMCID: PMC4261942.
He C, Sidoli S, Warneford-Thomson R, Tatomer DC, Wilusz JE, Garcia BA, Bonasio R. High-resolution mapping of RNA-binding regions in the nuclear proteome of embryonic stem cells. Molecular Cell 2016;64:416. PMID: 27768875; PMCID: PMC5222606.
Nicetto D, Donahue G, Jain T, Peng T, Sidoli S, Sheng L, Montavon T, Becker JS, Grindheim JM, Blahnik K, Garcia BA, Tan K, Bonasio R, Jenuwein T, Zaret KS. Loss of H3K9me3 heterochromatin at protein coding genes enables developmental lineage specification. Science 2019;363:294. PMID: 30606806; PMCID: PMC6664818.
Yan Q*, Shields EJ*, Bonasio R†, Sarma K†. Mapping native R-loops genome-wide with a targeted nuclease approach. Cell Reports 2019;179:953. PMID 31665646; PMCID PMC6870988.

4. Migratory routes of dendritic cells
In my graduate studies in immunology, I discovered that dendritic cells sample antigens in the periphery and, rather than migrating to primary lymphoid tissues, reenter the circulation to reach distant sites of action (Cavanagh 2005, Bonasio 2006). These discoveries challenged the long-held view that dendritic cells only traverse a unidirectional route from the blood to the peripheral tissues and from here to lymphoid organs, and suggested entirely new mechanisms by which central tolerance could be achieved and immunological memory reactivated. I also developed a new technique to covalently label T lymphocytes with quantum dots for in vivo visualization by intravital multiphoton microscopy (Bonasio 2007). Although less relevant for my current area of research, my studies in immunology demonstrate the breadth of my training and provide me with a valuable skill set in imaging, mouse genetics, and in vivo approaches that is typically lacking in more conventionally trained researchers in biochemistry and functional genomics.
Cavanagh LL*, Bonasio R*, Mazo IB, Halin C, Cheng G, van der Velden AW, Cariappa A, Chase C, Russell P, Starnbach MN, Koni PA, Pillai S, Weninger W, von Andrian UH. Activation of bone marrow-resident memory T cells by circulating, antigen-bearing dendritic cells. Nature Immunology 2005;6:1029–37. PMID: 16155571; PMCID: PMC1780273.
Bonasio R, Scimone ML, Schaerli P, Grabie N, Lichtman AH, von Andrian UH. Clonal deletion of autoreactive thymocytes by circulating dendritic cells homing to the thymus. Nature Immunology 2006;7:1092–100. PMID: 16951687.
Bonasio R, Carman CV, Kim E, Sage PT, Love KR, Mempel TR, Springer TA, von Andrian UH. Specific and covalent labeling of a membrane protein with organic fluorochromes and quantum dots. Proceedings of the National Academy of Sciences 2007;104:14753. PMID: 17785425; PMC1976196.

*Equal contributions
†Co-corresponding authors

Research Interest

The Bonasio Lab investigates the contribution of epigenetic gene regulation to brain function, with a focus on the role of noncoding RNAs. We study these fundamental biological processes at a mechanistic level in mouse stem cells and neurons and at a functional level the brain of traditional and less traditional model organisms, such as ants, fruit flies, and planarians.

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

Contribution to Science

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.

Contribution to Science

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.

Eric-Joyce

Eric F. Joyce, Ph.D.

Contribution to Science

We have generated custom Oligopaint probes to precisely target population-defined domains known as TADs in single cells and, using high- and super-resolution microscopy, found evidence for extensive heterogeneity across individual alleles. As this has implications for gene regulation, we discovered that the expression of genes at TAD boundaries are particularly sensitive to reduced cohesin levels in pathological cohesin dysfunction such as in cohesinopathies like Cornelia De Lange Syndrome. We further found that cohesin promotes stochastic boundary bypass between domains for proper expression of boundary-proximal genes. More recently, we found that co-depletion of NIPBL and WAPL, two opposing regulators of cohesin, rescues chromatin misfolding and gene misexpression, consistent with a model in which cohesin levels are balanced by its activity on chromatin.

Research Interest

Our laboratory studies the spatial organization of the genome. We use a combination of cellular, molecular, genetic, and computational tools to elucidate how the structure and position of chromosomes within the nucleus is established and inherited across cell divisions, and how dysfunctional organization contributes to genome instability and disease. We also develop and utilize new technologies that use fluorescent in situ hybridization (FISH) to interrogate chromosome structure at single-cell resolution.

1 … 9 10 11
Previous Next
Close
Test Caption
Test Description goes like this