Roger Greenberg, M.D., Ph.D.
University of Pennsylvania
The Perelman School of Medicine
Department of Cancer Biology
513 BRB II/III
421 Curie Blvd
Philadelphia, PA 19104
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.
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.
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