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.
•Discovery of thyroid hormone receptors and their mechanism of repression.
• Discovery of Nuclear Receptor Corepressor Complexes.
• Elucidation of physiological Roles of Nuclear Receptor Corepressors and HDAC3.
• Discovery of REV-ERB and mechanisms of circadian regulation of transcription and metabolism.
• Identification and characterization of PPAR g in Adipose Biology.
Research Interest
The goal of the Lazar lab is to understand the transcriptional regulation of circadian rhythms and metabolism both in normal physiology and in metabolic diseases such as diabetes and obesity. The focus is on nuclear receptors and HDAC3-containing corepressor complexes, whose functions are interrogated using a combination of genomic, proteomic, bioinformatic, and metabolic phenotyping methods. Of particular interest are the circadian REV-ERB nuclear receptors, which are transcriptional repressors that function in the circadian clock and coordinate biological rhythms of metabolism in liver, adipose, and other tissues. Another focus is on nuclear receptor PPARg, a key transcriptional link between obesity and diabetes which functions as the master regulator of adipocyte biology and whose ligands have potent antidiabetic activity. Nuclear receptors corepressors and HDAC3 are also of great interest as an integrators of the activities of nuclear receptors and other transcription factors, with tissue-specific functions that protect from challenges to the circadian, nutritional, and thermal environment.
We have contributed to the understanding of mechanisms that create and control cell-to-cell variability in gene expression. In particular, our work was amongst the first to use quantitative single molecule RNA detection techniques to describe the phenomenon of transcriptional bursts, in which we found that transcription is a pulsatile process consisting of pulses of activity interspersed with periods when the gene is completely inactive (Raj et al. PLOS Bio 2006, Leveque and Raj Nat Meth 2013a). We also contributed to the mathematical modeling of this field (Raj et al. PLOS Bio 2006). We have now shown how these pulses relate to homeostatic mechanisms that maintain transcript concentration despite changes in cell volume and DNA content (Padovan-Merhar et al. Mol Cell 2015). We have also shown that variability can be used as a tool for dissecting mechanisms of transcriptional control of molecules such as long non-coding RNA (Maamar et al. Genes and Dev. 2013).
Raj A, Peskin CS, Tranchina D, Vargas DY, Tyagi S. Stochastic mRNA synthesis in mammalian cells.PLoS Biol. 2006 Oct;4(10):e309. PubMed PMID: 17048983; PubMed Central PMCID: PMC1563489.
Levesque MJ, Raj A. Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation. Nat Methods. 2013 Mar;10(3):246-8. doi: 10.1038/nmeth.2372. Epub 2013 Feb 17. Erratum in: Nat Methods. 2013 May;10(5):445. PubMed PMID: 23416756; PubMed Central PMCID: PMC4131260.
Maamar H, Cabili MN, Rinn J, Raj A. linc-HOXA1 is a noncoding RNA that represses Hoxa1 transcription in cis. Genes Dev. 2013 Jun 1;27(11):1260-71. doi: 10.1101/gad.217018.113. Epub 2013 May 30. PubMed PMID: 23723417; PubMed Central PMCID: PMC3690399.
Padovan-Merhar O, Nair GP, Biaesch AG, Mayer A, Scarfone S, Foley SW, Wu AR, Churchman LS, Singh A, Raj A. Single Mammalian Cells Compensate for Differences in Cellular Volume and DNA Copy Number through Independent Global Transcriptional Mechanisms. Mol Cell. 2015 Apr 16;58(2):339-52. doi: 10.1016/j.molcel.2015.03.005. Epub 2015 Apr 9. PubMed PMID: 25866248; PubMed Central PMCID: PMC4402149.
We have contributed to the understanding of how cell-to-cell variability can lead to phenotypic consequences. Specifically, we showed that variability in transcription can lead to random cell fate decisions in bacteria (Maamar and Raj et al. Science 2008), and that variability in gene expression can lead to phenotypic variability in metazoan development (Raj and Rifkin et al. Nature 2010). More recently, we have linked gene expression variability to single cell non-genetic resistance mechanisms in melanoma (Shaffer et al. Nature, in press).
Maamar H, Raj A, Dubnau D. Noise in gene expression determines cell fate in Bacillus subtilis. Science. 2007 Jul 27;317(5837):526-9. Epub 2007 Jun 14. PubMed PMID: 17569828; PubMed Central PMCID: PMC3828679.
Raj A, Rifkin SA, Andersen E, van Oudenaarden A. Variability in gene expression underlies incomplete penetrance. Nature. 2010 Feb 18;463(7283):913-8. doi: 10.1038/nature08781. PubMed PMID: 20164922; PubMed Central PMCID: PMC2836165.
Shaffer SM, Dunagin M, Torborg S, Torre EA, Emert T, Krepler C, Beqiri M, Sproesser K, Brafford P, Xiao M, Eggan E, Anastopoulos IN, Vargas-Garcia CA, Singh A, Nathanson K, Heryn M, Raj A. Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance. Nature, in press.
We have contributed to methodological approaches to measuring expression and transcription in single cells via RNA fluorescence in situ hybridization (RNA FISH). First, we developed a method that greatly simplifies the detection of individual RNA molecules by RNA FISH (Raj et al. Nat Meth 2008). We have since pushed the method to high multiplexing in the detection of chromosome structure and gene expression simultaneously (Levesque and Raj, Nat Meth 2013a). We also have enabled the detection of single nucleotide variants on individual RNA molecules (Levesque et al. Nat Meth 2013b), which allows for mutation detection and measurements of allele-specific expression. Further, we have developed an ultra-fast variant of RNA FISH that enables use in diagnostic and point of care settings (Shaffer et al. PLOS ONE 2013).
Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods. 2008 Oct;5(10):877-9. doi: 10.1038/nmeth.1253. Epub 2008 Sep 21. PubMed PMID: 18806792; PubMed Central PMCID: PMC3126653.
Levesque MJ, Raj A. Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation. Nat Methods. 2013 Mar;10(3):246-8. doi: 10.1038/nmeth.2372. Epub 2013 Feb 17. Erratum in: Nat Methods. 2013 May;10(5):445. PubMed PMID: 23416756; PubMed Central PMCID: PMC4131260.
Levesque MJ, Ginart P, Wei Y, Raj A. Visualizing SNVs to quantify allele-specific expression in single cells. Nat Methods. 2013 Sep;10(9):865-7. doi: 10.1038/nmeth.2589. Epub 2013 Aug 4. PubMed PMID: 23913259; PubMed Central PMCID: PMC3771873.
Shaffer SM, Wu MT, Levesque MJ, Raj A. Turbo FISH: a method for rapid single molecule RNA FISH. PLoS One. 2013 Sep 16;8(9):e75120. doi: 10.1371/journal.pone.0075120. eCollection 2013. PubMed PMID: 24066168; PubMed Central PMCID: PMC3774626.
Research Interest
Our lab aims to develop a quantitative understanding of the molecular biology of the cell. Interests include chromosome structure and gene expression, non-coding RNA, and global regulation of gene expression. Applications include genetics, cancer and stem cells.
Contributions to Science
- Identifying Epigenetic Dependencies in Leukemia.
- CRISPR-based Genetic Screening Methods
- Identifying Transcriptional, Epi-transcriptional and Kinase Dependencies in Leukemia.
- Investigating Genetic Regulatory Pathways using CRISPR-based Screening Methods.
Research Interest
The physiological effects of cancer are a manifestation of the genetic abnormalities that cause the disease. While much progress has been made in the understanding of such genetic perturbations, scientists still struggle to effectively identify, understand, and treat cancer-causing mutations. This is due to the fast-paced evolution of the disease, and the accumulation of novel mutations that permit cell survival even in the harsh environment created by a therapeutic. CRISPR is a gene-editing technology that couples the elegance of base complementarity with the enzymatic activity of a DNA nuclease in order to introduce mutations into target loci. CRISPR technologies help advance our understanding of the genetic perturbations that contribute to cancer maintenance.
Promoter DNA methylation is generally linked to transcriptional repression. However, euchromatic DNA methylation frequently occurs in regions outside promoters, such as gene bodies or regions upstream of promoters. The function of such non-promoter DNA methylation was largely unclear. Combining mouse genetic models and in vitro neural stem cell (NSC) system with biochemical, epigenomic and bioinformatic analyses, my PhD research revealed a novel function of de novo DNA methyltransferase DNMT3A-mediated non-promoter DNA methylation in facilitating transcription of neurogenic genes in postnatal NSCs. In contrast to the conventional view that DNA methylation is only linked to gene silencing, this study shows that DNA methylation at non-promoter regions may promote transcription by functionally antagonizing Polycomb repression complex 2 (PRC2).
Wu H, Tao J, Sun YE. Regulation and function of mammalian DNA methylation patterns: a genomic perspective. Brief Funct Genomics. 2012 May;11(3):240-50.
Wu H, Coskun V, Tao J, Xie W, Ge W, Yoshikawa K, Li E, Zhang Y, Sun YE. Dnmt3a-dependent nonpromoter DNA methylation facilitates transcription of neurogenic genes. Science. 2010 Jul 23;329(5990):444-8.
Fan G, Martinowich K, Chin MH, He F, Fouse SD, Hutnick L, Hattori D, Ge W, Shen Y, Wu H, ten Hoeve J, Shuai K, Sun YE. DNA methylation controls the timing of astrogliogenesis through regulation of JAK-STAT signaling. Development. 2005 Aug;132(15):3345-56.
TET proteins are Fe2+ and 2-oxoglutarate-dependent dioxygenases capable of successively oxidizing 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Highly oxidized cytosine bases (i.e. 5fC and 5caC) are selectively recognized and excised by Thymine DNA glycosylase (TDG), and the resulting abasic site is restored to unmodified C through the base excision repair (BER) pathway. Thus, methylation, oxidation, and excision repair offer a biochemically-validated model of mammalian active DNA demethylation pathway. However, little was known about the genomic distribution and gene regulatory functions of TET enzymes. As a postdoctoral fellow in the laboratory of Dr. Yi Zhang, I determined where Tet1 proteins are located across the genome of mouse ESCs. This work was amongst the first to reveal genomic distribution of TET enzymes in the mammalian genome. I found that TET1 is preferentially enriched at CpG-rich sequences at promoters of both transcriptionally active genes and PRC2-repressed lineage-specific genes. Epigenomic and transcriptomic analyses of Tet1-depleted cells reveal that TET1 plays roles in both transcriptional activation and repression, and TET1 contributes to repression of poised developmental regulators in ESCs by maintaining DNA hypomethylation states to facilitate PRC2 binding.
Wu H, Zhang Y. Reversing DNA methylation: mechanisms, genomics, and biological functions. Cell. 2014 Jan 16;156(1-2):45-68.
Wu H, Zhang Y. Mechanisms and functions of Tet protein-mediated 5-methylcytosine oxidation. Genes Dev. 2011 Dec 1;25(23):2436-52.
Wu H, Zhang Y. Tet1 and 5-hydroxymethylation: a genome-wide view in mouse embryonic stem cells. Cell Cycle. 2011 Aug 1;10(15):2428-36.
Wu H, D’Alessio AC, Ito S, Xia K, Wang Z, Cui K, Zhao K, Sun YE, Zhang Y. Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells. Nature. 2011 May 19;473(7347):389-93.
A complete understanding of the function of TET enzymes requires new methods to determine the genome-wide distribution of oxidized 5mC bases (5hmC/5fC/5caC). I have developed affinity-enrichment-based (5hmC/5fC/5caC DIP-seq) genome-wide mapping methods and systematically charted the genomic architecture and dynamics of these new DNA modifications. 5hmC is preferentially enriched at transcriptionally inactive/poised promoters as well as gene bodies of actively transcribed genes. In addition, 5hmC is frequently localized near distally located enhancers and CTCF binding sites. Genome-wide mapping of 5fC and 5caC indicates that these highly oxidized bases also accumulate at distal active enhancers and PRC2-repressed developmental gene promoters when TDG is depleted, suggesting that TET/TDG-dependent active DNA demethylation occurs dynamically at both proximal and distal gene regulatory regions. To enable quantitative and high-resolution mapping of TET/TDG-dependent active DNA demethylation, I have recently developed a single-base resolution mapping method, termed Methylase-Assisted BS-seq (MAB-seq), to precisely locate and quantify 5fC and 5caC bases.
Wu H, Zhang Y. Charting oxidized methylcytosines at base resolution. Nat Struct Mol Biol. 2015 Sep;22(9):656-61.
Wu H*, Wu X*, Shen L, Zhang Y. Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing. Nat Biotechnol. 2014 Dec;32(12):1231-40.
Shen L*, Wu H*, Diep D, Yamaguchi S, D’Alessio AC, Fung HL, Zhang K, Zhang Y. Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics. Cell. 2013 Apr 25;153(3):692-706.
Wu H, D’Alessio AC, Ito S, Wang Z, Cui K, Zhao K, Sun YE, Zhang Y. Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells. Genes Dev. 2011 Apr 1;25(7):679-84.
Research Interest
DNA cytosine methylation (5-methylcytosine) is an evolutionarily conserved epigenetic mark and has a profound impact on transcription, development and genome stability. Historically, 5-methylcytosine (5mC) is considered as a highly stable chemical modification that is mainly required for long-term epigenetic memory. The recent discovery that ten-eleven translocation (TET) proteins can iteratively oxidize 5mC in the mammalian genome represents a paradigm shift in our understanding of how 5mC may be enzymatically reversed. It also raises the possibility that three oxidized 5mC bases generated by TET may act as a new class of epigenetic modifications.
Our laboratory uses high-throughput sequencing technologies, bioinformatics, mammalian genetic models, as well as synthetic biology tools to investigate the mechanisms by which proteins that write, read and erase oxidized 5mC bases contribute to mammalian development (particularly cardiovascular and neural lineages) and relevant human diseases. To achieve this goal, we are also interested in developing new genomic sequencing and programmable epigenome-modifying methods to precisely map and manipulate these DNA modifications in the complex mammalian genome.
1. Identification of transcriptional adaptors/coactivators Gcn5/Ada2/Ada3 and discovery of novel histone modifications and mechanisms in transcription and sperm genome opening
We discovered transcriptional “adaptors”, which we showed associate with DNA binding activators, a groundbreaking new model for transcriptional activation, to reveal how histone enzymatic modifiers are recruited to genes (Berger+, Cell1990, Cell1993). We revealed the importance of adaptor Gcn5 acetyltransferase activity in transcriptional activation (1998), unifying transcription and chromatin regulation. We discovered numerous novel histone modifications (PTMs), PTM cross-talk, and sequential histone PTMs in transcription, including histone phosphorylation/acetylation (2001) and ubiquitylation/deubiquitylation. We discovered (2017) that enhancer RNAs bind directly to CBP, the key metazoan acetyltransferase, to stimulate HAT activity in vitro and at enhancers in vivo. We showed (2019) that Gcn5 provides key histone acetylation to broadly open the mouse genome during spermatogenesis for extensive chromatin restructuring.
a. Wang L, Liu L. and Berger SL. (1998) Critical residues for histone acetylation by GCN5, functioning in Ada and SAGA complexes, are also required for transcriptional function in vivo. Genes & Development 12: 640-653. PMCID: PMC316586
b. Lo W-S…Shiekhattar R, and Berger SL. (2001) Snf1 is a histone kinase which works in concert with the histone acetyltransferase Gcn5 to regulate transcription. Science 293:1142-6. PMID:111498592
c. Bose DA, Donahue G, Reinberg D, Shiekhattar R, Bonasio R, Berger SL. (2017) RNA binding to CBP stimulates histone acetylation and transcription. Cell 168,135-149. PMCID: PMC5325706.
d. Luense LJ, Donahue G, Lin-Shiao E….Bartolomei M, Berger SL. (2019) Gcn5-mediated histone acetylation governs nucleosome dynamics in spermiogenesis. Developmental Cell 51:745-758.
2. Discovery of chromatin mechanisms controlling aging and senescence
We uncovered chromatin changes involved in aging and cellular senescence, indicating broad epigenome dysregulation. These include pioneering studies that histone acetylation drives aging in yeast (2009), disrupts the nuclear laminar chromatin in mammals, and are crucial to enhancer function in aging (2019). We showed these disruptions trigger both homeostatic genomic protection and cellular damage, and discovered nuclear autophagy pathways in senescence leading to inflammation in aging and cancer (2015,2017,2020). Our findings suggest potential epigenetic therapeutics to ameliorate age-associated disease.
a. Dang W…Kaeberlein M, Kennedy BK, and Berger SL. (2009) Histone H4 lysine-16 acetylation regulates cellular lifespan. Nature 459:802-7. PMCID: PMC2702157.
b. Dou Z…Adams PD^, and Berger SL^. (2015) Autophagy mediates degradation of nuclear lamina. Nature 527:105-9. PMCID: PMC4824414. (2017) Cytoplasmic chromatin triggers inflammation in senescence and cancer. Nature 550:402-406. PMID:28976970.
c. Sen P, Lan Y…Adams PD, Schultz DC, Berger SL. (2019) Histone acetyltransferase p300 induces de novo super-enhancers to drive cellular senescence. Molecular Cell 73:684-698. PMID:30773298.
d. Xu C, Wang L…Adams PA, Ott M, Tong W, Johansen T, Dou Z^, and Berger SL^. (2020) SIRT1 is downregulated by autophagy in senescence and aging. Nature Cell Biology 22:1170-1179.
3. Demonstration of chromatin mechanisms controlling memory and behavior and relevant to aging.
Our studies in mouse brain and memory show a pivotal role of the metabolic enzyme, ACSS2, in fueling “on-site” acetyl-CoA generation on chromatin for neuronal histone acetylation and gene expression in normal memory and in alcohol-fueled addiction memory (2017/19). Our work in human Alzheimer’s disease reveals that the cognitively normal aging brain is epigenetically protected compared to the AD brain (2018/20). In other research on brain, we pioneered investigation of eusocial ant caste-specific behavior for organismal-level chromatin regulation and epigenetics, owing to the remarkable fact that female ants of distinct social castes (such as queen, soldier, and forager) share an identical genome. We sequenced the first ant genomes and then profiled the first histone modification epigenomes (2010,Science) and pioneered Crispr genetics in ants (2017,Cell). Groundbreaking results indicate a critical role of histone modifications in altering ant brain function to instruct complex social behavior; we identified a “window”, early after hatching, to behavioral reprogramming via epigenetic manipulation (2016/2020). We linked regulation of caste behavior to remarkable aging disparity.
a. Simola DF…Reinberg D^, Liebig J^, Berger SL^. (2016) Epigenetic (re)programming of caste-specific behavior in the ant C. floridanus. Science 351:aac6633. PMID: 26722000, PMCID: PMC5057185.
b. Mews, P… Berger SL. (2017) Acetyl-CoA metabolism by ACSS2 regulates neuronal histone acetylation and hippocampal memory. Nature 546,381-386. PMCID: PMC5505514. Mews P, Egervari G^…Garcia, B, Berger SL^. (2019) Alcohol metabolism contributes to brain histone acetylation. Nature 574: 717-721.
c. Glastad K, Graham RJ, Ju L, Rossler J, Brady CM, and Berger SL (2020) Epigenetic regulator CoRest controls social behavior in ants. Molecular Cell 77:338-351.
d. Nativio R, Donahue G…Johnson FB^, Bonini NM^, Berger SL^ (2018) Dysregulation of the epigenetic landscape of normal aging in Alzheimer’s disease. Nature Neuroscience 21,497-505. Nativio R, Lan Y…Garcia BA, Trojanowski JQ, Bonini NM^, Berger SL^. (2020) An integrated multi-omics approach identifies epigenetic drivers associated with Alzheimer’s disease. Nature Genetics 52:1024-1035.
- Discovery of tumor suppressor p53 factor and histone modifications and their mechanisms including activating p53 acetylation, repressive p53 methylation, and novel chromatin pathways in p53-mediated transcriptional activation
Our work revealed new enzyme modifiers and post-translational modifications of p53 (including acetylation, methylation, and demethylation, 2006/7) regulating p53 activity. Our findings propelled broad efforts in the field to discover novel acetylation and methylation of transcription factors. We showed p53 methylation is generally repressive to its function, and showed repressive p53 methylation occurring in certain cancers bearing high levels of wild type p53. We discovered novel epigenetic pathways used by wild type and mutant p53 in regulating chromatin structure/function in normal and cancer cells, such as gain-of-function p53 mutants driving transcriptional activating and growth promoting histone modifications (2015). We showed that p53 and p63 establish new enhancers during stress and development. We found a novel role of p53 in promoting target gene association with nuclear speckles for transcriptional amplification (2021).
- a. Huang J…Jenuwein T, andBerger SL. (2006) Repression of p53 activity by Smyd2-mediated methylation. Nature 444:629-32. PMID:17108971. Huang J…Jenuwein T, and Berger SL. (2007) p53 is regulated by the lysine demethylase LSD1. Nature, 449:105-8.
- Bungard D…Thompson CB, Jones RG andBerger SL. (2010) Signaling kinase AMPK activates stress-promoted transcription via histone H2B phosphorylation. Science 329: 1201-5. PMCID: PMC3922052.
- Zhu J, Sammons MA, Donahue G, Dou Z…Arrowsmith CH, andBerger SL. (2015) Gain-of-function p53 mutants co-opt epigenetic pathways to drive cancer growth. Nature 525:206-11. PMCID: PMC4568559
- Alexander KA…Belmont A, Joyce EF, Raj A, and Berger SL. (2021) p53 mediates target gene association with nuclear speckles for amplified RNA expression. Molecular Cell 81:1666-1681.
- Investigation of epigenetic mechanisms in T and CART cell exhaustion and cancer immunotherapy
We established collaborations with Carl June (pioneer of CAR T cell therapy in cancer) and John Wherry (discovered key aspects of T cell exhaustion). We investigate epigenetic regulation in patient response to immunotherapy, and controlling T cell exhaustion in mouse models.
- Pauken KE…Berger SL, and Wherry EJ. (2016) Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade.Science 354,1160-1165. Khan O…Berger SL, and Wherry EJ. (2019) TOX transcriptionally and epigenetically programs CD8+ T cell exhaustion. Nature 571, 211.
- Fraietta JA…Berger SL, Bushman FD, June CH, and Melenhorst JJ. (2018) Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T-cells.Nature 555, 307-312.
- Chen Z…Berger SL, Wherry EJ, and Shi J. In vivo CRISPR screening identifies Fli1 as a transcriptional safeguard that restrains effector CD8 T cell differentiation during infection and cancer. Cell 184:1262.
- Good CR+, Kuramitsu S+, Aznar MA+…Young RM^,Berger SL^, June CH^ (2021) In vitro dysfunction model reveals the plasticity of patient CAR-T cells and identifies transcription factors whose modulation can restrain CAR-T cell exhaustion. Cell, 184:6081-6100.
Research Interest
Our lab focuses on mechanisms that regulate gene expression with a special emphasis on how the DNA-packaging structure of chromatin is manipulated during genomic processes. Our findings inform the study of cancer and other diseases, and ultimately drug discovery.
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.