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. We revealed the importance of adaptor Gcn5 acetyltransferase activity in transcriptional activation (1998), to unify understanding of transcription and chromatin regulation. We discovered numerous novel histone modifications, modification cross-talk, and sequential histone modifications in transcription, including histone phosphorylation/acetylation (2001) and ubiquitylation/deubiquitylation. In 2017, we discovered that enhancer RNAs bind directly to CBP, the key metazoan acetyltransferase, to stimulate HAT activity in vitro and at enhancers and promoters in vivo. We recently showed (2019) that Gcn5 provides key histone acetylation to broadly open the mouse genome during spermatogenesis for broad chromatin restructuring.
- 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
- Lo W-S, Duggan L, Belotserkovskya R, Emre T, Lane W, 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:11498592
- 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.
- 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 in yeast and mammals.
Our work uncovered chromatin changes involved in aging and cellular senescence, indicating broad dysregulation of the epigenome. These include pioneering studies demonstrating that histone acetylation drives aging in yeast (2009) and disruption of the nuclear lamina with its associated chromatin domains in mammals. We showed these disruptions trigger both homeostatic genomic protection and cellular damage, and discovery of nuclear autophagy pathways in senescence leading to inflammation in aging and cancer (2015, 2017, 2019, 2020). Our findings suggest potential epigenetic therapeutics to ameliorate age-associated disease.
- Dang W…Kaeberlein M, Kennedy BK, and Berger SL. (2009) Histone H4 lysine-16 acetylation regulates cellular lifespan. Nature 459:802-7. PMCID: PMC2702157.
- 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.
- 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.
- 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, Oct 9..
3. Demonstration of chromatin mechanisms controlling memory and behavior.
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 (2013). 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). We pioneered Crisper genetics in ants (2017).
- 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.
- Mews, P, Donahue G… 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.
- Glastad K, Graham RJ, Ju L, Rossler J, Brady CM, and Berger SL (2019) Epigenetic regulator CoRest controls social behavior in ants. Molecular Cell 77:338-351.
- 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.
4. 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, 2006/7/10)) regulating p53 activity. Our findings spurred broad efforts to discover novel transcription factor modifications. 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 (2019) establish new enhancers during stress and development.
- Huang J…Jenuwein T, and Berger SL. (2006) Repression of p53 activity by Smyd2-mediated methylation. Nature 444:629-32. PMID:17108971.
- Bungard D…Thompson CB, Jones RG and Berger 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, and Berger SL. (2015) Gain-of-function p53 mutants co-opt epigenetic pathways to drive cancer growth. Nature 525:206-11. PMCID: PMC4568559
- Lin-Shiao E, Lan Y…Sammons M, Ludwig K, and Berger SL. (2019) p63 establishes epithelial enhancers at critical craniofacial development genes. Science Advances, May 1; 5:eaaw0946.
5. Investigation of epigenetic mechanisms affecting cancer immunotherapy.
We established collaborations with Carl June (pioneered 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.
- 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.
- 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.
- Khan O…Berger SL, and Wherry EJ. (2019) TOX transcriptionally and epigenetically programs CD8+ T cell exhaustion. Nature 571, 211-218.
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.
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.
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.
Discovery of Rev-erba and mechanism of repression by nuclear receptors. I discovered receptors for thyroid hormone as well as the orphan NR that we named Rev-erba that is the subject of this proposal. My lab demonstrated that Rev-erbs bind to a unique DNA sequence as a monomer and to a related site as a dimer. We were among the first to recognize that NRs have a ligand-independent repression function, and we discovered the CoRNR box motif which explains why corepressors bind to unliganded but not liganded forms of nuclear receptors. Most recently we discovered that Rev-erba represses transcription at the genome both directly and indirectly, i.e., independent of its DNA-binding domain. Together, our studies have demonstrated the mechanism and physiological significance of repression by Rev-erba and other NRs.
Lazar MA, Hodin RA, Darling DS, Chin WW. A novel member of the thyroid/steroid hormone receptor family is encoded by the opposite strand of the rat c-erbA alpha transcriptional unit. Mol Cell Biol. 1989 Mar;9(3):1128-36. PubMed PMID: 2542765; PubMed Central PMCID: PMC362703.
Harding HP, Lazar MA. The monomer-binding orphan receptor Rev-Erb represses transcription as a dimer on a novel direct repeat. Mol Cell Biol. 1995 Sep;15(9):4791-802. Erratum in: Mol Cell Biol 1995 Nov;15(11):6479. PubMed PMID: 7651396; PubMed Central PMCID: PMC230723.
Hu X, Lazar MA. The CoRNR motif controls the recruitment of corepressors by nuclear hormone receptors. Nature. 1999 Nov 4;402(6757):93-6. PubMed PMID:10573424.
Zhang Y, Fang B, Emmett MJ, Damle M, Sun Z, Feng D, Armour SM, Remsberg JR, Jager J, Soccio RE, Steger DJ, Lazar MA. Discrete functions of nuclear receptor Rev-erba couple metabolism to the clock. Science 348:1488-1492, 2015. PubMed PMID: 26044300; PubMed Central PMCID: PMC4613749.
Nuclear Receptor Corepressor Complexes. My laboratory was first to purify an endogenous NR corepressor complex, and discovered the stoichiometric presence of the epigenome modifying enzyme HDAC3. We demonstrated that HDAC3 polypeptide itself has little intrinsic enzymatic activity, and its deacetylation function requires interaction with a region of NCoR or SMRT that we termed the DAD (“Deacetylase Activating Domain”), and that the catalytic activity of HDAC3 requires the DAD in vivo. We also generated NCoR mutant mice to show for the first time that corepressors and HDAC3 were critical for normal adult physiology. These studies have demonstrated the repression mechanisms and physiological functions of nuclear receptor corepressors.
Guenther MG, Lane WS, Fischle W, Verdin E, Lazar MA (corresponding), Shiekhattar R. A core SMRT corepressor complex containing HDAC3 and TBL1, a WD40-repeat protein linked to deafness. Genes Dev. 2000 May 1;14(9):1048 57. PubMed PMID: 10809664; PubMed Central PMCID: PMC316569.
Guenther MG, Barak O, Lazar MA. The SMRT and N-CoR corepressors are activating cofactors for histone deacetylase 3. Mol Cell Biol. 2001 Sep;21(18):6091-101. PubMed PMID: 11509652; PubMed Central PMCID: PMC87326.
Alenghat T, Meyers K, Mullican SE, Leitner K, Adeniji-Adele A, Avila J, Buƒáan M, Ahima RS, Kaestner KH, Lazar MA. Nuclear receptor corepressor and histone deacetylase 3 govern circadian metabolic physiology. Nature. 2008 Dec 18;456(7224):997-1000. doi: 10.1038/nature07541. Epub 2008 Nov 26. PubMed PMID: 19037247; PubMed Central PMCID: PMC2742159.
You SH, Lim HW, Sun Z, Broache M, Won KJ, Lazar MA. Nuclear receptor co-repressors are required for the histone-deacetylase activity of HDAC3 in vivo. Nat Struct Mol Biol. 2013 Feb;20(2):182-7. doi: 10.1038/nsmb.2476. Epub 2013 Jan 6. PubMed PMID: 23292142; PubMed Central PMCID: PMC3565028.
PPARg in Adipose Biology. In 1994, we reported that PPARg is predominantly expressed in adipose tissue and induced during adipocyte differentiation. We found that phosphorylation regulates PPARg activity and in vivo insulin sensitivity, and that adipose PPARg is required for normal fat development in vivo. We were first to characterize the PPARg cistrome in mouse adipocytes, and demonstrate a role for nearby binding of C/EBPa. We have also shown that antidiabetic TZD ligands activate PPARg bound at enhancers, while transrepression is due redistribution of coactivators away from enhancer sites controlled by other factors. Most recently we demonstrated that SNPs regulate adipose tissue PPARg binding, function, and response to TZDs. In sum, we identified PPARg as an important transcription factor in adipocytes, and have uncovered its mechanisms of action at the genome.
Chawla A, Schwarz EJ, Dimaculangan DD, Lazar MA. Peroxisome proliferator-activated receptor (PPAR) gamma: adipose-predominant expression and induction early in adipocyte differentiation. Endocrinology. 1994 Aug;135(2):798-800. PubMed PMID: 8033830.
Lefterova MI, Zhang Y, Steger DJ, Schupp M, Schug J, Cristancho A, Feng D, Zhuo D, Stoeckert CJ Jr, Liu XS, Lazar MA. PPARgamma and C/EBP factors orchestrate adipocyte biology via adjacent binding on a genome-wide scale. Genes Dev. 2008 Nov 1;22(21):2941-52. doi: 10.1101/gad.1709008. PubMed PMID: 18981473; PubMed Central PMCID: PMC2577797.
Step SE, Lim HW, Marinis JM, Prokesch A, Steger DJ, You SH, Won KJ, Lazar MA. Anti-diabetic rosiglitazone remodels the adipocyte transcriptome by redistributing transcription to PPARg-driven enhancers. Genes Dev. 2014 May 1;28(9):1018-28. doi: 10.1101/gad.237628.114. PubMed PMID: 24788520; PubMed Central PMCID: PMC4018489.
Soccio RE, Chen ER, Rajapurkar SR, Safabakhsh P, Steger DJ, Marinis JM, Dispirito JR, Briggs ER, Fang B, Everett LJ, Lim HW, Won KJ, Wu Y, Civelek M, Voight BF, Lazar MA. Genetic variation determines PPARg function and antidiabetic drug response in vivo. Cell 162:33-44, 2015. PMCID: PMC4493773 [Available on 2016-07-02].
Resistin. In 2001, we discovered the polypeptide hormone resistin as an adipocyte-specific, secreted protein whose gene expression was down-regulated by TZD treatment. We also discovered a family of other Resistin-Like Molecules (RELMs). We showed ectopic resistin exacerbated insulin resistance while genetic deletion of resistin improved glucose metabolism. We found that resistin expression in adipocytes requires a PPARg binding site that is present in the mouse genome but absent in humans (e), where resistin is secreted mainly from monocytes in response to inflammatory stimuli. Humanizing mice for resistin exacerbated insulin resistance due to inflammation. In sum, our discovery of resistin and its role in insulin resistance have contributed in a major way to current understanding of how adipokines and inflammation impact metabolism.
Steppan CM, Bailey ST, Bhat S, Brown EJ, Banerjee RR, Wright CM, Patel HR, Ahima RS, Lazar MA. The hormone resistin links obesity to diabetes. Nature. 2001 Jan 18;409(6818):307-12. PubMed PMID: 11201732.
Steppan CM, Brown EJ, Wright CM, Bhat S, Banerjee RR, Dai CY, Enders GH, Silberg DG, Wen X, Wu GD, Lazar MA. A family of tissue-specific resistin-like molecules. Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):502-6. PubMed PMID:11209052; PubMed Central PMCID: PMC14616.
Banerjee RR, Rangwala SM, Shapiro JS, Rich AS, Rhoades B, Qi Y, Wang J, Rajala MW, Pocai A, Scherer PE, Steppan CM, Ahima RS, Obici S, Rossetti L, Lazar MA. Regulation of fasted blood glucose by resistin. Science. 2004 Feb 20;303(5661):1195-8. PubMed PMID: 14976316.
Qatanani M, Szwergold NR, Greaves DR, Ahima RS, Lazar MA. Macrophage-derived human resistin exacerbates adipose tissue inflammation and insulin resistance in mice. J Clin Invest. 2009 Mar;119(3):531-9. doi: 10.1172/JCI37273. Epub 2009 Feb 2. PubMed PMID: 19188682; PubMed Central PMCID: PMC2648673.
Transcriptional Regulation of Circadian Rhythms and Metabolism. Rev-erba is expressed with a large amplitude circadian rhythm in most cells and represses the activating clock gene BMAL1 via NCoR/HDAC3. We discovered that Rev-erba protein activity is modulated by heme ligand as well as by proteasomal degradation facilitated by lithium. We demonstrated that Rev-erba binds to the genome rhythmically with NCoR/HDAC3 leading to circadian modulation of the epigenome on a genome-wide scale and that loss of Rev-erbs a and b abolishes circadian rhythms. We also were first to characterize circadian enhancers in liver, and discovered the mechanism whereby Rev-erbs and other factors control multiple distinct phases of circadian gene expression. Genetic loss of the Rev-erbs, NCoR, or HDAC3 leads to massive hepatosteatosis in large part via a non-enzymatic function of HDAC3. We are currently interrogating the enzyme-independent functions of HDAC3 in liver. In brown adipose tissue, we discovered that Rev-erba controls circadian rhythm of cold tolerance and heat generation. Together, our studies have demonstrated circadian modulation of the epigenome and its relation to metabolism in health and disease.
Feng D, Liu T, Sun Z, Bugge A, Mullican SE, Alenghat T, Liu XS, Lazar MA. A circadian rhythm orchestrated by histone deacetylase 3 controls hepatic lipid metabolism. Science. 2011 Mar 11;331(6022):1315-9. doi: 10.1126/science.1198125. PubMed PMID: 21393543; PubMed Central PMCID: PMC3389392.
Bugge A, Feng D, Everett LJ, Briggs ER, Mullican SE, Wang F, Jager J, Lazar MA. Rev-erba and Rev-erbb coordinately protect the circadian clock and normal metabolic function. Genes Dev. 2012 Apr 1;26(7):657-67. doi: 10.1101/gad.186858.112. PubMed PMID: 22474260; PubMed Central PMCID: PMC3323877.
Gerhart-Hines Z, Feng D, Emmett MJ, Everett LJ, Loro E, Briggs ER, Bugge A, Hou C, Ferrara C, Seale P, Pryma DA, Khurana TS, Lazar MA. The nuclear receptor Rev-erba controls circadian thermogenic plasticity. Nature. 2013 Nov 21;503(7476):410-3. doi: 10.1038/nature12642. Epub 2013 Oct 27. PubMed PMID: 24162845; PubMed Central PMCID: PMC3839416.
Fang B, Everett LJ, Jager J, Briggs E, Armour SM, Feng D, Roy A, Gerhart-Hines Z, Sun Z, Lazar MA. Circadian enhancers coordinate multiple phases of rhythmic gene transcription in vivo. Cell. 2014 Nov 20;159(5):1140-52. doi:10.1016/j.cell.2014.10.022. PubMed PMID: 25416951; PubMed Central PMCID: PMC4243056.
My laboratory focuses on the transcriptional and epigenomic regulation of metabolism by nuclear receptors and their coregulators. Our identification of the nuclear heme receptor Rev-erba and its corepressor complex, including histone deacetylase 3 (HDAC3), have uncovered fundamental principles of molecular clocks and the circadian regulation of metabolism, as well as the tissue-specificity of coregulator function and epigenomic modifications. Our pioneering studies of PPARg and adipocyte biology, including discovery of the hormone resistin, have linked basic mechanisms of gene transcription to physiology and metabolic diseases. This work has important implications for endocrinology, diabetes, and metabolism.
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.
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.
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.
Current areas of interest within the lab include: (1) Defining the functional importance of epigenetic regulators in leukemia, (2) Development and optimization of AsCas12a for multiplex genetic dropout screening, and (3) Developing new functional genomic tools.
While whole exome sequencing of the leukemia cancer genome revealed many oncogene mutations, few of these genetic alterations lead to directly actionable therapeutic opportunities. A major objective of the lab is to annotate and dissect these genetic vulnerabilities in leukemia. To approach this, we use our highly developed domain-focused CRISPR genetic knockout screening technology, where CRISPR-mediated mutagenesis is directed to gene sequences encoding critical protein domains. This method generates a larger fraction of functional null-alleles, which increase the severity in a negative selection-based genetic screen. In contrast to RNA interference-based methods or prior CRISPR-based screening approaches, this new method is not only more efficient than other screening approaches, but also has the potential to evaluate protein domain function directly from genetic screening, and may allow high-throughput identification of protein domains that are suitable drug targets in cancer. Coupling functional genomics screening, biochemical assays, and pre-clinical mouse models, we investigate the aberrant transcription signaling networks of leukemia and explore them as potential therapeutic opportunities. Since genetic screenings are only as successful as the underlying technology, a major focus of the lab is to further optimize and expand our screening toolbox. Projects are underway to engineer different Cas proteins for multiplex genetic screening using a variety of methods, including structure-guided rational design and directed evolution. Our ultimate goal is to uncover complex genetic interactions in leukemia that are therapeutically tractable.
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.
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.