Matt Weitzman, Ph.D.

Work in my lab addresses the dynamic interactions between viruses and host cells when their genomes are in conflict. My lab pioneered the study of cellular damage sensing machinery as an intrinsic defense to virus infection. We have studied the DNA damage responses with a range of human DNA viruses and identified distinct ways that they manipulate signaling networks and DNA repair processes. Studying DNA damage as part of the cellular response to infection has opened up a new area in the biology of virus-host interactions. It has also provided a platform for interrogating cellular pathways involved in recognition and processing of DNA damage. This work revealed that the MRN complex is the mammalian sensor of DNA breaks and viral genomes, and that it is required for efficient activation of ATM/ATR damage signaling.

Stracker, TH, Carson, CT and Weitzman, MD. (2002) Adenovirus oncoproteins inactivate the Mre11-Rad50-NBS1 DNA repair complex. Nature, 418, 348-352.
Carson, CT, Schwartz, RA, Stracker, TH, Lilley, CE, Lee, DV and Weitzman, MD (2003). The Mre11 complex is required for ATM activation and the G2/M checkpoint.  EMBO J, 22,6610-6620.
Lilley, CE, Carson, CT, Muotri, AR, Gage, FH and Weitzman, MD (2005). DNA repair proteins affect the HSV-1 lifecycle.  Proc Natl Acad Sci USA, 102, 5844-5849.
Lilley, CE, Chaurushiya, MS, Boutell, C, Everett, RD, and Weitzman, MD (2011). The intrinsic antiviral defense to incoming HSV-1 genomes includes specific DNA repair proteins and is counteracted by the viral protein ICP0. PLoS Pathog 7:e1002084. PMC3116817
Chaurushiya, MS, Lilley, CE, Aslanian, A, Meisenhelder, J, Scott, DC, Landry, S, Ticau, S, Boutell, C, Yates, JR, Schulman, BA, Hunter, T and Weitzman, MD (2012). Viral E3 ubiquitin-mediated degradation of a cellular E3: viral mimicry of a cellular phosphorylation mark targets the RNF8 FHA domain.  Mol Cell 46, 79-90. PMC3648639

I have had a long-standing interest in viral and host proteins that bind DNA and chromatin. As a postdoc I used biochemical approaches to identify a recognition sequence for the Rep protein of AAV within the site-specific integration site on chromosome 19 (AAVS1). I demonstrated that Rep proteins can mediate interaction between cellular and viral DNA to promote targeted integration. We have recently employed proteomic approaches to identify proteins associated with viral DNA genomes during infection, as well as the modifications that occur to chromatin on the host genome. We have analyzed histone post-translational modifications during virus infection and shown how these are altered by viruses. We recently discovered that the histone-like protein VII encoded by Adenovirus for packaging of its genome, can also affect the composition of cellular chromatin by retaining danger signals to overcome immune signaling. We are now interested in looking at how viruses impact genome and nuclear architecture and the effects this has on gene expression.

Weitzman, MD, Kyöstiö, SRM, Kotin, RM and Owens, RA (1994). Rep proteins of adeno-associated virus (AAV) mediate a complex formation between AAV DNA and the AAV integration site on human chromosome 19.  Proc Natl Acad Sci USA, 91, 5808-5812.
Kulej, K, Avgousti, DC, Weitzman, MD and Garcia, BA (2015). Characterization of histone post-translational modifications during virus infection using mass spectrometry-based proteomics. Methods 90, 8-20.
Avgousti, DC, Herrmann, C, Sekulic, N, Kulej, K, Petrescu, J, Molden, RC, Pancholi, NJ, Reyes, ED, Seeholzer, SH, Black, BE, Garcia, BA and Weitzman, MD (2016). A core viral protein binds host nucleosomes to sequester immune danger signals. Nature 535, 173-177. PMC4950998
Kulej, K, Avgousti, DC, Sidoli, S, Herrman C, Della Fera, AN, Kim ET, Garcia, BA and Weitzman, MD (2017). Time-resolved global and chromatin proteomics during Herpes Simplex Virus Type 1 (HSV-1) infection. Mol Cell Proteomics 16, S92-S107.
Reyes, RD, Kulej, K, Akhtar, LN, Avgousti, DC, Pancholi, NJ, Kim, ET, Bricker, D, Koniski, S, Seeholzer, SH, Isaacs, SN, Garcia, BA, and Weitzman, MD. Identifying host factors associated with DNA replicated during virus infection. (in press).

My lab is interested in cellular responses that restrict virus replication. APOBEC3 proteins belong to a family of cytidine deaminases that provide a line of defense against retroviruses and endogenous mobile retroelements. We were the first to show that human APOBEC3A (A3A) is a catalytically active cytidine deaminase, with a preference for ssDNA. We demonstrated that A3A is a potent inhibitor of endogenous retroelements such as LINE1, and also blocks replication of single-stranded parvoviruses such as AAV and MVM. We have also shown how the SAMHD1 protein limits replication of the DNA virus HSV-1. We discovered ways that cellular DNA repair proteins can act as species-specific barriers through their interaction with viral proteins.

Chen, H, Lilley, CE, Yu, Q, Lee, DV, Chou, J, Narvaiza, I, Landau, NR and Weitzman, MD (2006). APOBEC3A is a potent inhibitor of adeno-associated virus and retrotransposons.  Curr Bio 16, 480-485.
Narvaiza, I, Linfesty, DC, Greener, BN, Hakata, Y, Pintel, DJ, Logue, E, Landau, NR, and Weitzman, MD (2009). Deaminase-independent inhibition of parvoviruses by the APOBEC3A cytidine deaminase.  PLoS Pathog 5, e1000439. PMC2678267
Richardson, SR, Narvaiza, I, Planegger, RA, Weitzman, MD and Moran, JV (2014). APOBEC3A deaminates transiently exposed single-strand DNA that arises during LINE-1 retrotransposition.  eLife 3, e02008. PMC4003774
Kim, ET, White, TE, Brandariz-Nunez, A, Diaz-Griffero, F, and Weitzman, MD (2013). SAMHD1 restricts herpes simplex virus type 1 (HSV-1) in macrophages by limiting DNA replication.  J Virol 87, 12949-12956. PMC3838123
Lou, DI, Kim, ET, Shan, S, Meyerson, NR, Pancholi, NJ, Mohni, KM, Enard, D, Petrov, DA, Weller, SK, Weitzman, MD*, and Sawyer, SL (2016). An intrinsically disordered region of the DNA repair protein Nbs1 is a species-specific barrier to Herpes Simplex Virus 1 in primates.  Cell Host & Microbe 20, 178-188. (*Co-corresponding author)

Proteins that mutate viral genetic material must also be carefully regulated to prevent deleterious effects on the host genome. While studying antiviral functions for A3A we discovered that the enzyme can also act on the cellular genome, inducing DNA breaks and cell cycle arrest. We suggested therefore that APOBEC proteins cause genomic instability and contribute to malignancy, and we are now studying how they are regulated to prevent inappropriate mutations. This body of work demonstrates how studying virus-host interactions can lead to insights into fundamental processes that impact cellular genomic integrity. We have recently found A3A upregulated in a subset of human leukemias and demonstrated how this provides vulnerability for targeted cancer therapies.

Landry, S, Narvaiza, I, Linfesty, DC and Weitzman, MD (2011). APOBEC3A can activate the DNA damage response and cause cell cycle arrest.  EMBO Reports 12, 444-450. PMC3090015
Narvaiza, I, Landry, S, and Weitzman, MD (2012). APOBEC3 proteins and genome stability: The high cost of a good defense? Invited Extraview in Cell Cycle 11, 33-38.
Green, AM, Landry, S, Budagyan, K, Avgousti, D, Shalhout, S, Bhagwat, AS and Weitzman, MD (2016). APOBEC3A damages the cellular genome during DNA replication.  Cell Cycle 15, 998-1008. PMC4889253
Green, AM, Budagyan, K, Hayer, KE, Reed, MA, Savani, MR, Wertheim, GB and Weitzman, MD (2017). Cytosine deaminase APOBEC3A sensitizes leukemia cells to inhibition of the DNA replication checkpoint. Cancer Research (in press)

Research Interests

Our lab aims to understand host responses to virus infection, and the cellular environment encountered and manipulated by viruses. We study multiple viruses in an integrated experimental approach that combines biochemistry, molecular biology, genetics and cell biology. We have chosen viral models that provide tractable systems to investigate the dynamic interplay between viral genetic material and host defense strategies. We have used proteomic approaches to probe the dynamic interactions that take place on viral and cellular genomes during infection, and have uncovered ways that viruses manipulate histones and chromatin as they take control of cellular processes. The pathways illuminated are key to fighting diseases of viral infection, provide insights into fundamental processes that maintain genome instability, and have implications for the development of efficient viral vectors for gene therapy.

Roberto Bonasio, Ph.D.

Research Interest

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

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

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

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

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

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

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

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

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

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

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

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

Research Interest

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

Elizabeth Heller, Ph.D.

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

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

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

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

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

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

Research Interest

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

Eric F. Joyce, Ph.D.

Visualizing the genome with Oligopaint FISH probes. Typically, studies of chromosome positioning have been stymied by the lack of affordable, high-resolution FISH probes, which are usually targeted to only a few loci at a time. Moreover, not only are conventional technologies not a practical source of probe for use in high-throughput methodologies, they fall short of revealing the location of whole chromosomes or specific sub-chromosomal regions in interphase nuclei. As a postdoctoral fellow, I co-developed Oligopaint FISH probes, which, in contrast to conventional probes, are computationally designed, synthesized on microarrays, and generated via PCR amplification. This strategy provides precise control over the sequences they target and allows for single and multicolor imaging at a fraction of the cost of conventional probes.

Beliveau B.J.*, Joyce E.F.*, Apostolopoulos N., Yilmaz F., Fonseka C.Y., McCole R.B., Chang Y., Li J.B., Senaratne T.N., Williams B.R., Rouillard J.M., Wu CT. (2012). Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes. PNAS. 109(52). PMID: 23236188. (*equal authorship)
Beliveau B.J., Boettiger A.N., Avendaño M.S., Jungmann R., McCole R.B., Joyce E.F., Kim-Kiselak C., Bantignies F., Fonseka C.Y., Erceg J., Hannan M.A., Hoang H.G., Colognori D., Lee J.T., Shih W.M., Yin P., Zhuang X., Wu CT. (2015). Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes. Nature Communications. 6(7147). PMID: 25962338.

High-throughput FISH-based screening for novel architectural factors. The least understood aspects of spatial genome organization are the mechanisms that determine where a gene or genomic region is localized in the cell nucleus. As a postdoctoral fellow, I spearheaded the development of Hi-FISH, a fully automated FISH-based imaging pipeline to quantitatively determine the position of multiple loci in the nucleus. Using this tool, we were able to conduct the first FISH-based genome-wide RNAi screen for factors important for nuclear organization in Drosophila. The screen targeted two heterochromatic sequences, located on different chromosomes, and revealed a complex network of genes that either promote or antagonize interchromosomal associations between these regions, shifting our viewpoint of chromosome interactions towards a more dynamic process. In particular, we isolated the condensin II complex as a major organizing factor that antagonizes chromosome pairing and heterochromatin clustering during interphase, consistent with a general role in antagonizing interchromosomal interactions. Intriguingly, our identification of condensin II as an anti-pairing factor is in line with the intrachromosmal functions of compaction and chromatin looping being a mechanism by which long-range interactions are inhibited. The implications of this model are especially profound with respect to DNA repair, replication, and gene expression.

Joyce E.F., Williams B.R., Xie T., Wu CT. (2012). Identification of genes that promote or antagonize somatic homolog pairing using a high-throughput FISH-based screen. PLoS Genetics. 8(5). PMID: 22589731.
Senaratne T.N., Joyce E.F., Nguyen S.C., Wu CT. (2016). Investigating the Interplay between Sister Chromatid Cohesion and Homolog Pairing in Drosophila Nuclei. PLoS Genetics. 12(8). PMID: 27541002.
Joyce E.F.. Toward high-throughput and multiplexed imaging of genome organization. (2017) Assay and Drug Development Technologies. Jan 15. PMID: 28092459.

Inheritance of chromosome positioning. Chromosome positioning plays important roles in genome stability and gene function. However, it remains unclear how this information gets transmitted from mother to daughter cell and whether genome organization, in general, is a component of heritable information through meiosis.  To address these questions, we examined nuclear organization across different developmental time points in the Drosophila germline, from early embryogenesis to adulthood. Using our custom Oligopaint FISH technology, we were able to target multiple regions across the genome and found that the spatial organization of the genome differs between the germline and the soma from the earliest moments of development. It is currently unclear how different tissues could acquire such different organizational fates in the early embryo; however, we favor a model in which germline nuclei actively suppress or delay the mechanisms that drive specific interchromosomal interactions in the soma. This model is consistent with our screen results, described above, that argue for a controlled process reflecting genes that promote as well as those that antagonize these types of interactions.

Joyce E.F., Apostolopoulos N., Beliveau B.J., Wu CT. (2013). Germline progenitors escape the widespread phenomenon of homolog pairing during Drosophila development. PLoS Genetics. 9(12). PMID: 24385920.
Joyce E.F., Erceg J., Wu CT. (2016). Pairing and anti-pairing: a balancing act in the diploid genome. Current Opinion in Genetics & Development. 37. PMID: 27065367.

Recently, my group has successfully scaled the Oligopaint design to label large genomic regions, including whole chromosomes, which we have shown can also be multiplexed to visualize different chromatin states and gene activity (Nguyen and Joyce in prep.). We anticipate this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes, providing a novel battery of assays to better characterize how chromatin is packaged and spatially positioned in the nucleus and how these events impact genome integrity. Using a candidate approach, we also isolated Cap-H2, a subunit of the Condensin II complex, as a factor that controls chromosome size and chromosome intermixing in Drosophila (Rosin and Joyce in prep.). These analyses show that Oligopaints are robust enough to detect changes in large-scale chromosome organization at single cell resolution. They also show that condensin II is a strong candidate for a complex that drives CT formation in diploid cells, which would represent the first molecule confirmed in a metazoan to have such a function.

Research Interest

Our laboratory studies the spatial organization of the genome, with implications for gene regulation, genome integrity, and diseases such as cancer, aging, and neurodegenerative disorders. We use Drosophila and mammalian systems in combination with cellular, molecular, genetic, and computational tools to elucidate how the structure and position of chromosomes within the nucleus is established and inherited across cell divisions.

Mitchell A. Lazar, M.D., Ph.D.

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, Bućan 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.

Research Interest

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.

Mia Levine, Ph.D.

The evolution of young genes via de novo- and duplication- based mechanisms
Evolutionary mechanisms of innovation at the molecular level are numerous. Codons diverge, regulatory elements arise and degenerate, and new genes are born. These signatures of adaptive evolutionary change are frequently species-restricted. My PhD research identified very young genes that harbor no homology to exons in related genomes. In contrast to classic mechanisms of novel gene formation like gene duplication, these de novo genes arise instead from fortuitous sequence evolution at noncoding DNA. These genes exhibited testis-biased expression and signatures of adaptive evolution, implicating male germline processes as potent agents of selection of these rare mutations. This publication was the first to describe such de novo genes that have since been documented in a wide array of taxa, including humans. My postdoctoral research focused instead on gene duplication as a potent mechanism of adaptive diversification. A shared domain structure between parent and daughter proteins facilitated my goal to identify lineage-specific innovations in proteins that package DNA. Prior to my research, the Heterochromatin Protein 1 (HP1) gene family was thought to encode between 2 and 5 members across eukaryotes. I discovered 22 new HP1 members in Drosophila. These 22 paralogs were all born less than 20 million years ago. Nevertheless, the number of HP1 genes per species remains relatively constant. This revolving door of gene replacement implicates conserved, currently undefined chromatin functions encoded by unconserved components recurrently generated by gene duplication.

Levine, M.T., C. D. Jones, A. D. Kern, H. A. Lindfors, and Begun, D.J. (2006) Novel genes derived from noncoding DNA in Drosophila melanogaster are frequently X-linked and exhibit testis-biased expression.Proceedings of the National Academy of Sciences 103: 9935-9939. PMCID: PMC1502557.
Levine, M.T., McCoy, C. Vermaak. D., LeeY.C.G, Hiatt, M.A., Matsen, F.A., and H.S. Malik (2012) Phylogenomic analysis reveals dynamic evolutionary history of the Drosophila Heterochromatin Protein 1 (HP1) gene family.  PLOS Genetics8(6): e1002729. PMCID: PMC3380853.

Adaption to novel environments at DNA packaging proteins

melanogaster, like humans, evolved in Africa but more recently invaded the New World and established populations from tropical to temperate climates. These geographically structured populations are panmictic (randomly mating), so any genotypic or phenotypic differences observed are likely the product of fitness variation across environments, i.e., natural selection. This spatially-structured system therefore presents a unique opportunity to elucidate the molecular basis of adaptation. I have taken both whole genome- and single locus- approaches. In one report, genomic DNA fromD. melanogastertropical and temperate populations from both Australia and the US were hybridized to whole-genome tiling arrays from which we inferred geographic sequence divergence (allele frequency variation) based on geographically structured differences in probe intensities. Our analysis demonstrated that a remarkably large fraction of the D. melanogaster genome has been targeted by spatially-varying positive selection. Our whole-genome analysis also uncovered many previously unsuspected biological functions associated with adaptation to novel environments. One of the most intriguing of these functions was chromatin binding. In light of the extensive data on the environment sensitivity of chromatin dynamics, I was especially interested in the unexplored role that chromatin-remodeling factors play in adaptation to novel habitat. Under one model, chromatin-remodeling factors evolve to maintain chromatin structure that is perturbed by environmental fluctuations. I focused primarily on the Polycomb Group genechameau. I discovered a linear relationship between latitude and allele frequency at several SNPs in both the US and Australian populations, which represent independent colonization events. Moreover, an amino acid-changing SNP predicted variation in tolerance to freezing temperatures. These data strongly implicated the action of natural selection and introduced chromatin-remodeling factors as a potentially rich source of adaptive genetic variation. Inspired by the observation that chromatin-based gene regulation can span more than one promoter, I also tested the hypothesis that adaptive expression variation across latitudinal gradients spans physically linked genes. I found that gene “neighborhoods” (of up to 15 genes), rather than single genes, exhibit adaptive transcriptional profiles, consistent with the notion that chromatin factors regulate adaptive expression variation across space.
Turner, L.T., Levine, M.T., and Begun, D.J. (2008). Genomic analysis of adaptive differentiation in Drosophila melanogaster.Genetics 179: 475-485. PMCID: PMC2390623.
Levine, M.T. and Begun, D.J. (2008). Evidence of spatially varying selection at four chromatin-remodeling loci inDrosophila melanogaster. Genetics 179: 455-473. PMCID: PMC2390624.
Levine, M.T., Eckert, M., and D.J. Begun (2011) Whole genome expression plasticity across tropical and temperateDrosophila melanogaster populations from eastern Australia. Molecular  Biology and Evolution 28: 249–256. PMCID: PMC3002243.

Evolutionary and functional diversification of essential DNA packaging proteins
Conserved nuclear proteins support conserved nuclear processes. Yeast and humans, for example, share essential, homologous chromatin proteins that package eukaryotic DNA and support shared, essential functions like chromosome segregation and telomere integrity. These cellular processes, however, also rely on unconserved molecular machinery. A surprisingly large fraction of essential genes that encode chromatin proteins evolve rapidly. My dissertation documented early evidence of this paradoxical phenomenon. The Dosage Compensation Complex (DCC) is responsible for equalizing X-linked gene dosage via chromatin remodeling of the single male X chromosome.  Loss of function at DCC genes is lethal. I discovered population genetic evidence of positive selection at four of the five DCC complex components. Continuing this theme during my postdoctoral research, I uncovered the essential function of the Heterochromatin Protein 1 paralog, HP1E. HP1E is required for faithful segregation of paternal DNA during the first embryonic mitosis. Nevertheless, a subset of Drosophila species apparently persists without HP1E. I discovered that in D. melanogaster not all paternal chromosomes are equally vulnerable to chromatin bridging during the first embryonic mitosis—the heterochromatin-rich sex chromosomes are more likely to mis-segregate than the large autosomes. Intriguingly, over evolutionary time major rearrangements of these same sex chromosomes co-occur with the pseudogenization of HP1E in the obscura group of Drosophila. These data support a model under which karyotype evolution rendered dispensable a once-essential gene. My findings thus provided a neat hypothesis to resolve the apparent paradox of HP1E’s essentiality in D. melanogaster together with its loss in related species.

Levine, M.T., Holloway, A.K., Arshad, U., and Begun, D.J. (2007) Pervasive and largely lineage-specific adaptive protein evolution in the dosage compensation complex of Drosophila melanogaster.Genetics 177: 1959–1962. PMCID: PMC2147993.
Levine, M.T. and H.S. Malik (2013) A rapidly evolving genomic toolkit of Drosophila heterochromatin.Fly 7: 137-141. PMCID: PMC4049844.
Levine, M.T., Vander Wende, H., and H.S. Malik (2015) Mitotic fidelity requires transgenerational action of a testis-restricted HP1.eLife 4: e07378. PMCID: PMC4491702

Research Interest

Chromatin proteins package our genomic DNA. Essential, highly conserved cellular processes rely on this genome compartmentalization, yet many chromatin proteins are wildly unconserved over evolutionary time. We study the biological forces that drive chromatin protein evolution and the functional consequences for chromosome segregation, telomere integrity, and genome defense.

Ronen Marmorstein, Ph.D.

My laboratory has pioneered the structure-function analysis of histone acetyltransferases (HATs) and continues to make seminal contributions in this area.  Specifically, my laboratory determined the first crystal structure of a type A HAT and characterized its mechanism of catalysis, and the first to describe the mode of histone substrate binding by a HAT. My laboratory has extended our studies to the broader family of N- acetlyltransferases including the non-histone lysine acetyltrasnferases (KATs) and the N-amino acetyltransferases (NATs).  We have uncovered important molecular signatures that distinguish HATs, KATs and NATs. My laboratory has also contributed to the development of acetyltransferase inhibitors. The vast majority of the human proteome is acetylated in a functionally important manner and alterations occur in human diseases.  This suggests that protein acetylation may rival protein phosphorylation as a biologically important protein modification and that KATs and NATs represent important therapeutic targets.

Lasko, L.M., Jakob, C.G., Edalji, R.P., Qiu W., Montgomery D., Digiammarino .EL., Hansen T.M., Risi R.M., Frey R., Manaves V., Shaw B., Algire M., Hessler P., Lam L.T., Uziel T., Faivre E., Ferguson D., Buchanan F.G., Martin R.L., Torrent M., Chiang G.G., Karukurichi K., Langston J.W., Weinert B.T., Choudhary C., de Vries P., Van Drie J.H., McElligott D., Kesicki E., Marmorstein R., Sun C., Cole P.A., Rosenberg SH, Michaelides M.R., Lai A., Bromberg K.D. Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours. (2017) Nature, 550:128-132. PMID:28953875; PMCID:PMC6050590
Gottlieb, L. and Marmorstein, R. Structure of human NatA and its regulation by the Huntingtin interacting protein HYPK. (2018) Structure 26:925-935. PMID: 29754825: PMCID: PMC6031454
Deng, S., Magin, R.S., Wei, X., Pan, B., Petersson, E.J. and Marmorstein, R., Structure and mechanism of acetylation by the N-terminal dual enzyme NatA/Naa50 complex. (2019) Structure, 27: 1057-1070. PMID:31155310; PMCID:6610660
Deng, S., McTiernan, N., Wei, X., Arnesen, T. and Marmorstein, R. Molecular basis for N-terminal acetylation by human NatE and its modulation by HYPK, (2020) Nature Comm., 11: 14584-14587. PMID32042062: PMCID: PMC7010799

My laboratory is studying the molecular basis for how chromatin is assembled and maintain by histone chaperone complexes.  We have focused on the binding and histone deposition of H3/H4 complexes by the ASF1 and VPS75 proteins and the multisubunit HIRA complex, which specifically deposits the histone H3 variant, H3.3, in a replication independent manner.  Histone H3.3 is deposited at active genes, after DNA repair and in certain forms of heterochromatin in non-proliferating senescent cells, and recurrent H3.3 mutations are found in pediatric glioblastoma and misregulation of H3.3-specific activities in tumor growth and leukemia exemplifies the necessity for proper regulation of H3.3-specific deposition pathways. Together with the Peter Adams laboratory we have pioneered a molecular understanding of the HIRA complex highlighting the particular importance of the HIRA and Ubn1 subunits of H3.3-specific activities.

Ricketts, M.D., Frederick, B., Hoff. H., Tang, Y., Schultz, D.C. Rai, T.S., Vizioli, M.G. Adams, P.D. and Marmorstein, R. Ubinuclein-1 confers histone H3.3-specific binding specificity by the HIRA histone chaperone complex. (2015) Nature Communications. 6:7711-. PMID: 26159857: PMCID: PMC4509171
Haigney, A., Ricketts, M. D. and Marmorstein, R. Dissecting the Molecular Roles of Histone Chaperones in Histone Acetylation by Type B Histone Acetyltransferases (HAT-B), (2015) J. Biol. Chem., 290:30648-30657. PMID: 26522166: PMCID: PMC4683284
Ray-Gallet, D., Ricketts, M.D., Sato, Y., Gupta, K., Boyarchuk, E., Senda, T., Marmorstein, R., and Almouzni, G. Functional activity of the H3.3 histone chaperone complex HIRA requires trimerization of the HIRA subunit. (2018) Nat. Commun. 9:3103. PMID:30082790: PMCID: PMC6078998
Ricketts, M.D., Dasgupta, N., Fan, J., Han, J., Gerace, M., Tang, Y., Black, B.E., Adams, P.D. and Marmorstein, R. The HIRA histone chaperone complex subunit UBN1 harbors H3/H4 and DNA binding activity. (2019) J. Biol. Chem., 294: 9239-9259. PMID:31040182; PMCID: PMC6556585

My laboratory has leveraged our expertise in biochemistry and X-ray crystallography with small molecule screening for structure-based Inhibitor development for therapy of melanoma and other cancers.  There is a particular interest in melanoma and the laboratory had developed inhibitors to several important oncogenic kinases in melanoma including BRAF, PI3K, PAK1 and S6K1. The laboratory has also targeted the oncoproteins E7 and E6 from human papillomavirus (HPV), the causative agent of a number of epithelial cancers, and a significant portion of head and neck cancers. These studies have important implications for therapy.

Qin, J., Rajaratnam, R., Feng, L., Salami, J., Barber-Rotenberg, J.S., Domsic, J., Reyes-Uribe, P., Liu, H., Dang, W., Berger, S.L., Villanueva, J., Meggers, E. and Marmorstein, R.  Development of organometallic S6K1 inhibitors. (2015) J. Med. Chem. 58:305-314. PMID: 25356520; PMCID: PMC4289024
Grasso, M., Estrada, M.A., Ventocilla, C., Samanta, M., Maksimoska, J., Villanueva, J., Winkler, J.D. and Marmorstein, R. Chemically linked vemurafenib inhibitors promote an inactive BRAFV600E conformation. (2016) ACS Chem. Biol. 11: 2876-2888. PMID: 27571413: PMCID: PMC5108658
Emtage, R.P., Schoeberger, M.J. Fergusion, K.M., and Marmorstein, R. Intramolecular autoinhibition of Checkpoint Kinase 1 is mediated by conserved basic motifs of the C-terminal Kinase Associated-1 domain. (2017) J. Biol. Chem. 292:19024-19033. PMID:28972186: PMCID: PMC5704483
Grasso, M., Estrada, M.A., Berrios, K.N., Winkler, J.D. and Marmorstein, R. N-(7-Cyano-6-(4-fluro-3-(2-(3-(trifluoromethyl)phenyl)acetamido)phenoxy)benzo[d]thiazol-2-yl)cyclopropanecarboxamide (TAK632) promotes inhibition of BRAF through the induction of inhibited dimers. (2018) J. Med. Chem. 61:5034-5046. PMID: 29727562: PMCID: PMC6540792

My laboratory has more recently studied the connection between metabolism with cancer signaling and chromatin regulation, with a particular focus on the acetyl-CoA metabolism and metabolite acylation enzymes such as ATP citrate lyase (ACLY). Our studies uncovered the molecular mechanism of ACLY and provided a molecular scaffold for the structure-based development of ACLY inhibitors for therapy of cancer and metabolic and cardiovascular disorders.

Bazilevsky, G.A., Affronti, H.C., Wei, X., Campbell, S.L., Wellen, K.E. and Marmorstein. R. ATP-citrate lyase multimerization is required for coenzyme-A substrate binding and catalysis, (2019) J. Biol. Chem. 294:7529-7268. PMID: 30877197; PMCID: PMC6509486
Wei, X., Schultz, K., Bazilevsky, G.A., Vogt, A. and Marmorstein R. Molecular basis of acetyl-CoA production by ATP-citrate lyase. (2020) Nature Structural & Molecular Biology 27:33-41. PMID: 31873304

Research Interest

The Marmorstein laboratory studies the molecular mechanisms of (1) epigenetic regulation (2) protein post- and co-translational modification with a particular focus on protein acetylation, and (3) enzyme signaling in cancer and metabolism.  The laboratory uses a broad range of biochemical, biophysical and structural research tools (X-ray crystallography and cryo-EM) to determine macromolecular structure and mechanism of action.  The laboratory also uses high-throughput small molecule screening and structure-based design strategies to develop protein-specific small-molecule probes to interrogate protein function and for preclinical studies.

Jennifer E. Phillips-Cremins, Ph.D.

Research Interest

Epigenomics and Systems Neurobiology Lab: The Cremins Lab investigates the link between three-dimensional organization of genomes and the establishment and maintenance of neural cell function. We employ systems level experimental and computational approaches to (1) create high-resolution 3-D genome architecture maps and (2) integrate 3-D architecture maps with genome-wide maps of epigenetic modifications and gene expression. Current work is focused on understanding the role for higher-order chromatin organization during differentiation of embryonic stem cells into neurons, during reprogramming of neurons into induced pluripotent stem cells and in models of neurodegenerative disease.

Arjun Raj, Ph.D.

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.

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