Posts classified under: Program Members

Penn Epigenetics > Program Members

Roberto Bonasio, Ph.D.

Contributions to Science

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

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

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

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

*Equal contributions
†Co-corresponding authors

Research Interest

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

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

Contribution to Science

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

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

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

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

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

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

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

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

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

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

Research Interest

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

Elizabeth Heller, Ph.D.

Contribution to Science

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

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

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

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

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

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

Research Interest

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

Eric-Joyce

Eric F. Joyce, Ph.D.

Contribution to Science

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

Research Interest

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

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

Contribution to Science

•Discovery of thyroid hormone receptors and their mechanism of repression.

• Discovery of Nuclear Receptor Corepressor Complexes.

• Elucidation of physiological Roles of Nuclear Receptor Corepressors and HDAC3.

• Discovery of REV-ERB and mechanisms of circadian regulation of transcription and metabolism.

• Identification and characterization of PPAR g in Adipose Biology.

Research Interest

The goal of the Lazar lab is to understand the transcriptional regulation of circadian rhythms and metabolism both in normal physiology and in  metabolic diseases such as diabetes and obesity. The focus is on nuclear receptors and HDAC3-containing corepressor complexes, whose functions are interrogated using a combination of genomic, proteomic, bioinformatic, and metabolic phenotyping methods.  Of particular interest are the circadian REV-ERB nuclear receptors, which are transcriptional repressors that function in the circadian clock and coordinate biological rhythms of metabolism in liver, adipose, and other tissues. Another focus is on nuclear receptor PPARg, a key transcriptional link between obesity and diabetes which functions as the master regulator of adipocyte biology and whose ligands have potent antidiabetic activity. Nuclear receptors corepressors and HDAC3 are also of great interest as an integrators of the activities of nuclear receptors and other transcription factors, with tissue-specific functions that protect from challenges to the circadian, nutritional, and thermal environment.

Mia Levine, PhD

Mia Levine, Ph.D.

Contribution to Science

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.

Contribution to Science

1. 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- acetyltransferases including the non-histone lysine acetyltransferases (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.
a. 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
b. Deng, S., Pan, B., Gottlieb, L. and Marmorstein, R. Molecular basis for N-terminal alpha-synuclein acetylation by human NatB. (2020) eLife. 9: e57491. PMID32885784
Deng, S., Gottlieb, L., Pan, B., Supplee, J., Wei, Xuepeng, Petersson, E.J. and Marmorstein, R., Molecular mechanism of N-terminal acetylation by the ternary NatC complex. (2021) Structure, S0969-2126. PMID34019809
Gottlieb, L., Guo, L., Shorter, J. and Marmorstein, R. N-alpha-acetylation of Huntingtin protein increases its propensity to aggregate. (2021) J. Biol. Chem., 31: 101363-. PMID34732320

2. 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 multi-subunit 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 dysregulation 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.
a. 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 Commun. 6:7711-. PMID: 26159857: PMCID: PMC4509171
b. 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
c. 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
d. 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

3. 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.
a. 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
b. 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
c. 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
d. 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

4. 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.
a. 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
b. 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
Wei, X. and Marmorstein, R., Reply to: Acetyl-CoA is produced by the citrate synthetase homology module of ATP-citrate lyase. (2021) Nat. Struct. Mol. Biol., 28: 639-641. PMID34294921
Wei, X., Kixmoeller, K., Baltrusaitis, E., Yang, X. and Marmorstein, R. Allosteric role of a structural NADP+ molecule in glucose-6-phosphate dehydrogenase activity, (2022) Proc. Nat. Acad. Sci. USA, 119(29): e2119695119

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.

Contributions to Science

The Cremins Lab focuses on higher-order genome folding and how chromatin works through long-range, spatial mechanisms to govern neural specification and synaptic plasticity in healthy and diseased neural circuits. We have developed molecular and computational technologies to create kilobase-resolution maps of chromatin folding and have built synthetic architectural proteins to engineer loops with light, together catalyzing new understanding of the genome’s structure-function relationship. We applied our technologies to discover that topologically associating domains (TADs), nested subTADs, and loops undergo marked reconfiguration during neural lineage commitment, somatic cell reprogramming, neuronal activity stimulation, and in models of repeat expansion disorders. We have demonstrated that loops induced by neural circuit activation, engineered through synthetic architectural proteins, and miswired in fragile X syndrome (FXS) are tightly connected to transcription, thus providing early insight into the genome’s structure-function relationship. Moreover, we have also demonstrated that cohesin-mediated loop extrusion can position the location of human replication origins which fire in early S phase, revealing a role for genome structure beyond gene expression in DNA replication. Recently, we have discovered that nearly all unstable short tandem repeat tracts in trinucleotide expansion disorders are localized to the boundaries between TADs, suggesting they are hotspots for pathological instability. We have identified that Mb-scale H3K9me3 domains decorating autosomes and the X chromosome in FXS are exquisitely sensitive to the length of the CGG STR tract. H3K9me3 domains spatially connect via inter-chromosomal interactions to silence synaptic genes and stabilize STRs prone to instability on autosomes. Together, our work uncovers a link between subMegabase-scale genome folding and genome function in the mammalian brain, thus providing the foundation upon which we will dissect the functional role for chromatin mechanisms in governing defects in synaptic plasticity and long-term memory in currently intractable and poorly understood neurological disorders.

Research Interest

The Cremins lab aims to understand how chromatin works through long-range physical folding mechanisms to encode neuronal specification and long-term synaptic plasticity in healthy and diseased neural circuits. We pursue a multi-disciplinary approach integrating data across biological scales in the brain, including molecular Chromosome-Conformation-Capture sequencing technologies, single-cell imaging, optogenetics, genome engineering, induced pluripotent stem cell differentiation to neurons/organoids, and in vitro and in vivo electrophysiological measurements.

Our long-term scientific goal is to dissect the fundamental mechanisms by which chromatin architecture causally governs genome function and, ultimately, long-term synaptic plasticity and neural circuit features in healthy mammalian brains as well as during the onset and progression of neurodegenerative and neurodevelopmental disease states

Our long-term mentorship goal is to develop a diverse cohort of next-generation scientific thinkers and leaders cross-trained in molecular and computational approaches. We seek to create a positive, high-energy environment with open and honest communication to empower individuals to discover and refine their purpose and grow into the best versions of themselves.

Arjun-Raj

Arjun Raj, Ph.D.

Contribution to Science

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.

Junwei Shi, Ph.D.

Contributions to Science

Contributions to Science

  1. Identifying Epigenetic Dependencies in Leukemia.
  2. CRISPR-based Genetic Screening Methods
  3. Identifying Transcriptional, Epi-transcriptional and Kinase Dependencies in Leukemia.
  4. Investigating Genetic Regulatory Pathways using CRISPR-based Screening Methods.

Research Interest

The physiological effects of cancer are a manifestation of the genetic abnormalities that cause the disease. While much progress has been made in the understanding of such genetic perturbations, scientists still struggle to effectively identify, understand, and treat cancer-causing mutations. This is due to the fast-paced evolution of the disease, and the accumulation of novel mutations that permit cell survival even in the harsh environment created by a therapeutic. CRISPR is a gene-editing technology that couples the elegance of base complementarity with the enzymatic activity of a DNA nuclease in order to introduce mutations into target loci. CRISPR technologies help advance our understanding of the genetic perturbations that contribute to cancer maintenance.

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