Posts classified under: University Of Pennsylvania

Penn Epigenetics > Program Members > University Of Pennsylvania

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.

Doris Wagner, Ph.D.

Contribution to Science

Topics of study
The role and Regulation of SWI/SNF chromatin remodeling complexes in plants.
ATP-dependent chromatin remodeling can change the chromatin state by using the energy derived from ATP hydrolysis to alter histone/DNA interactions. We uncovered key roles for plant SWI/SNF subfamily remodelers in stem cell maintenance and in overcoming Polycomb repression for induction of the floral homeotic genes during flower patterning. Finally, activity of one of the SWI/SNF remodelers, BRM, is modulated directly by signaling components of the stress hormone ABA for drought tolerance.
Kwon, C.S., Chen, C., and Wagner, D. (2005). WUSCHEL is a primary target for transcriptional regulation by SPLAYED in dynamic control of stem cell fate in Arabidopsis. Genes Development 19, 992-1003.
Bezhani, S., Winter, C., Hershman, S., Wagner, J.D., Kennedy, J.F., Kwon, C.S., Pfluger, J., Su, Y., and Wagner, D. (2007). Unique, Shared, and Redundant Roles for the Arabidopsis SWI/SNF Chromatin Remodeling ATPases BRAHMA and SPLAYED. Plant Cell 19, 403-416.
Han, S.K., Sang, Y., Rodrigues, A., BIOL425F2010, B., Rodriquez, P.L. and Wagner, D. (2012) The SWI2/SNF2 chromatin remodeling ATPase BRAHMA represses Abscisic Acid Responses in the Absence of the Stress Stimulus in Arabidopsis, Plant Cell 24 4892-4906.
Wu, M.F., Sang, Y., Bezhani, S., Yamaguchi, N., Han, S.K., Li, Z., Su, Y., Slewinski, T.L., and Wagner, D. (2012). SWI2/SNF2 chromatin remodeling ATPases overcome polycomb repression and control floral organ identity with the LEAFY and SEPALLATA3 transcription factors. Proceedings of the National Academy of Sciences of the United States of America 109, 3576-3581. http://www.eurekalert.org/pub_releases/2015-10/uop-ph101315.php
Peirats-Llobet, M., Han, S.K., Gonzalez-Guzman, M., Jeong, C.W., Rodriguez, L., Belda-Palazon, B., Wagner, D*., and Rodriguez, P.L*. (2016). A Direct Link between Abscisic Acid Sensing and the Chromatin-Remodeling ATPase BRAHMA via Core ABA Signaling Pathway Components. Molecular Plant 9, 136-147. * corresponding authors
The switch to flower formation, a major developmental switch critical for reproductive success.
Plants generate different types of lateral organs (leaves, then branches and finally flowers) post-embryonically from stem cell descendants at the shoot apex. When flowers form is critical for plant reproductive success. Our research has established that the plant specific helix-turn-helix transcription factor LEAFY is a key regulator of the onset of flower formation. We showed that the regulatory network downstream of LFY is comprised of a set of interlocking feed-forward loops that together control the timing of the upregulation of the direct LFY target APETALA1, a commitment factor of floral fate. We further found that LFY directly alters the hormone environment in newly formed primordia to promote floral fate.
Yamaguchi, A., Wu, M.F., Yang, L., Wu, G., Poethig, R.S., and Wagner, D. (2009). The microRNA-regulated SBP-Box transcription factor SPL3 is a direct upstream activator of LEAFY, FRUITFULL, and APETALA1. Developmental Cell 17, 268-278. (faculty 1000 recommended)
Winter, C.M., Austin, R.S., Blanvillain-Baufume, S., Reback, M.A., Monniaux, M., Wu, M.F., Sang, Y., Yamaguchi, A., Yamaguchi, N., Parker, J.E., J.E., Parcy, F., Jensen, S.T., Li, H., Wagner, D. (2011). LEAFY Target Genes Reveal Floral Regulatory Logic, cis Motifs, and a Link to Biotic Stimulus Response. Developmental Cell 20, 430-443.
Yamaguchi, N., Winter, C., Wu, M-F., Kanno, Y., Yamaguchi, A., Seo, M., and Wagner, D. (2014) Gibberellin acts positively then negatively to control onset of flower formation in Arabidopsis. Science 344, 638-41. https://stke.sciencemag.org/content/7/325/ec127); http://www.eurekalert.org/pub_releases/2014-05/uop-phh050814.php
Zhu, Y., Klasfeld, S., Jeong, C.W., Jin, R., Goto, K., Yamaguchi, N., and Wagner, D. TERMINAL FLOWER 1-FD complex target genes and competition with FLOWERING LOCUS T. Nature Communications, 11, 5118. https://www.eurekalert.org/pub_releases/2020-10/uop-dpg100920.php
Jin, R., Klasfeld, S., Garcia, M.F., Xiao, J., Han, S.K., Konkol, A., Zhu, Y., and Wagner, D.. LEAFY is a pioneer transcription factor and licenses cell reprogramming to floral fate. 2021  Nature Communications, 12(1):626. https://www.eurekalert.org/pub_releases/2021-01/uop-fe012721.php
Organogenesis and differentiation in response to hormonal cues
We have recently become interested in the question how hormone responses control organogenesis and differentiation. We have identified key targets of a master transcriptional regulator of the auxin hormone response, AUXIN RESPONSE FACTOR5/MONOPTEROS during organogenesis (flower primordium initiation) and uncovered an auxin hormone triggered chromatin state switch. In addition, we have elucidated how BRM together with leaf maturation transcription factors triggers leaf differentiation by lowering response to the hormone cytokinin.
Yamaguchi, N., Wu, M.-F., Winter, C., Berns, M., Nole-Wilson, S., Yamaguchi, A., Coupland, G., Krizek, B., and Wagner, D. (2013) Auxin-mediated Initiation of the Flower Primordium. Developmental Cell 24, 1–12. (faculty 1000 recommended)
Efroni, I., Han, S.K., Kim, H.Y., Wu, M.F., Sang, Y., Hong, J.C., Eshed, Y*., and Wagner, D*. (2013). Regulation of leaf maturation by chromatin-mediated modulation of hormonal responses. Developmental Cell. 24, 438-445. *corresponding authors
Wu, M-F, Yamaguchi, N., Xiao J., Bargmann, B. Estelle, M., Sang, Y. and Wagner, D. (2015) Auxin-regulated chromatin switch directs acquisition of flower primordium founder fate. eLife;4:e09269 http://www.eurekalert.org/pub_releases/2015-10/uop-ph101315.php
Chung, Y., Zhu, Y., Wu,M.-F., Simonini, S., Armenta-Medina, A.,  Jin, R.,  Østergaard, L.,  Gillmor, C.S., and Wagner, D. (2019) Auxin Response factors promote organogenesis by chromatin-mediated repression of the pluripotency gene SHOOTMERISTEMLESS. Nature Communications 10, 886.
Polycomb silencing and recruitment in plants
Cell identity depends on silencing of unnecessary or detrimental gene expression programs. To understand the regulation of epigenetic gene silencing we have elucidated how Polycomb Repressive Complexes are targeted to developmental genes in plants. We have uncovered a genome-encoded recruitment mechanism very similar to that previously described in the fruitfly. Our studies pave the way to future epigenetic manipulation of desirable plant traits.
Bossi, F., Fan, J., Xiao, J., Chandra, L., Shen, M., Dorone, Y., Wagner, D., and Rhee, S.Y. (2017). Systematic discovery of novel eukaryotic transcriptional regulators using sequence homology independent prediction. BMC Genomics 18, 480.
Xiao, J., Jin, R., Yu, X., Shen, M., Wagner, J.D., Pai, A., Song, C., Zhuang, M., Klasfeld, S., He, C., Santos, A. M., Helliwell, C., Pruneda-Paz, J. L., Kay, S. A., Lin, X., Cui, S., Garcia, M. F., Clarenz, O., Goodrich, J., Zhang, X., Austin, R. S., Bonasio, R., Wagner, D. (2017). Cis and trans determinants of epigenetic silencing by Polycomb repressive complex 2 in Arabidopsis. Nat Genet. 49 (10) 1546-1552  preview in NG. https://www.eurekalert.org/pub_releases/2017-08/uop-pbs082117.php
Sun, B., Zhou, Y., Cai, J., Shang, E., Yamaguchi, N., Xiao, J., Looi, L.S., Wee, W.Y., Gao, X., Wagner, D. and Ito, T. (2019). Integration of transcriptional repression and Polycomb-mediated silencing of WUSCHEL in floral meristems. Plant Cell.
Bieluszewski, T., Xiao, J., Yang, Y., and Wagner, D. (2021). PRC2 activity, recruitment, and silencing: a comparative perspective. Trends Plant Sci 26, 1186-1198.
Lee, U.S., Bieluszewski, T., Xiao, J., Yamaguchi, A., and Wagner, D. (2022). H2A.Z contributes to trithorax activity at the AGAMOUS locus. Mol Plant 15, 207-210.

Research Interest

(Re)programming Cell Identity and Function in the Context of Chromatin

Our research focuses on the reprogramming of cell identity and function during developmental transitions and in response to environmental inputs in plants. Reprogramming offers a window of opportunity to unravel the regulatory logic that underlies cell fate and function as existing programs are shut down and new one’s are put in place. Plants are an excellent experimental system to address this question as they tailor their final form and cell function to a changing environment to optimize growth and survival. We have shown that master transcriptional regulators, hormone response and chromatin state together orchestrate cell fate reprogramming in plants.

Hao-Wu

Hao Wu, Ph.D.

Contribution to Science

Promoter DNA methylation is generally linked to transcriptional repression. However, euchromatic DNA methylation frequently occurs in regions outside promoters, such as gene bodies or regions upstream of promoters. The function of such non-promoter DNA methylation was largely unclear. Combining mouse genetic models and in vitro neural stem cell (NSC) system with biochemical, epigenomic and bioinformatic analyses, my PhD research revealed a novel function of de novo DNA methyltransferase DNMT3A-mediated non-promoter DNA methylation in facilitating transcription of neurogenic genes in postnatal NSCs. In contrast to the conventional view that DNA methylation is only linked to gene silencing, this study shows that DNA methylation at non-promoter regions may promote transcription by functionally antagonizing Polycomb repression complex 2 (PRC2).

Wu H, Tao J, Sun YE. Regulation and function of mammalian DNA methylation patterns: a genomic perspective. Brief Funct Genomics. 2012 May;11(3):240-50.
Wu H, Coskun V, Tao J, Xie W, Ge W, Yoshikawa K, Li E, Zhang Y, Sun YE. Dnmt3a-dependent nonpromoter DNA methylation facilitates transcription of neurogenic genes. Science. 2010 Jul 23;329(5990):444-8.
Fan G, Martinowich K, Chin MH, He F, Fouse SD, Hutnick L, Hattori D, Ge W, Shen Y, Wu H, ten Hoeve J, Shuai K, Sun YE. DNA methylation controls the timing of astrogliogenesis through regulation of JAK-STAT signaling. Development. 2005 Aug;132(15):3345-56.

TET proteins are Fe2+ and 2-oxoglutarate-dependent dioxygenases capable of successively oxidizing 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Highly oxidized cytosine bases (i.e. 5fC and 5caC) are selectively recognized and excised by Thymine DNA glycosylase (TDG), and the resulting abasic site is restored to unmodified C through the base excision repair (BER) pathway. Thus, methylation, oxidation, and excision repair offer a biochemically-validated model of mammalian active DNA demethylation pathway. However, little was known about the genomic distribution and gene regulatory functions of TET enzymes. As a postdoctoral fellow in the laboratory of Dr. Yi Zhang, I determined where Tet1 proteins are located across the genome of mouse ESCs. This work was amongst the first to reveal genomic distribution of TET enzymes in the mammalian genome. I found that TET1 is preferentially enriched at CpG-rich sequences at promoters of both transcriptionally active genes and PRC2-repressed lineage-specific genes. Epigenomic and transcriptomic analyses of Tet1-depleted cells reveal that TET1 plays roles in both transcriptional activation and repression, and TET1 contributes to repression of poised developmental regulators in ESCs by maintaining DNA hypomethylation states to facilitate PRC2 binding.

Wu H, Zhang Y. Reversing DNA methylation: mechanisms, genomics, and biological functions. Cell. 2014 Jan 16;156(1-2):45-68.
Wu H, Zhang Y. Mechanisms and functions of Tet protein-mediated 5-methylcytosine oxidation. Genes Dev. 2011 Dec 1;25(23):2436-52.
Wu H, Zhang Y. Tet1 and 5-hydroxymethylation: a genome-wide view in mouse embryonic stem cells. Cell Cycle. 2011 Aug 1;10(15):2428-36.
Wu H, D’Alessio AC, Ito S, Xia K, Wang Z, Cui K, Zhao K, Sun YE, Zhang Y. Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells. Nature. 2011 May 19;473(7347):389-93.

A complete understanding of the function of TET enzymes requires new methods to determine the genome-wide distribution of oxidized 5mC bases (5hmC/5fC/5caC). I have developed affinity-enrichment-based (5hmC/5fC/5caC DIP-seq) genome-wide mapping methods and systematically charted the genomic architecture and dynamics of these new DNA modifications. 5hmC is preferentially enriched at transcriptionally inactive/poised promoters as well as gene bodies of actively transcribed genes. In addition, 5hmC is frequently localized near distally located enhancers and CTCF binding sites. Genome-wide mapping of 5fC and 5caC indicates that these highly oxidized bases also accumulate at distal active enhancers and PRC2-repressed developmental gene promoters when TDG is depleted, suggesting that TET/TDG-dependent active DNA demethylation occurs dynamically at both proximal and distal gene regulatory regions. To enable quantitative and high-resolution mapping of TET/TDG-dependent active DNA demethylation, I have recently developed a single-base resolution mapping method, termed Methylase-Assisted BS-seq (MAB-seq), to precisely locate and quantify 5fC and 5caC bases.

Wu H, Zhang Y. Charting oxidized methylcytosines at base resolution. Nat Struct Mol Biol. 2015 Sep;22(9):656-61.
Wu H*, Wu X*, Shen L, Zhang Y. Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing. Nat Biotechnol. 2014 Dec;32(12):1231-40.
Shen L*, Wu H*, Diep D, Yamaguchi S, D’Alessio AC, Fung HL, Zhang K, Zhang Y. Genome-wide analysis reveals TET- and TDG-dependent 5-methylcytosine oxidation dynamics. Cell. 2013 Apr 25;153(3):692-706.
Wu H, D’Alessio AC, Ito S, Wang Z, Cui K, Zhao K, Sun YE, Zhang Y. Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells. Genes Dev. 2011 Apr 1;25(7):679-84.

Research Interest

DNA cytosine methylation (5-methylcytosine) is an evolutionarily conserved epigenetic mark and has a profound impact on transcription, development and genome stability. Historically, 5-methylcytosine (5mC) is considered as a highly stable chemical modification that is mainly required for long-term epigenetic memory. The recent discovery that ten-eleven translocation (TET) proteins can iteratively oxidize 5mC in the mammalian genome represents a paradigm shift in our understanding of how 5mC may be enzymatically reversed. It also raises the possibility that three oxidized 5mC bases generated by TET may act as a new class of epigenetic modifications.
Our laboratory uses high-throughput sequencing technologies, bioinformatics, mammalian genetic models, as well as synthetic biology tools to investigate the mechanisms by which proteins that write, read and erase oxidized 5mC bases contribute to mammalian development (particularly cardiovascular and neural lineages) and relevant human diseases. To achieve this goal, we are also interested in developing new genomic sequencing and programmable epigenome-modifying methods to precisely map and manipulate these DNA modifications in the complex mammalian genome.

Shelley L. Berger, Ph.D.

Contributions to Science

1.  Identification of transcriptional adaptors/coactivators Gcn5/Ada2/Ada3 and discovery of novel histone modifications and mechanisms in transcription and sperm genome opening

We discovered transcriptional “adaptors”, which we showed associate with DNA binding activators, a groundbreaking new model for transcriptional activation, to reveal how histone enzymatic modifiers are recruited to genes (Berger+, Cell1990, Cell1993). We revealed the importance of adaptor Gcn5 acetyltransferase activity in transcriptional activation (1998), unifying transcription and chromatin regulation. We discovered numerous novel histone modifications (PTMs), PTM cross-talk, and sequential histone PTMs in transcription, including histone phosphorylation/acetylation (2001) and ubiquitylation/deubiquitylation. We discovered (2017) that enhancer RNAs bind directly to CBP, the key metazoan acetyltransferase, to stimulate HAT activity in vitro and at enhancers in vivo.  We showed (2019) that Gcn5 provides key histone acetylation to broadly open the mouse genome during spermatogenesis for extensive chromatin restructuring.

a.  Wang L, Liu L. and Berger SL. (1998) Critical residues for histone acetylation by GCN5, functioning in Ada and SAGA complexes, are also required for transcriptional function in vivo. Genes & Development 12: 640-653. PMCID: PMC316586

b.  Lo W-S…Shiekhattar R, and Berger SL.  (2001) Snf1 is a histone kinase which works in concert with the histone acetyltransferase Gcn5 to regulate transcription. Science 293:1142-6.  PMID:111498592

c.  Bose DA, Donahue G, Reinberg D, Shiekhattar R, Bonasio R, Berger SL. (2017)  RNA binding to CBP stimulates histone acetylation and transcription. Cell 168,135-149. PMCID: PMC5325706.

d.  Luense LJ, Donahue G, Lin-Shiao E….Bartolomei M, Berger SL. (2019) Gcn5-mediated histone acetylation governs nucleosome dynamics in spermiogenesis. Developmental Cell 51:745-758.

2. Discovery of chromatin mechanisms controlling aging and senescence

We uncovered chromatin changes involved in aging and cellular senescence, indicating broad epigenome dysregulation. These include pioneering studies that histone acetylation drives aging in yeast (2009), disrupts the nuclear laminar chromatin in mammals, and are crucial to enhancer function in aging (2019). We showed these disruptions trigger both homeostatic genomic protection and cellular damage, and discovered nuclear autophagy pathways in senescence leading to inflammation in aging and cancer (2015,2017,2020). Our findings suggest potential epigenetic therapeutics to ameliorate age-associated disease.

a.  Dang W…Kaeberlein M, Kennedy BK, and Berger SL. (2009) Histone H4 lysine-16 acetylation regulates cellular lifespan. Nature 459:802-7. PMCID: PMC2702157.

b.  Dou Z…Adams PD^, and Berger SL^. (2015) Autophagy mediates degradation of nuclear lamina. Nature 527:105-9. PMCID: PMC4824414.   (2017) Cytoplasmic chromatin triggers inflammation in senescence and cancer. Nature 550:402-406.  PMID:28976970.

c.  Sen P, Lan Y…Adams PD, Schultz DC, Berger SL. (2019) Histone acetyltransferase p300 induces de novo super-enhancers to drive cellular senescence. Molecular Cell 73:684-698. PMID:30773298.

d.  Xu C, Wang L…Adams PA, Ott M, Tong W, Johansen T, Dou Z^, and Berger SL^.  (2020)  SIRT1 is downregulated by autophagy in senescence and aging.  Nature Cell Biology 22:1170-1179.

3. Demonstration of chromatin mechanisms controlling memory and behavior and relevant to aging. 

Our studies in mouse brain and memory show a pivotal role of the metabolic enzyme, ACSS2, in fueling “on-site” acetyl-CoA generation on chromatin for neuronal histone acetylation and gene expression in normal memory and in alcohol-fueled addiction memory (2017/19). Our work in human Alzheimer’s disease reveals that the cognitively normal aging brain is epigenetically protected compared to the AD brain (2018/20). In other research on brain, we pioneered investigation of eusocial ant caste-specific behavior for organismal-level chromatin regulation and epigenetics, owing to the remarkable fact that female ants of distinct social castes (such as queen, soldier, and forager) share an identical genome. We sequenced the first ant genomes and then profiled the first histone modification epigenomes (2010,Science) and pioneered Crispr genetics in ants (2017,Cell). Groundbreaking results indicate a critical role of histone modifications in altering ant brain function to instruct complex social behavior; we identified a “window”, early after hatching, to behavioral reprogramming via epigenetic manipulation (2016/2020).  We linked regulation of caste behavior to remarkable aging disparity.

a.  Simola DF…Reinberg D^, Liebig J^, Berger SL^.  (2016)  Epigenetic (re)programming of caste-specific behavior in the ant C. floridanus. Science 351:aac6633. PMID: 26722000, PMCID: PMC5057185.

b.  Mews, P… Berger SL. (2017) Acetyl-CoA metabolism by ACSS2 regulates neuronal histone acetylation and hippocampal memory. Nature 546,381-386. PMCID: PMC5505514.  Mews P, Egervari G^…Garcia, B, Berger SL^. (2019) Alcohol metabolism contributes to brain histone acetylation. Nature 574: 717-721.

c.  Glastad K, Graham RJ, Ju L, Rossler J, Brady CM, and Berger SL (2020) Epigenetic regulator CoRest controls social behavior in ants.  Molecular Cell 77:338-351.

d.  Nativio R, Donahue G…Johnson FB^, Bonini NM^, Berger SL^ (2018) Dysregulation of the epigenetic landscape of normal aging in Alzheimer’s disease. Nature Neuroscience 21,497-505.  Nativio R, Lan Y…Garcia BA, Trojanowski JQ, Bonini NM^, Berger SL^.  (2020) An integrated multi-omics approach identifies epigenetic drivers associated with Alzheimer’s disease.  Nature Genetics 52:1024-1035.

  1. Discovery of tumor suppressor p53 factor and histone modifications and their mechanisms including activating p53 acetylation, repressive p53 methylation, and novel chromatin pathways in p53-mediated transcriptional activation

Our work revealed new enzyme modifiers and post-translational modifications of p53 (including acetylation, methylation, and demethylation, 2006/7) regulating p53 activity. Our findings propelled broad efforts in the field to discover novel acetylation and methylation of transcription factors. We showed p53 methylation is generally repressive to its function, and showed repressive p53 methylation occurring in certain cancers bearing high levels of wild type p53. We discovered novel epigenetic pathways used by wild type and mutant p53 in regulating chromatin structure/function in normal and cancer cells, such as gain-of-function p53 mutants driving transcriptional activating and growth promoting histone modifications (2015).  We showed that p53 and p63 establish new enhancers during stress and development. We found a novel role of p53 in promoting target gene association with nuclear speckles for transcriptional amplification (2021).

  1. a. Huang J…Jenuwein T, andBerger SL. (2006) Repression of p53 activity by Smyd2-mediated methylation.  Nature 444:629-32. PMID:17108971.  Huang J…Jenuwein T, and Berger SL.  (2007) p53 is regulated by the lysine demethylase LSD1.  Nature, 449:105-8.
  2. Bungard D…Thompson CB, Jones RG andBerger SL. (2010) Signaling kinase AMPK activates stress-promoted transcription via histone H2B phosphorylation. Science 329: 1201-5. PMCID: PMC3922052.
  3. Zhu J, Sammons MA, Donahue G, Dou Z…Arrowsmith CH, andBerger SL. (2015) Gain-of-function p53 mutants co-opt epigenetic pathways to drive cancer growth. Nature 525:206-11. PMCID: PMC4568559
  4. Alexander KA…Belmont A, Joyce EF, Raj A, and Berger SL. (2021) p53 mediates target gene association with nuclear speckles for amplified RNA expression. Molecular Cell 81:1666-1681.
  1. Investigation of epigenetic mechanisms in T and CART cell exhaustion and cancer immunotherapy

We established collaborations with Carl June (pioneer of CAR T cell therapy in cancer) and John Wherry (discovered key aspects of T cell exhaustion).  We investigate epigenetic regulation in patient response to immunotherapy, and controlling T cell exhaustion in mouse models.

  1. Pauken KE…Berger SL, and Wherry EJ.  (2016) Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade.Science 354,1160-1165.  Khan O…Berger SL, and Wherry EJ.  (2019) TOX transcriptionally and epigenetically programs CD8+ T cell exhaustion.  Nature 571, 211.
  2. Fraietta JA…Berger SL, Bushman FD, June CH, and Melenhorst JJ. (2018) Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T-cells.Nature 555, 307-312.
  3. Chen Z…Berger SL, Wherry EJ, and Shi J.  In vivo CRISPR screening identifies Fli1 as a transcriptional safeguard that restrains effector CD8 T cell differentiation during infection and cancer.  Cell 184:1262.
  4. Good CR+, Kuramitsu S+, Aznar MA+…Young RM^,Berger SL^, June CH^ (2021) In vitro dysfunction model reveals the plasticity of patient CAR-T cells and identifies transcription factors whose modulation can restrain CAR-T cell exhaustion. Cell, 184:6081-6100.

Research Interest

Our lab focuses on mechanisms that regulate gene expression with a special emphasis on how the DNA-packaging structure of chromatin is manipulated during genomic processes. Our findings inform the study of cancer and other diseases, and ultimately drug discovery.

Ken Zaret

Kenneth S. Zaret, Ph.D.

Research Interest

Ken’s laboratory discovered “pioneer factors” that bind to silent chromatin, endow the competence for cell differentiation, and promote cellular reprogramming. Recently, his lab found broad chromatin domains that can resist pioneer factor binding and serve as impediments to cellular reprogramming; these domains appear to help commit cells to particular fates. Finally, his lab has unveiled how inductive signaling in the embryo leads to chromatin modifications that affect cell fate choices, thereby identifying specific enzymatic targets for small molecules to modulate cell fate control.

Marisa Bartolomei, Ph.D.

Contributions to Science

The work in my laboratory focuses on elucidating the mechanisms governing genomic imprinting in mammals. Imprinted genes number in the hundreds, are largely located in domains and are expressed from a single parental allele. This monoallelic gene expression pattern is set in the gametes and maintained during development using epigenetic mechanisms such as DNA methylation and posttranslational histone modifications. Genomic imprinting is an excellent model for studying epigenetic gene regulation during mammalian development. We have used mouse models with mutations in cis-acting regulatory sequences and trans-acting epigenetic factors to study imprinted gene regulation, including examining tissue-specific effects and higher order chromatin structure and architecture. Historically we conducted in depth analyses of the H19–Igf2 imprinted locus but have more recently expanded to Grb10–Ddc1 locus to reveal cis-acting regulatory elements. For elucidating establishment and maintenance of imprinted gene expression we have studied most of the imprinted loci and incorporated genome-wide approaches and mutations in the DNA methylation machinery. Specifically, we have studied the role of oxidase TET1 in reprogramming of iPSCs and genomic imprints, more recently expanding to study reprogramming of the male germline using a series of Tet1 mutant mice. We have also studied X inactivation in many of these model systems.

Additionally, we use mouse models to study the epigenetic consequences of environmental perturbations such as in utero exposure to endocrine disrupting compounds (EDCs) and Assisted Reproductive Technologies (ART). With respect to EDCs, we have used a mouse model to show that BPA exerts an abnormal metabolic, skeletal health and behavior phenotypes, largely observed in males. In these models we have focused on placenta as well as fetal and postnatal phenotypes. For the ART mouse model, we have studied the long-term outcomes of procedures used in assisted reproduction and have observed sex-specific metabolic and cardiovascular phenotypes and behavioral perturbations as mice age. Moreover, we have shown that embryo culture in the most significant procedure with respect to conferring abnormal DNA methylation profiles in ART-conceived offspring. Finally, we have employed high throughput technologies to study DNA methylation, transcription, chromatin structure and proteomics in a variety of cell types.

Research Interest

The research in the Bartolomei laboratory focuses epigenetic control of genomic imprinting. They also study how the environment can perturb genomic imprinting and other epigenetic processes important in reproduction and health.

Zhaolan (Joe) Zhou, Ph.D.

Contribution to Science

1. Rett syndrome is a debilitating neurodevelopmental disorder caused by mutations in the gene encoding methyl-CpG binding protein 2 (MeCP2). One characteristic feature of Rett syndrome is the regression of learned motor and language skills after 6-18 months of normal development. The onset of the disease coincides with synaptic maturation driven by sensory experience in humans. As a postdoctoral fellow, I started to pursue the role of MeCP2 in neuronal activity-dependent gene regulation and investigated the possibility that defective experience-dependent synaptic maturation underlies the pathogenesis of Rett syndrome. I found that MeCP2 is selectively phosphorylated in the brain in a neuronal activity-dependent manner and this event mediates dendritic morphogenesis and spine maturation. Sponsored by an NIH Pathway to Independence Award (K99/R00), I went on and led a research team finding that MeCP2 regulates gene expression programs and neuronal development in a brain region, neuronal cell type, and age-specific manner. My lab also found MeCP2 plays a necessary and sufficient role in GABAergic interneurons to maintain neural network integrity, providing the cellular origin driving auditory event related potentials (ERPs) and paving the way to investigate Rett syndrome pathogenesis in defined cell types of interest.

a. Zhou Z, Hong E, Cohen S, Zhao W, Ho SY, Chen W, Savner E, Hu L, Steen J, Weitz C and Greenberg ME* (2006). Brain-specific phosphorylation of MeCP2 regulates activity-dependent Bdnf transcription, dendritic growth, and spine maturation. Neuron, 52: 255–269. PMCID: PMC3962021.

b. Wang IT, Reyes AR, and Zhou Z* (2013). Neuronal morphology in MeCP2 mouse models is intrinsically variable and depends on age, cell type, and Mecp2 mutation. Neurobiology of Disease, 58C: 3-12. PMCID: PMC3748238.

c. Zhao YT, Goffin D, Johnson BS and Zhou Z* (2013). Loss of MeCP2 function is associated with distinct gene expression changes in the striatum. Neurobiology of Disease, 59C: 257-266. PMCID: PMC3790640.

d. Goffin D, Brodkin ES, Blendy JA, Siegel SJ and Zhou Z* (2014). Cellular origins of auditory event-related potential deficits in Rett syndrome. Nature Neuroscience, 17(6): 804-806. PMCID: PMC4038660.

2. The field of Rett syndrome research has been greatly benefited and advanced through studies of knockout, conditional knockout and conditional rescue mouse models of MeCP2. However, nearly one-third of Rett syndrome mutations are missense in the methyl-CpG binding domain (MBD) of MeCP2. To specifically address the contribution of these missense mutations to the pathogenesis of Rett Syndrome and provide the research community with clinically relevant mouse models, I led a research team and developed the first allelic series of knockin models that faithfully recapitulate patient mutations, MeCP2 T158A, T158M and R106W. We found that those missense mutations impair the binding of MeCP2 to methylated DNA in vivo, concomitantly reduce MeCP2 protein stability, and lead to behavioral phenotypes resembling Rett syndrome. Notably, we uncovered that stabilizing MeCP2 T158M protein or elevating MeCP2 T158M expression rescues Rett-like phenotypes in vivo, providing a novel therapeutic strategy to treat Rett syndrome. Though studies of these mouse models, we also discovered that event-related neuronal responses are stalled in maturation by the loss-of-function of Mecp2, and auditory ERPs represent robust, sensitive, and quantitative biomarkers informing phenotypic progression, at least in mouse models. Furthermore, we demonstrated both cell and non-cell autonomous effects of MeCP2 on gene expression and the existence of post-transcriptional compensation to MeCP2 loss. These findings allowed us to postulate that MeCP2 functions as an architectural protein organizing chromatin in 3-dimentional fashion to facilitate precise gene transcription.

a. Goffin D, Allen M, Amorim M, Zhang L, Wang I-TJ, Reyes A-RS, Mercado-Berton A, Ong C, Cohen S, Hu L, Blendy JA, Carlson G,Siegel S, Greenberg ME and Zhou Z* (2012). Rett Syndrome mutation MeCP2 T158A mutation disrupts DNA binding, protein stability and ERP responses. Nature Neuroscience, 15: 274-283. PMCID: PMC3267879.

b. Lamonica JM, Kwon DY, Goffin D, Fenik P, Johnson BS, Cui Y, Guo H, Veasey S and Zhou Z* (2017). Elevating expression of MeCP2 T158M rescues DNA binding and Rett syndrome-like phenotypes. Journal of Clinical Investigation, 127 (5): 1889-1904. PMCID: PMC5409785.

c. Johnson BS, Zhao Y, Fasolino M, Lamonica JM, Kim YJ, Georgakilas G, Wood KH, Bu D, Cui Y, Goffin D, Vahedi G, Kim TH and Zhou Z* (2017). Biotin tagging of MeCP2 reveals contextual insights into the Rett syndrome transcriptome. Nature Medicine, 23(10): 1203-1214. PMCID: PMC5630512.

d. Connolly DR, Zhou Z* (2019). Genomic insights into MeCP2 function: A role for the maintenance of chromatin architecture. Current Opinion in Neurobiology, 59:174-179. PMCID: PMC6889049.

3. While I was in the process of studying MeCP2 phosphorylation and trying to identify its up-stream kinase, several human genetic studies linked mutations in the X-linked gene encoding cyclin-dependent kinase-like 5 (CDKL5) to atypical Rett syndrome, a variant with early-onset epileptic features. While several in vitro biochemistry and cell culture studies began to support an interaction between CDKL5 and MeCP2, the experimental evidence remained contentious. I decided to take a genetic approach to investigate CDKL5 function in vivo, and therefore, led a research team to the development and characterization of the first knockout mouse model of CDKL5 deficiency disorder (CDD). We found that CDKL5 dysfunction disrupts many key signal transduction pathways and long-range neural circuit communication, leading to autistic-like phenotypes in mice. We have also carried out conditional knockout studies to dissect the cellular origin and uncovered a crucial role for CDKL5 in glutamatergic neurons for learning and memory, in GABAergic neurons for social interaction and repetitive behaviors. Through temporal manipulation of CDKL5 expression in vivo, we uncovered that CDKL5 is essential for post-developmental brain function, and importantly, CDD-related behavioral and synaptic phenotypes are reversible after the early stage of brain development. We have now developed and contributed multiple mouse models of CDD to the community, demonstrated the potential for symptomatic reversal, at least in mouse models, and paved the way for mechanistic and therapeutic studies of CDD.

a. Wang IT, Allen M, Goffin D, Zhu X, Fairless AH, Brodkin ES, Siegel SJ, Marsh ED, Blendy JA, and Zhou Z* (2012). Loss of CDKL5 disrupts kinome profile and ERP response leading to autistic-like phenotypes in mice. Proc Natl Acad Sci USA.109: 21516-21521. PMCID: PMC3535652.

b. Tang S, Wang I-T, Yue C, Takano H, Terzic B, Pance K, Lee JY, Cui Y, Coulter DA* and Zhou Z* (2017). Loss of CDKL5 in glutamatergic neurons disrupts hippocampal microcircuitry and leads to memory impairment in mice. Journal of Neuroscience, 37(31): 7420-7437. PMCID: PMC5546111.

c. Terzic B, Cui Y, Edmondson AC, Tang S, Sarmiento N, Zaitseva D, Marsh ED, Coulter DA and Zhou Z* (2021): X-linked cellular mosaicism underlies age-dependent occurrence of seizure-like events in mouse models of CDKL5 deficiency disorder. Neurobiology of Disease, 148: 105176. doi: 10.1016/j.nbd.2020. 105176. PMCID: PMC7856307.

d. Terzic B, Davatolhagh MF, Ho Y, Tang S, Liu Y-T, Xia Z, Cui Y, Fuccillo MV and Zhou Z* (2021): Temporal manipulation of Cdkl5 reveals essential post-developmental functions and reversible CDKL5 deficiency disorder-related deficits. Journal of Clinical Investigation, 131(20): e143655, 2021. PMCID: PMC8516470.

4. The genetic underpinnings of mental health disorders are highly complex, involving multifaceted interactions between risk genes and the environment. It is known that environmental factors such as adverse early life events confer significantly greater susceptibility to psychiatric conditions later in life. But, the epigenetic mechanisms by which environmental factors interact with genetic programs in the nervous system remain poorly understood. Sponsored by the Biobehavioral Research Award for Innovative Scientist (BRAINS) from NIMH, I led a research team conceived a novel Cre-dependent biotinylation strategy and developed a series of genetically modified mice that allow for genome wide profiling of DNA methylation, histone modifications and RNA expression from cell types of interest while overcoming extensive cellular heterogeneity in the brain. We found that long genes genetically implicated in autism harbor broad enhancer-like chromatin domains (BELD) and causally link chromatin genes to autism etiology. My research team have also developed a computational pipeline to identify integrated epigenetic code in cell types of interest and in response to environmental stimuli. To investigate the causal role of stress-induced epigenetic changes to behavioral maladaptation, my research team have also adopted a modified CRISPR/Cas9 strategy to alter DNA methylation and histone acetylation at loci of interest. Notably, we recently found that chronic stress impacts higher-order chromatin architecture and identified YY1 as a regulator of stress-induced maladaptive behavior, thus providing a molecular target that mediates gene-environment interactions in the context of stress-related neuropsychiatric conditions.

a. Wood KH, Johnson BS, Welsh SA, Lee JY, Cui Y, Krizman E, Brodkin ES, Blendy JA, Robinson MB, Bartolomei MS and Zhou Z* (2016): Tagging of Methyl-CpG-binding Domain Proteins Reveals Different Spatiotemporal Expression and Supports Distinct Functions. Epigenomics, 4: 455-473. PMCID: PMC4880565.

b. Kwon DY, Zhao YT, Lamonica JM and Zhou Z* (2017). Locus-specific histone deacetylation using a synthetic CRISPR-Cas9-based HDAC. Nature Communications, 8:15315. Doi:10.1038/ncomms15315. PMCID: PMC5437308.

c. Zhao YT, Kwon DY, Johnson BS, Fasolino M, Lamonica JM, Kim YJ, Zhao BS, He C, Vahedi G, Kim TH and Zhou Z* (2018). Long genes linked to autism harbor broad enhancer-like chromatin domains. Genome Research, 28:933-942. PMCID: PMC6028126.

d. Kwon DY, Xu B, Hu P, Zhao Y-T, Beagan JA, Nofziger JH, Cui Y, Phillips-Cremins JE, Blendy JA, Wu H, Zhou Z* (2022): Neuronal YY1 in the prefrontal cortex regulates transcriptional and behavioral responses to chronic stress. Nature Communications, 13: 55. doi.org/10.1038/s41467-021-27571-3. PMCID: PMC8748737.

Research Interest

The Zhou laboratory is interested in understanding the epigenetic mechanisms that integrate environmental factors with the genetic code to govern brain development and function, elucidating the pathophysiology of specific neurodevelopmental disorders with known genetic causes such as Rett syndrome and CDKL5 deficiency, and illuminating the pathogenesis of selective neuropsychiatric disorders with complex genetic traits such as autism and major depression. We use a variety of cutting-edge genomic technologies, together with cellular and physiological assays in genetically modified mice, to pursue our interests, and aim to ultimately translate our findings into therapeutic development to improve treatment for neurodevelopmental and neuropsychiatric disorders.

Irfan A. Asangani, Ph.D.

Research Interest

The Asangani Lab is affiliated with the Department of Cancer Biology at the Perelman School of Medicine, University of Pennsylvania, Philadelphia. Our lab investigates how epigenetic regulators, such as chromatin modifying enzymes and chromatin-associated proteins, cooperate with transcription factors to orchestrate transcriptional addiction in cancer cells. We aim to translate this knowledge into clinical tools by developing novel diagnostic, prognostic, and therapeutic strategies.

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