Kavitha Sarma, Ph.D.

R-loop biology.
R-loops are RNA containing chromatin structures that have the potential to be powerful regulators of epigenetic gene expression. Many questions about where R-loops form, what protein factors function to modulate these structures in vivo, and their mechanisms in gene regulation remain unanswered. As an independent investigator, I drew on my considerable expertise in protein and RNA biochemistry to initiate projects to understand the function and mechanisms of R-loops. We developed a novel technique that we named, “MapR”, that combines the specificity of RNase H for DNA:RNA hybrids with the sensitivity, speed, and convenience of the CUT&RUN approach, whereby targeted genomic regions are released from the nucleus by micrococcal nuclease (MNase) and sequenced directly, without the need for affinity purification. MapR is an antibody independent, recombinant protein-based technology that is fast, sensitive, and easy to implement (Yan et al, 2019). Also, we have recently improved MapR to increase resolution and confer strand specificity (Wulfridge and Sarma, 2021) and have used proximity proteomics to identify the R-loop proteome (Yan et al, 2022). We discovered a previously unappreciated role for zinc finger and homeodomain proteins in R-loop regulation and identified ADNP, a high confidence autism spectrum disorder gene, as an R-loop resolver.

Yan Q*, Shields, EJ*, Bonasio R†, Sarma K†. 2019. Mapping native R-loops genome-wide using a targeted nuclease approach. Cell Rep, 29(5):1369-1380. PMC6870988.
Yan Q, Sarma K. 2020. MapR: A method for identifying native R-loops genome-wide. Curr Protoc Mol Biol. 130(1):e113. PMC6986773.
Wulfridge P, and Sarma K. 2021. A nuclease- and bisulfite-based strategy captures strand-specific R-loops genome-wide. eLife 10:e65146. PMC7901872.
Yan Q*, Wulfridge P*, Doherty J, Fernandez-Luna JL, Real PJ, Tang HY, Sarma K. 2021. Proximity labeling identifies a repertoire of site-specific R-loop modulators. Nature Commun. 13(1):53. PMCID: PMC8748879. ‘*’- equal contribution.

Non-coding RNA regulation.
As a post-doctoral fellow, I applied my expertise in biochemistry to investigate the significance of interactions between proteins and long non-coding RNAs (lncRNAs) in the context of X chromosome inactivation. I discovered a novel use for locked nucleic acids (LNAs) in the study of lncRNA function. The mechanism of Xist RNA spreading had remained an open question in the field since its discovery in the 1990s. Using LNAs, I found that Xist RNA utilizes both repetitive and unique elements within itself to spread uniformly over the entire X chromosome to silence it (Sarma et al, 2010). Furthermore, combining the use of LNAs with a high throughput sequencing approach (CHART) allowed us to identify regions of the genome that were bound by Xist RNA (Simon et al, 2013). The use of LNAs was key in discovering that Xist RNA utilizes distinct mechanisms to coat the inactive X chromosome at different developmental stages. This technology has been patented and commercialized and is now being used to functionally characterize several different non-coding RNAs that play important roles in cellular homeostasis. During this time, I also contributed to examining the interactions between the PRC2 complex and Xist RNA (Cifuentes-Rojas et al, 2014), and discovered ATRX to be a high affinity RNA binding protein that interacts with Xist RNA to regulate PRC2 enrichment on the inactive X chromosome (Sarma et al, 2014).

Sarma K, Levasseur P, Aristarkhov A, Lee JT. 2010. Locked nucleic acids (LNAs) reveal sequence requirements and kinetics of Xist RNA localization to the X chromosome. Proc Natl Acad Sci U S A 107:22196-22201. PMCID: PMC3009817.
Simon MD*, Pinter SF*, Fang R*, Sarma K, Rutenberg-Schoenberg M, Bowman SK, Kesner BA, Maier VK, Kingston RE, Lee JT. 2013. High-resolution Xist binding maps reveal two-step spreading during X-chromosome inactivation. Nature 504:465-469. PMCID: PMC3904790.
Cifuentes-Rojas C, Hernandez AJ, Sarma K, Lee JT. 2014. Regulatory interactions between RNA and polycomb repressive complex 2. Mol Cell 55:171-185. PMCID: PMC4107928.
Sarma K, Cifuentes-Rojas C, Ergun A, Del Rosario A, Jeon Y, White F, Sadreyev R, Lee JT. 2014. ATRX regulates binding of PRC2 to Xist RNA and Polycomb targets. Cell 159:1228. PMID: 28898627.

Histone methyltransferases and Polycomb proteins.
I have spent a large part of my career working toward understanding the role of various chromatin factors in epigenetic gene regulation. As a graduate student, I purified and characterized some of the first reported histone lysine methyltransferases (HKMTs) such as PR-Set7, Set9, and the Polycomb Repressive Complex 2 (PRC2). During this time, I also developed and characterized reagents and tools that were critical to advance research in this field. My discovery that the mammalian homolog of the drosophila polycomblike protein (PHF1), a PRC2 accessory factor, facilitates the activity of the PRC2 complex provided first evidence of the role of accessory proteins in the function of the PRC2 complex (Sarma et al, 2008). Recent publications by several independent groups underscore the importance of PHF1 and other polycomblike proteins in PRC2 function. As a post-doctoral fellow, I discovered that the chromatin remodeler ATRX regulates PRC2 localization genome-wide (Sarma et al, 2014). In my own lab, we showed that ATRX-RNA interactions specify PRC2 targeting to a subset of polycomb targets (Ren et al, 2020). We also discovered that ATRX mutations in the histone binding or the helicase domains have distinct effects on PRC2 localization and neuronal differentiation (Bieluszewska et al, 2022).

Sarma K, Margueron R, Ivanov A, Pirrotta V, Reinberg D. 2008. Ezh2 requires PHF1 to efficiently catalyze H3 lysine 27 trimethylation in vivo. Mol Cell Biol 28:2718-2731. PMCID: PMC2293112.
Sarma K, Cifuentes-Rojas C, Ergun A, Del Rosario A, Jeon Y, White F, Sadreyev R, Lee JT. 2014. ATRX regulates binding of PRC2 to Xist RNA and Polycomb targets. Cell 159:1228. PMID: 28898627.
Ren W*, Medeiros N*, Warneford-Thomson R, Wulfridge P, Yan Q, Bian J, Sidoli S, Garcia BA, Skordalakes E, Joyce E, Bonasio R, Sarma K. 2020. Disruption of ATRX-RNA interactions uncovers roles in ATRX localization and PRC2 function. Nat Commun. 6;11(1):2219. PMID: 32376827
Bieluszewska A, Wulfridge P, Doherty J, Ren W, Sarma K. (2022). ATRX histone binding and helicase activities have distinct roles in neuronal differentiation. Nucleic Acids Res. PMC in progress

Research Interest

The Sarma lab focuses on elucidating the molecular mechanisms of RNA mediated epigenetic gene regulation in eukaryotes. Using various cell culture models including mouse embryonic stem cells, neuronal differentiation systems, and cancer models, we employ biochemical and various -omics tools to examine the mechanisms of RNAs and their interactions with chromatin and epigenetic modifiers in gene regulation. We have become increasingly interested in RNA containing chromatin structures called R-loops and how they are misregulated in neurodevelopmental disorders and cancers and their impact on genomic processes that can drive disease. Most recently, we developed high throughput sequencing methods to profile native R-loops and identified R-loop interactors using a proximal proteomic approach.

Montserrat Anguera, Ph.D.

1. Functional characterization of long noncoding RNAs that regulate gene expression during development and in adulthood. As a postdoctoral fellow, I began investigating epigenetic mechanisms and modeling these mechanisms of gene expression using conditional knockout mouse models. I demonstrated that the Tsx gene does not encode for a protein but instead is transcribed as a long noncoding RNA, similar to other neighboring genes. I generated a conditional knockout mouse for Tsx and discovered that Tsx RNA has functions beyond the germline, affecting stem cell growth, expression of neighboring genes that regulate X-chromosome inactivation, and also behavior in a gender-specific fashion. This work provided a conceptual foundation for experimentally testing for coding potential of noncoding RNAs and for their functional characterization. As an associate professor, my lab continues to study epigenetic mechanisms of gene expression involving long noncoding RNAs from the X chromosome. We have also investigated sex-specific gene expression profiles during human placental development using in vitro pluripotent stem cell derived model systems. In addition, my lab continues to have a long-standing interest in functional characterization of novel X-linked long noncoding RNAs. We discovered LNCRHOXF1 which functions during early placental development.

  • Anguera, M.C., Ma, W., Clift, D., Namekawa, S., Kelleher, R.J, and Lee, J.T. (2011). “Tsx produces a long noncoding RNA and has general functions in the germline, stem cells, and brain.” PLoS Genet. 7 (9):1-14. PMCID: PMC3164691
  • Ian Penkala, Camille Syrett, Jianle Wang, and Montserrat C. Anguera. (2016). “LNCRHOXF1: a long noncoding RNA from the X-chromosome that suppresses viral response genes during development of the early human placenta”. Mol Cell Biol. Apr 11, 2016, PMID: 27066803
  • Camille Syrett, Isabel Sierra, Corbett Berry, Daniel Beiting, and Montserrat C. Anguera. (2018) “Sex-specific gene expression during differentiation of human pluripotent stem cells to trophoblast progenitors”. Stem Cells Dev. July 11; PMID: 29993333

2. Defining abnormal X-Chromosome Inactivation maintenance in human pluripotent stem cells. During my postdoc, I also investigated human X-Chromosome Inactivation (XCI) in the pluripotent state to specifically determine whether reprogramming human female fibroblasts would reactive the inactive X chromosome. I discovered that the reprogramming process generated a heterogeneous mixture of X-Inactivation cells, distinguishable by their expression pattern for the long noncoding RNA XIST. I made the critical observation that loss of XIST RNA expression in human pluripotent stem cells results in the acquisition of cancer-like phenotypes. This work was the first to demonstrate that human female pluripotent stem cells are potentially dangerous in a clinical setting. These experiments are the basis for my current research program investigating how abnormal expression from the inactive X, resulting from aberrant XCI maintenance, is involved in female-biased autoimmunity, where X-silencing is compromised.

  • Anguera, M.C., Sadreyev, R., Zhang, Z., Szanto, A., Sheridan, S., Haggerty, S., Jaenisch, R., Gimbelbrandt, A., Mitalipova, M., and Lee, J.T. (2012). “Molecular signatures and epigenetic stability of human induced pluripotent stem cells.” Cell Stem Cell 11(1):75-90. PMCID: PMC3587778
  • Lessing, D., Anguera, M.C., and Lee, J.T. (2013). “X Chromosome Inactivation and Epigenetic Responses to Cellular Reprogramming.” Annu Rev Genomics Hum Genet. 14:85-110.

3. Mechanisms of X-Chromosome Inactivation in lymphocytes and other immune cells. As an associate professor at Penn, I have continued investigating the molecular mechanisms of X-Chromosome Inactivation, and how altered dosage of X-linked genes contributes to sex-biased disease. As auto-antigen driven activation of lymphocytes drives autoimmune disease we first investigated the epigenetic status of the inactive X in female lymphocytes from humans and mice, and made the remarkable discovery that these cells do not maintain X-Chromosome Inactivation in the same way as other female somatic cells. We were the first to discover that the inactive X has euchromatic features in female lymphocytes, which may underlie the female-bias in autoimmune disorders including lupus. Our research also demonstrated that T and B cells, upon antigen-mediated stimulation, exhibit relocalization of Xist RNA and heterochromatic marks to the inactive X chromosome, which we have termed ‘dynamic XCI maintenance’. My lab is investigating the molecular details of dynamic XCI maintenance in T and B cells, and has identified protein factors that are required for localization of epigenetic modifications to the inactive X.

  • Wang, J., Syrett, C.M., Kramer, M. Basu, A., Atchison, M. & Anguera, M.C. (2016). “Unusual maintenance of X-chromosome Inactivation predisposes female lymphocytes for increased expression from the inactive X”. Proc Natl Acad Sci, Mar. 21, 2016. PMCID: PMC4833277
  • Syrett, C.M., Sindhava, V., Hodawadekar, S., Myles, A., Liang, G., Zhang, Y., Nandi, S., Cancro, M., Atchison, M., & Anguera, M.C. (2017) “Loss of Xist RNA from the inactive X during B cell development is restored in a dynamic YY1-dependent two-step process in activated B cells”. Plos Genetics, Oct 9; 13 (10). PMCID: PMC5648283
  • Camille Syrett, Vishal Sindhava, Isabel Sierra, Aimee Dubin, Michael Atchison, and Montserrat C. Anguera (2018). “Diversity of Epigenetic Features of the inactive X-chromosome in NK cells, dendritic cells, and macrophages”. Frontiers in Immunology. Dec 14. PMCID: PMC6331414.
  • Sierra I, Anguera MC. (2019). “Enjoy the silence: X-chromosome inactivation diversity in somatic cells.” Curr Opin Genet Dev. 2019 Apr;55:26-31. Epub 2019 May 17. Review. PubMed PMID: 31108425; PubMed Central PMCID: PMC6759402.

4. Impact of autoimmune disease on X-Chromosome Inactivation mechanisms
We continue to investigate diseases exhibiting a female bias that also have abnormal increased expression of X-linked genes. My lab has focused on the autoimmune disorder systemic lupus erythematosus (SLE), which has a strong female-bias and exhibits aberrant over-expression of the X-linked genes CXCR3, CD40LG, TLR7, and FOXP3 in lymphocytes. We made the remarkable discovery that female SLE patients and mouse models with lupus-like disease have perturbations with markers of the inactive X, most notably XIST/Xist RNA localization and heterochromatic modifications H3K27me3 and H2AK119ubiquitin. Our work also found female-specific X-linked gene signatures of SLE patients. We are actively continuing this work in an effort to understand how transcription from the inactive X contributes to autoimmune disease susceptibility and disease severity, and also investigate mouse models of spontaneous lupus disease to examine how disease onset impacts XCI maintenance and X-linked gene expression.

  • Syrett CM, Paneru B, Sandoval-Heglund D, Wang J, Banerjee S, Sindhava V, Behrens EM, Atchison M, & Anguera M.C. (2019) “Altered X-chromosome Inactivation in T cells may promote sex-biased autoimmune diseases”. JCI Insight, Apr 4;4(7). PMCID: PMC6483655.
  • Syrett CM, Sierra I, Beethem ZT, Dubin AH, Anguera MC. “Loss of epigenetic modifications on the inactive X chromosome and sex-biased gene expression profiles in B cells from NZB/W F1 mice with lupus-like disease.” J Autoimmun. (2020) Feb;107:102357. Epub 2019 Nov 25. PubMed PMID: 31780316; PubMed Central PMCID: PMC7237307.
  • Pyfrom S, Paneru B, Knoxx, J, Posso S, Buckner J, Cancro M, Anguera MC. “The dynamic epigenetic regulation of the inactive X chromosome in healthy human B cells is dysregulated in lupus patients”. Proc Natl Acad Sci. (2021) June;Vol.118.
  • Jiwrajka N, Anguera MC. “The X in SeX-Biased Immunity and Autoimmune Rheumatic Disease.” J Exp Med. (2022). Review. doi 10.1084/jem.20211487.

Research Interest

My research program focuses on epigenetic gene regulation that underlies sex differences in development and disease. In particular, I am interested in understanding how gene expression from the X-chromosome is regulated to ensure dosage compensation between males and females, and how these mechanisms become altered in diseases exhibiting a sex-bias, such as autoimmunity. We investigate the female-specific epigenetic process of X-chromosome Inactivation (XCI). The X-chromosome contains genes necessary for cell proliferation, metabolism, cognition, reproduction, and has the highest density of immunity-related genes. Thus, gene expression from the X-chromosome is critical for cellular identity, and XCI ensures proper dosage of these genes in females. Abnormal expression of X-linked genes is a feature of many diseases exhibiting a sex-bias, including cancer, cardiovascular disease, and autoimmunity. I have a broad background in epigenetics, spanning the metabolism of one-carbon units that generate methyl groups that alter gene expression, to the epigenetic regulatory mechanisms involved in XCI. My independent research program at the University of Pennsylvania continues to investigate the epigenetic mechanisms that regulate X-linked gene expression, and how alterations result in female-biased disease. My laboratory has undertaken and completed studies establishing a new field of research, defining novel epigenetic pathways involving the X-chromosome that impact human development, immune responses during development, and lymphocyte function. We routinely use RNA and DNA fluorescence in situ hybridization, immunofluorescence experiment, and allele-specific RNA sequencing, which enables single-cell resolution of the epigenetic characteristics of the inactive X and transcriptional activity. Our long-term goal is to understand how dosage compensation of X-linked genes is maintained in lymphocytes, how perturbations with this silencing contribute to female-biased autoimmune disease, and how XCI maintenance mechanisms can be targeted to correct aberrant X-linked gene expression.

Ongoing Projects:
R01 AI134834
Anguera (PI)
6/01/2018-5/31/2023
Gene regulation from the inactive X in activated B cells

Lupus Mechanisms and Targets Award
Anguera (PI)
6/1/2020-5/30/2023
Targeting the inactive X for correcting dosage imbalances in lupus

Jonathan A. Epstein, M.D.

1. We have demonstrated that engineered T cells (CART cells) can be used to target activated fibroblasts in the diseased heart with resulting improvement in cardiac function. This work has received significant attention (highlighted in the NEJM, Scientific American, the NYTimes and elsewhere) and has opened the field of CART cells to the possibility of providing new treatments for fibrotic disorders.
Rurik JG, Tombácz I, Yadegari A, Méndez Fernández PO, Shewale SV, Kimura T, Younoss SO, Papp TE, Tam YK, Mui BL, Albelda SM, Pure E, June CH, Aghajanian H, Weissman D, Parhiz H, Epstein JA. CAR T cells produced in vivo to treat cardiac injury. Science. 2022 Jan 7;375(6576):91-96. doi: 10.1126/science.abm0594
Rurik JG, Aghajanian H, Epstein JA. Immune cells and immunotherapy for cardiac injury and repair. Circ. Res. 2021 May 28; 128(11):1766-1779. Review
Epstein JA, Rosenthal N, Feldman AM. Teasing the Immune System to Repair the Heart. N Engl J Med. 2020 Apr 23;382(17):1660-1662. Review
Aghajanian H, Kimura T, Rurik JG, Hancock AS, Leibowitz MS, Li L, Scholler J, Monslow J, Lo A, Han W, Wang T, Bedi K, Morley MP, Linares Saldana RA, Bolar NA, McDaid K, Assenmacher CA, Smith CL, Wirth D, June CH, Margulies KB, Jain R, Puré E, Albelda SM, Epstein JA. Targeting cardiac fibrosis with engineered T cells. Nature. 2019 Sep;573(7774):430-433. PMCID: PMC6752964

2. We have elucidated the role of epigenetics in heart development and we have applied this knowledge to explore novel therapies for adult cardiac disease including ischemia-reperfusion and congestive heart failure. In particular, we demonstrated that histone deacetylase inhibitors can prevent pathologic cardiac hypertrophy, which was counter to the prevailing dogma at the time. More recently, we have shown that nuclear architecture (how chromatin is packaged in three dimensions in the nucleus) contributes to cell identity, gene expression, and developmental competence.
Smith, C.L., Poleshko, A., Epstein, J.A. The nuclear periphery is a scaffold for tissue-specific enhancers. Nucleic Acids Res. 2021; 49: 6181-6195. PMCID: PMC8216274
Poleshko, A., Shah, P.P., Gupta, M., Babu, A., Moreley, M., Manderfield, L.J., Ifkovits, J.L., Dubois, N., Morrisey, E.E., Lazar, M.A., Smith, C.L., Epstein, J.A., Jain, R. Genome–nuclear lamina interactions regulate progenitor cell lineage restriction during cardiogenesis. Cell. 2017 Oct 7. (co-senior author and mentor of first author). PMCID: PMC5683101
Kook H, Lepore JJ, Gitler AD, Lu MM, Wing-Man Yung W, Mackay J, Zhou R, Ferrari V, Gruber P, Epstein JA. Cardiac hypertrophy and histone deacetylase-dependent transcriptional repression mediated by the atypical homeodomain protein Hop. The Journal of Clinical Investigation. 2003;112(6):863-71. PMCID: PMC193673.
Chen F, Kook H, Milewski R, Gitler AD, Lu MM, Li J, Nazarian R, Schnepp R, Jen K, Biben C, Runke G, Mackay JP, Novotny J, Schwartz RJ, Harvey RP, Mullins MC, Epstein JA. Hop is an unusual homeobox gene that modulates cardiac development. Cell. 2002;110(6):713-23.

3. My laboratory has contributed to our understanding of how the neural crest influences cardiac development and to the embryologic and genetic basis of congenital heart disease. Neural crest contribution to the heart was described in the early 1980s by Kirby and colleagues, and our laboratory extended these observations to mammalian systems, performed genetic fate-mapping in mouse models, identified candidate genes for congenital heart defects and described molecular pathways including Notch, Fgf, Wnt and Hippo that regulate cross-talk between neural crest, endothelium and second heart field derivatives in the heart.
Manderfield LJ, Engleka KA, Aghajanian H, Gupta M, Yang S, Li L, Baggs JE, Hogenesch JB, Olson EN, Epstein JA. Pax3 and hippo signaling coordinate melanocyte gene expression in neural crest. Cell Reports. 2014;9(5):1885-95. PMCID: PMC4267159.
Manderfield LJ, High FA, Engleka KA, Liu F, Li L, Rentschler S, Epstein JA. Notch activation of Jagged1 contributes to the assembly of the arterial wall. Circulation. 2012;125(2):314-23. PMCID: PMC3260393.
Katz TC, Singh MK, Degenhardt K, Rivera-Feliciano J, Johnson RL, Epstein JA, Tabin CJ. Distinct compartments of the proepicardial organ give rise to coronary vascular endothelial cells. Developmental Cell. 2012;22(3):639-50. PMCID: PMC3306604.
de la Pompa JL, Epstein JA. Coordinating tissue interactions: Notch signaling in cardiac development and disease. Developmental Cell. 2012;22(2):244-54. PMCID: PMC3285259.

4. We described the molecular basis for cardiovascular manifestations in Type 1 Neurofibromatosis using mouse and zebrafish models, including valvular pulmonic stenosis, endothelial defects and cardiac hypertrophy. We generated a zinc-finger knockout of the Nf1 genes in zebrafish, and demonstrated behavioral defects amenable to small molecule drug screening.
Wolman MA, de Groh ED, McBride SM, Jongens TA, Granato M, Epstein JA. Modulation of cAMP and ras signaling pathways improves distinct behavioral deficits in a zebrafish model of neurofibromatosis type 1. Cell Reports. 2014;8(5):1265-70. PMCID: PMC5850931
Shin J, Padmanabhan A, de Groh ED, Lee JS, Haidar S, Dahlberg S, Guo F, He S, Wolman MA, Granato M, Lawson ND, Wolfe SA, Kim SH, Solnica-Krezel L, Kanki JP, Ligon KL, Epstein JA, Look AT. Zebrafish neurofibromatosis type 1 genes have redundant functions in tumorigenesis and embryonic development. Disease Models & Mechanisms. 2012;5(6):881-94. PMCID: PMC3484870.
Ismat FA, Xu J, Lu MM, Epstein JA. The neurofibromin GAP-related domain rescues endothelial but not neural crest development in Nf1 mice. The Journal of Clinical Investigation. 2006;116(9):2378-84. PMCID: PMC1533876.
Gitler AD, Zhu Y, Ismat FA, Lu MM, Yamauchi Y, Parada LF, Epstein JA. Nf1 has an essential role in endothelial cells. Nature Genetics. 2003;33(1):75-9. PMCID: PMC3079412.

5. We were among the first to show that guidance molecules known to regulate the migration of axons in the central nervous system can also regulate angiogenesis and cardiac development. We have shown that members of the semaphorin and plexin families help to pattern the outflow tract of the heart, the pulmonary veins and coronary arteries, and the peripheral vasculature.
Epstein JA, Aghajanian H, Singh MK. Semaphorin signaling in cardiovascular development. Cell Metabolism. 2015;21(2):163-73. Review
Aghajanian H, Choi C, Ho VC, Gupta M, Singh MK, Epstein JA. Semaphorin 3d and semaphorin 3e direct endothelial motility through distinct molecular signaling pathways. The Journal of Biological Chemistry. 2014;289(26):17971-9. PMCID: PMC4140303.
Degenhardt K, Singh MK, Aghajanian H, Massera D, Wang Q, Li J, Li L, Choi C, Yzaguirre AD, Francey LJ, Gallant E, Krantz ID, Gruber PJ, Epstein JA. Semaphorin 3d signaling defects are associated with anomalous pulmonary venous connections. Nature Medicine. 2013;19(6):760-5. PMCID: PMC3746328.
Gitler AD, Lu MM, Epstein JA. PlexinD1 and semaphorin signaling are required in endothelial cells for cardiovascular development. Developmental Cell. 2004;7(1):107-16.

Research Interest

The Epstein laboratory studies molecular mechanisms of cardiovascular development and stem cell biology, and the implications of these mechanisms for understanding human disease. The lab has a longstanding interest in the genetic causes of congenital heart disease and transcriptional regulation of cell fate determination. Most recently, we have focused on epigenetics, including the role of histone deacetylases in cardiac development and adult heart function. Aims of current projects include gaining an understanding of the three-dimensional packaging of DNA and chromatin in the nucleus (“nuclear architecture”), and the regulation of cell differentiation by protein complexes that tether regions of the genome to the nuclear periphery. The lab has pioneered the concept that interactions between the nuclear lamina and the chromatin contribute to the regulation of entire gene programs that define cardiac cell types.

Rajan Jain, M.D.

Our work is broadly characterized by three major themes.

First, we are defining the molecular factors regulating 3D chromatin spatial positioning. We have shown that lamina-chromatin interactions (lamina-associated domains or LADs) mediate cardiomyocyte differentiation during cardiac development, establishing physiologic relevance of spatial positioning. More recently, we have shown that pathogenic LMNA variants associated with cardiomyopathy compromise genome organization at the nuclear lamina in cardiomyocytes but not in hepatocytes or adipocytes. The disruption in lamina-chromatin interactions is targeted and results in the misexpression of non-myocyte genes in mutant cardiomyocytes. Finally, we have generated an atlas of lamina-chromatin interactions across multiple human cell types and defined a new, intermediate LAD state. Collectively, my work has shown that 3D spatial positioning regulates cardiac myocyte cellular identity and contributes to phenotypes observed in human laminopathies. We are now working to reveal the molecular mechanisms underlying spatial positioning, using genome-wide screens, mouse and human iPSC models, high resolution imaging and genomics. We work closely with the Joyce and Lakadamyali laboratories. This work has been supported by my NIH New Innovator award (DP2HL147123) and expanded by our interactions with the 4D Nucleome Consortium (U01DA052715).

A second major area of investigation in the laboratory is understanding how Bromodomain and Extraterminal Domain (BET) proteins regulate cardiac cell fate and state. I first became interested in BET proteins as an undergraduate in the Tjian laboratory (PMID: 12620225). In my program, we are examining the cell type specific roles of individual BET proteins in cardiac cell types, with a particular focus on BRD4. We have shown that BRD4 gains locus-specificity in adult cardiac myocytes by interacting with GATA4. Most recently, my group discovered a novel role for BRD4 in genome folding by regulating stability of cohesin on chromatin. Loss of BRD4 in neural crest results in phenotypes resembling human syndromes associated with mutations in cohesin and associated proteins which we mechanistically attribute to the role of BRD4 in genome folding. Our data is amongst the first demonstrating the functional relevance of genome folding to cellular identity. We are extending this work to identify other regulators of genome folding, their mechanism of action and relevance to development and disease. The work is supported by an NHLBI R01 (HL139783).

Our work is further advanced by a third theme in longstanding collaborative work with the Raj lab to elucidate determinants of cellular plasticity, which may inform our understanding of transdifferentiation. My group has shown demonstrated the lineage relationships in various tissues and organs, both in vivo and in vitro. Taken together, our studies suggest that lineage plasticity exists and helps maintain tissue homeostasis. This is funded through a Transformative Research Award (R01GM137425).

My lab strives to explain our science to the lay public:

https://www.youtube.com/watch?v=fthq4chhrGo&list=LL&index=1&t=1s

https://www.youtube.com/watch?v=t2uOlbohsNo&list=LL&index=2&t=271s

Research Interest

Our research program is driven by the central hypothesis that three-dimensional (3D) genome organization orchestrates the establishment and maintenance of cardiac cellular identity. Uncovering the molecular mechanisms that guide nuclear architecture will transform our understanding of how coordinated gene expression is regulated and cellular identity is achieved. Progenitor cells progressively restrict their lineage potential during cardiac development. The mechanisms that establish and maintain cellular identity through mitosis and over a lifespan underlie health. Compromised differentiation and identity have been linked to several diseases. Models underlying fate determination and identity often focus on transcription factors and/or niche signals. Current paradigms fail to reconcile how the interplay between a finite number of morphogens and lineage specific transcription factors result in 200+ cell types with distinct and stable identities. We posit that nuclear architecture represents a critical mechanism for achieving coordinated regulation of hundreds of genes underlying cellular identity by governing their accessibility or availability. My interdisciplinary training and research program in developmental biology and epigenetics, background as a practicing cardiologist, collaborative network have allowed me to build a strong body of work demonstrating that nuclear architecture regulates cellular identity in development and disease. We aim to decipher the rules governing spatial positioning of loci and genome folding, and to reveal their importance to cardiac development and disease.

Klaus Kaestner, Ph.D., M.S.

Research Interest

The Kaestner lab employs modern mouse genetic approaches, such as gene targeting, tissue-specific and inducible gene ablation, to understand the molecular mechanisms of organogenesis and physiology of the liver, pancreas and gastrointestinal tract. We also employ next-generation sequencing explore the differences between the transcriptome and epigenome of normal vs diseased tissues.
The prevalence of Diabetes Mellitus has reached epidemic proportions world-wide, and is predicted to increase rapidly in the years to come, putting a tremendous strain on health care budgets in both developed and developing countries. There are two major forms of diabetes and both are associated with decreased beta-cell mass. No treatments have been devised that increase beta-cell mass in vivo in humans, and transplantation of beta-cells is extremely limited due to lack of appropriate donors. For these reasons, increasing functional beta-cell mass in vitro, or in vivo prior to or after transplantation, has become a “Holy Grail” of diabetes research. Our previous studies clearly show that adult human beta-cells can be induced to replicate, and – importantly – that cells can maintain normal glucose responsiveness after cell division. However, the replication rate achieved was still low, likely due in part to the known age-related decline in the ability of the beta-cell to replicate. We propose to build on our previous findings and to develop more efficacious methods to increase functional beta-cell mass by inducing replication of adult beta-cells, and by restoring juvenile functional properties to aged beta-cells. We will focus on mechanisms derived from studies of non-neoplastic human disease as well as age-related phenotypic changes in human beta-cells.
We are determining  the mechanisms of age-related decline in beta-cell function and replicative capacity, by mapping the changes in the beta-cell epigenome that occur with age. Selected genes will then be targeted using cutting-edge and emerging technologies such as Crispr-activation and inhibition systems that are already established or are being developed in our laboratories. The research team combines clinical experience with expertise in molecular biology and extensive experience in genomic modification aimed at enhancing beta-cell replication. By basing interventions on changes found in human disease and normal aging, this approach will increase the chances that discoveries made can be translated more rapidly into clinically relevant protocols.

Doris Wagner, Ph.D.

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, Ph.D.

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.

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. Cell184:6081-6100.

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.

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.

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 H19Igf2 imprinted locus but have more recently expanded to Grb10Ddc1 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.

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