Posts classified under: Neuroepigenetics Interest Group

Penn Epigenetics > Faculty > Neuroepigenetics Interest Group
Dana Silverbush headshot

Dana Silverbush, PhD

Research Interest

A major obstacle to effective cancer therapy is the co-existence of multiple cell states with distinct molecular and phenotypical profiles within a tumor (intra-tumoral heterogeneity). The ability of tumor cells to transition between these states (tumor plasticity) has been shown to mediate treatment resistance and disease progression. As a result, malignancies with high rates of heterogeneity and plasticity—such as glioma, acute myeloid leukemia, pancreatic, and lung cancers—remain highly treatment-refractory. Notably, intra-tumoral heterogeneity encompasses genetic, epigenetic, transcriptional, and phenotypic components, and is increasingly thought to be driven by an interplay of genetic and epigenetic factors. The aim of the Silverbush lab is to map the genetic and epigenetic patterns driving transcriptomic heterogeneity and tumor plasticity, leveraging these patterns to enhance diagnostic and prognostic accuracy. To achieve this, we develop single-cell multi-omic assays and corresponding analysis solutions, employing them to explore intra-tumoral heterogeneity and tumor plasticity. The lab consists of both a wetlab component and a computational component, and we welcome students with diverse backgrounds and interested in either or both.

Kahlilia Blanco headshot

Kahlilia Morris-Blanco, Ph.D.

Research Interest

Dr. Morris-Blanco’s laboratory investigates epigenetic mechanisms involved in stroke pathophysiology by examining the interplay between spatial and temporal epigenetic dynamics, transcriptional regulation, and mitochondrial function in the post-stroke brain. Using both in vitro and in vivo experimental stroke models, they employ gene-specific and genome wide assessments of epigenomic organization, single-cell omics, metabolomics, and functional assessments of mitochondria and neuroprotection. Dr. Morris-Blanco is especially interested in using these mechanistic studies to develop novel treatment strategies, with the goal of translating epigenetic therapies to the clinic.

Richard Phillips, M.D., Ph.D.

Contributions to Science

  1. Identification of oncogenic pathways as therapeutic targets in epigenetically driven gliomas

I investigated the mechanism of action of a menin-inhibitor, MI-2, in H3K27M gliomas following up on studies which identified a menin-inhibitor (MI-2) as the top ‘hit’ in a chemical screen in a new model of this disease (Funato et. al, 2014, Science). Menin is an epigenetic regulator which we hypothesized may be specifically required in the setting of H3K27M induced epigenetic dysregulation. I demonstrated that MI-2 exhibits anti-glioma activity in H3K27M mutant and H3 wild-type glioma subtypes and showed menin is not the relevant molecular target for this drug in gliomas. Instead, using an integrated approach employing genetic, biochemical and metabolomic methods, I discovered the direct molecular target of MI-2 in glioma as lanosterol synthase, a cholesterol biosynthesis enzyme; revealing a novel metabolic vulnerability in glioma and more broadly implicating cholesterol homeostasis as an attractive pathway to target in this malignancy. 

Phillips RE, Yang Y, Smith R, Thompson B, Yamasaki T, Soto-Feliciano Y, Funato K, Liang Y, Garcia-Bermudez J, Wang X, Garcia B, Yamasaki K, McDonald J, Birsoy K, Tabar V, Allis CD. Target identification reveals lanosterol synthase as a vulnerability in glioma. Proc Natl Acad Sci U S A. 2019 Apr 16;116(16):7957-7962. PMCID: PMC6475387

Phillips RE, Soshnev AA, Allis CD. Epigenomic Reprogramming as a Driver of Malignant Glioma. Cancer Cell. 2020 Aug 31:S1535-6108(20)30419-0.

  1. Development of novel therapeutics in glioma targeting epigenetic mechanisms

We identified the chromatin regulator EZH2 as a context-specific dependency in H3K27M gliomas and through computational modeling of existing, non-brain penetrant EZH2 inhibitor scaffolds, and I led a collaboration which devised a chemical strategy resulting in the discovery of the first brain-penetrant small molecule targeting EZH2 for brain tumors.

A Chemical Strategy toward Novel Brain-Penetrant EZH2 Inhibitors. Liang R, Tomita D, Sasaki Y, Ginn J, Michino M, Huggins DJ, Baxt L, Kargman S, Shahid M, Aso K, Duggan M, Stamford AW, DeStanchina E, Liverton N, Meinke PT, Foley MA, Phillips RE. ACS Med Chem Lett. 2022 Feb 10;13(3):377-387. PMCID: PMC4981478

  1. Treatment approaches for management of brain tumors

Medulloblastoma is the most common primary brain tumor in children and management of extra-neural recurrence is a controversial and difficult-to-treat clinical scenario in Neuro-Oncology. We demonstrated efficacy of a combination standard-dose chemotherapy regimen which obviated the need for high-dose chemotherapy and stem cell transplantation (which both have significant potential side-effects) to induce long-term remission in this clinical entity.

Phillips RE, Curran KJ, Khakoo Y. Management of late extra-neural recurrence of medulloblastoma without high-dose chemotherapy. J Neurooncol. 2015 Sep;124(3):523-4. PMCID: PMC4981478

RESEARCH INTEREST

Coordinated epigenetic regulation enables cells to adopt specific gene expression programs to orchestrate normal differentiation and maintain cell fate. Gliomas are the most common type of brain cancer and exome-sequencing data has identified mutations in epigenetic regulators as a major driver of these tumors. However, it remains incompletely understood how these epigenetic drivers rewire the chromatin landscape and how this epigenetic dysregulation alters cellular phenotypes such as differentiation and immune evasion. In the Phillips Lab, we employ a number of cutting-edge techniques – from the development of forward genetics tools (i.e. CRISPR-Cas9 screening technology), epigenomic profiling, using neural stem cell models, and patient-derived models of glioma – to elucidate how epigenetic mechanisms contribute to gliomagenesis. Our long term research goal is to understand the how epigenetic pathways are rewired in brain cancer during tumorigenesis, therapy, and evasion of immunity.

Erica Korb, Ph.D

Contribution to Science

The role of chromatin in neurodevelopmental disorders and disease.

Epigenetic regulation plays a critical role in many neurodevelopmental disorders, including Autism Spectrum Disorder (ASD). In particular, many such disorders are the result of mutations in genes that encode chromatin modifying proteins. However, while these disorders share many features, it is unclear whether they also share gene expression disruptions resulting from the aberrant regulation of chromatin. We examined 5 chromatin modifiers that are all linked to ASD despite their different roles in regulating chromatin. Using RNA-sequencing, we identified a transcriptional signature that is shared between multiple neurodevelopmental syndromes, helping to elucidate the link between epigenetic regulation and the underlying cellular mechanisms that result in ASD.

During the COVID-19 shut-down, we sought to apply our understanding of histone biology to better understand the ability of SARS-CoV-2 to evade the immune system. In rare cases, viral proteins dampen antiviral responses by mimicking critical regions of human histone proteins particularly those containing posttranslational modifications required for transcriptional regulation. We found that the SARS-CoV-2 protein encoded by ORF8 (Orf8) functions as a histone mimic of the ARKS motifs in histone 3. Orf8 is associated with chromatin, binds to numerous histone-associated proteins, and is itself acetylated at this site. Orf8 expression disrupts multiple critical histone post-translational modifications including H3K9ac, H3K9me3, and H3K27me3 and promotes chromatin compaction while Orf8. Further, SARS-CoV-2 infection in human cells and patient lung tissue cause these same disruptions to chromatin acting through the Orf8 histone mimic motif. These findings define a mechanism through which SARS-CoV-2 disrupts host cell epigenetic regulation.

In my postdoctoral work in the lab of Dr. C. David Allis, I studied Fragile X syndrome (FXS). FXS is the most common genetic cause of intellectual disability and autism and is caused by loss of function of fragile X mental retardation protein (FMRP). I found that a disproportionate number of FMRP targets encode transcriptional regulators, particularly chromatin-associated proteins. In addition, I discovered that the loss of FMRP results in widespread chromatin misregulation and aberrant transcription. Finally, I demonstrated that the small molecule inhibitor Jq1 which blocks chromatin binding of the BET family of proteins alleviated many of the transcriptional changes and behavioral phenotypes associated with FXS. Through this work, I elucidated a novel causative mechanism of epigenetic disruption underlying FXS and demonstrated that targeting transcription may provide new treatment approaches. This work was published in Cell and included in a patent.

  • Kee, J., Thudium, S., Renner, D.M., Glastad, K., Palozola, K., Zhang, Z., Li, Y., Lan, Y., Cesare, J., Poleshko, A., Kiseleva, A.A., Truitt, R., Cardenas-Diaz, F. L., Zhang, X., Xie, X., Kotton, D. N., Alysandratos, K. D., Epstein, J.A., Shi, P.Y., Yang, W., Morrisey, E., Garcia, B. A., Berger, S. L., Weiss, S. R., Korb, E. SARS-CoV-2 protein encoded by ORF8 contains a histone mimic that disrupts chromatin regulation. Nature. (PMC in progress)
  • Thudium S, Palozola K, L’Her E, Korb E. Identification of a transcriptional signature found in multiple models of ASD and related disorders. Genome Research. (PMC in progress)
  • Korb, E., Herre, M., Zucker-Scharff, I., Allis, C.D., Darnell, RB. 2017. Excess translation of epigenetic regulators contributes to Fragile X Syndrome and is alleviated by Bd4 inhibition. Cell. (PMC5740873)
  • Inquimbert, P., Moll, M., Latremoliere, A., Tong, C.K., Wang, J., Sheehan, G.F., Smith, B.M., Korb, E., Athie, M.C.P., Babaniyi, O., Ghasemlou, N., Yanagawa, Y., Allis, C.D., Hof, P.R., Scholz, J. 2018. NMDA Receptor activation underlies the loss of spinal dorsal horn neurons and the transition to persistent pain after peripheral nerve injury. Cell Rep. (PMC62761118)

Epigenetic regulation of information storage in the brain

While chromatin regulation is crucial for the mechanisms underlying memory formation, the role of many chromatin-associated proteins in the context of neuronal function remains unclear. During my postdoctoral fellowship, I focused on Brd4, which binds acetylated histones. Despite the increasing use of Brd4 inhibitors as therapeutics, it had never been examined in the brain. I found that specific synaptic signals which leads to enhanced binding of Brd4 to histones to activate transcription of key neuronal genes that underlie memory formation. The loss of Brd4 function affects synaptic protein content, which results in memory deficits in mice and decreases seizure susceptibility. Thus, Brd4 provides a critical and previously uncharacterized link between neuronal activation and the transcriptional responses that occur during memory formation. This work has implications for the use of BET inhibitors in clinical settings and in possible treatments for epilepsy.

I also contributed to work examining additional mechanisms linking epigenetic regulation of transcription to behavioral responses to experience. This work from our collaborators in the lab of Dr. Eric Nestler (Ichan School of Medicine at Mount Sinai), examined the role of the ACF chromatin remodeling complex in depression. I investigated mechanisms controlling activity-dependent changes in expression of ACF. Together, these projects advanced our understanding of the link between neuronal signaling and epigenetic regulation underlying animal behavior both in normal conditions and in the context of mental health disorders. Finally, as an independent investigator, we published a review on the links between chromatin and plasticity mechanisms in the brain.

  • Korb, E., Herre, M., Zucker-Scharff, I., Darnell, RB., Allis, C.D. 2015. BET protein Brd4 activates transcription in neurons and BET inhibitor Jq1 blocks memory in mice. Nat. Neuro. (PMC4752120)
  • Herre, M., Korb, E. The chromatin landscape of neuronal plasticity. Curr. Opin. Neurobiol. (PMID: 31174107)
  • Sun, H., Damez-Werno, D.M., Scobie, K.M., Shao, N., Dias, C., Rabkin, J., Koo, J.W., Korb, E., Bagot, R.C., Ahn, F.H., Cahill, M., Labonte, B., Mouzon, E., Heller, E.A., Cates, H., Golden, S.A., Gleason, K., Russo, S.J., Andrews, S., Neve, R., Kennedy, P.J., Maze, I., Dietz, D.M., Allis, C.D., Turecki, G., Varga-Weisz, P., Tamminga, C., Shen, L., Nestler. E.J. 2015. ACF chromatin remodeling complex mediates stress-induced depressive-like behavior. Nat. Med. (PMC4598281)

Linking the synapse to the nucleus.

During my graduate school research, I sought to elucidate previously unexamined mechanisms regulating learning and memory. I focused on a protein that is critical for memory formation and synaptic plasticity, the activity-regulated cytoskeletal protein. Arc expression is robustly induced by activity, and Arc protein localizes both to active synapses and the nucleus. While its synaptic function had been examined in great detail, it was not clear why or how Arc is localized to the nucleus. I identified distinct regions of Arc that control its localization, including a nuclear localization signal, a nuclear retention domain, and a nuclear export signal. Arc localization to the nucleus regulates transcription, PML nuclear bodies, synaptic strength, and homeostatic plasticity. This was the first demonstration that Arc was important in regulating transcription and one of the first indications that PML bodies play an important role in neurons.

Our lab has undertaken projects examining other pathways that link external signals to responses within the nucleus. As part of a collaboration with Dr. Steven Josefowicz at Weill Cornell Medical School, we examined a previously unexplored histone modification, histone H3.3 serine 31 phosphorylation (H3.3S31ph). We demonstrated that in neurons H3.3S31ph is rapidly and robustly induced in neurons in response to synaptic stimulation. This was critical in expanding finding to additional cell types and systems beyond an immune cell response and was recently published in Nature.

  • Korb, E., Wilkinson, C. L., Delgado, R.N., Lovero, K.L., Finkbeiner, S. 2013. Arc in the nucleus regulates PML-dependent GluA1 transcription and homeostatic plasticity. Nat. Neuro. 16(7), 874-83. (PMC3703835)
  • Korb, E., Finkbeiner, S. 2011. Arc in synaptic plasticity: from gene to behavior. Trends Neurosci. 34, 591-8. (PMC3207967)
  • Korb, E., Finkbeiner, S. 2013. PML in the Brain: From Development to Degeneration. Frontiers in Molecular and Cellular Oncology. 17, 242. (PMC3775456)
  • Armache, A., Yang, S., Martinez de Paz, A., Robbins, L.E., Durmaz, C., Yeong, J.Q., Ravishankar, A., Daman, A.W., Ahimovic, D.J., Klevorn, T., Yue, Y., Arslan, T., Lin, S., Panchenko, T., Hrit, J., Wang, M., Thudium, S., Garcia, B.A., Korb, E., Armache, K., Rothbart, S.B., Hake, S.B., Allis, C.D., Li H., Josefowicz, S.Z. 2020. Histone H3.3 phosphorylation amplifies stimulation-induced transcription. Nature. 583(7818), 852-857. (PMC75175895)

Research Interest

The Korb lab works at the intersection of neuroscience and epigenetics. Epigenetic regulation is extremely important in neuronal function and contributes to the creation of new memories, our ability to adapt to our environment, and numerous neurological disorders. We try to understand how the world around us can influence gene expression in our neurons to allow us to learn, adapt, and become the people we are today.
In the lab, we focus on chromatin and its role in neuronal function. Chromatin is the complex of DNA and proteins called histones, which package our DNA into complex structures and control access to our genes. To study the role of histones in neuronal function and in disorders such as autism, we combine methods such as microscopy, bioinformatics, biochemistry, behavioral testing, and more. We have multiple areas of research in the lab, all focused on the study of chromatin and how it regulates neuronal function and neurodevelopmental disorders.

Kavitha Sarma, Ph.D.

Contributions to Science

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.

Elizabeth Heller, Ph.D.

Contribution to Science

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

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

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

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

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

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

Research Interest

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

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.

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.

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