Erica Korb, Ph.D

The role of chromatin in neurological disorders.
Disruption of epigenetic mechanisms can lead to a wide range of disorders. Neurons appear to be particularly sensitive to these changes and epigenetic misregulation contributes to many neurological disorders, from autism to chronic pain. We demonstrated that Fragile X syndrome (FXS), the most common genetic cause of intellectual disability and autism, results in part from changes to the epigenome. Furthermore, targeting the resulting transcriptional deficits can successfully reverse phenotypes in a mouse model of the disease. We also contributed to work on chronic pain and depression that explore how underlying mechanisms are linked to epigenetic disruption.
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. (PMID: 28823556)
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 horm neurons and the transition to persistent pain after peripheral nerve injury. Cell Rep.(PMID: 29847798)
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. (PMID: 26390241)
Epigenetic regulation in information storage in the brain
Epigenetic regulation of transcription in neurons is crucial for the mechanisms underlying memory formation and the response to an ever-changing environment. Such cellular responses occur in part through regulation of the chromatin landscape, such as through modifications to the histone proteins that regulate gene activation. However, the link between neuronal stimulation and the resulting changes in histone modifications that activate transcription in neurons is not fully understood. We worked on elucidating mechanisms of epigenetic regulation of transcription that link neuronal inputs to behavioral responses. These projects help advance our understanding of how the brain uses the epigenome to continually adapt to its environment throughout the lifetime of an animal.
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. (PMID: 26301327)
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. (PMID: 23749147)
Korb, E., Finkbeiner, S. 2011. Arc in synaptic plasticity: from gene to behavior. Trends Neurosci. 34, 591-8. (PMID: 21963089)
Korb, E., Finkbeiner, S. 2013. PML in the Brain: From Development to Degeneration. Frontiers in Molecular and Cellular Oncology. 17, 242. (PMID: 2406991)

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.

George Burslem, Ph.D.

Targeted Protein Degradation
Over the last 20 years, the Crews lab has pioneered the concept of targeted protein degradation as a powerful approach to understand and modulated biological systems. By co-opting the ubiquitin proteasome system (UPS), heterobifunctional small molecules can induce the degradation of target proteins within a cellular context. Proteolysis Targeting Chimera (PROTACs) contain two distinct chemical recruiting elements tethered by a linker; one for an E3 ligase and one for the target protein. We were able to demonstrate that this approach is capable of inducing the degradation of transmembrane receptor tyrosine kinases despite the fact that they are not usually UPS substrates. This enable us to develop compounds with enhanced activity against FLT-3 ITD as a potential therapeutic for acute myeloid leukaemia. Additionally, PROTACs represent a useful tool for the study of protein function which we demonstrated, in collaboration with the Druker lab, enabled the study of kinase independent roles of BCR-Abl in chronic myeloid leukaemia stem cells.

  • Burslem, G.M., and Crews, C.M. (2017). Small-Molecule Modulation of Protein Homeostasis. Chemical Reviews 117, 11269-11301.
  • Burslem, G.M., Schultz, A.R., Bondeson, D.P., Eide, C.A., Savage Stevens, S.L., Druker, B.J., and Crews, C.M. (2019). Targeting BCR-ABL1 in Chronic Myeloid Leukemia by PROTAC-mediated Targeted Protein Degradation. Cancer Research, 79, 4744-4753
  • Burslem, G.M., Smith, B.E., Lai, A.C., Jaime-Figueroa, S., McQuaid, D.C., Bondeson, D.P., Toure, M., Dong, H., Qian, Y., Wang, J., et al. (2018). The Advantages of Targeted Protein Degradation Over Inhibition: An RTK Case Study. Cell Chemical Biology 25, 67-77.
  • Bondeson, D.P., Smith, B.E., Burslem, G.M., Buhimschi, A.D., Hines, J., Jaime-Figueroa, S., Wang, J., Hamman, B.D., Ishchenko, A., and Crews, C.M. (2018). Lessons in PROTAC Design from Selective Degradation with a Promiscuous Warhead. Cell Chemical Biology 25, 78-87.
  • Burslem, G.M., Song, J., Chen, X., Hines, J., and Crews, C.M. (2018). Enhancing Antiproliferative Activity and Selectivity of a FLT-3 Inhibitor by Proteolysis Targeting Chimera Conversion. Journal of the American Chemical Society 140, 16428-16432.

Inhibition of the HIF-1α/p300 Protein-Protein Interaction
The transcription factor, hypoxia inducible factor 1α (HIF-1 α), plays a central role in the cellular response to hypoxia. It does so by forming a complex with the transcriptional co-activator and epigenetic regulator, p300. Since solid tumours rapidly deplete their oxygen supply and become hypoxic, inhibition of the HIF‐1α–p300 interaction represents an attractive approach for their treatment. Using proteomimetic, phage display and bionic protein approaches, we were able to identify several inhibitors of this crucial protein-protein interaction, including the first biophysically characterized small molecule inhibitor.

  • Burslem, G.M., Kyle, H.F., Nelson, A., Edwards, T.A., and Wilson, A.J. (2017). Hypoxia inducible factor (HIF) as a model for studying inhibition of protein-protein interactions. Chemical Science, 8, 4188-4202.
  • Burslem, G.M., Kyle, H.F., Breeze, A.L., Edwards, T.A., Nelson, A., Warriner, S.L., and Wilson, A.J. (2014). Small-Molecule Proteomimetic Inhibitors of the HIF-1α–p300 Protein–Protein Interaction. ChemBioChem, 15, 1083-1087.
  • Burslem, G.M., Kyle, H.F., Breeze, A.L., Edwards, T.A., Nelson, A., Warriner, S.L., and Wilson, A.J. (2016). Towards “bionic” proteins: replacement of continuous sequences from HIF-1α with proteomimetics to create functional p300 binding HIF-1α mimics. Chemical Communications, 52, 5421-5424.
  • Kyle, H.F., Wickson, K.F., Stott, J., Burslem, G.M., Breeze, A.L., Tiede, C., Tomlinson, D.C., Warriner, S.L., Nelson, A., Wilson, A.J., et al. (2015). Exploration of the HIF-1α/p300 interface using peptide and Adhiron phage display technologies. Molecular BioSystems, 11, 2738-2749.
  • Burslem, G.M., Kyle, H.F., Prabhakaran, P., Breeze, A.L., Edwards, T.A., Warriner, S.L., Nelson, A., and Wilson, A.J. (2016). Synthesis of highly functionalized oligobenzamide proteomimetic foldamers by late stage introduction of sensitive groups. Organic & Biomolecular Chemistry, 14, 3782-3786.

Research Interest

The Burslem lab is interested in developing chemical tools to understand and modulate lysine post-translational modifications, specifically acetylation and ubiquitination. The laboratory is particularly interested in novel pharmacological approaches to modulate post-translational modifications which regulate gene expression and protein stability.

Colin Conine, Ph.D.

Research Interest

The functions of small RNAs in fertility, inheritance, and development.

Liling Wan, Ph.D.

Molecular link between histone acetylation and oncogenic gene activation Histone acetylation is a chromatin mark generally associated with gene activation, yet the molecular mechanisms underlying this correlative relationship remain incompletely understood. I led a collaborative study in which we identified a novel ‘reader’ for histone acetylation named ENL. We showed in leukemia cells that ENL interacts with histone acetylation via the well-conserved YEATS domain, and in so doing, helps to recruit and stabilize its associated transcriptional machinery to drive transcription of leukemogenic genes. By determining the structure of ENL in complex with an acetylated histone peptide, we and our collaborators demonstrated that disrupting the reader function reduced chromatin recruitment of ENL-associated transcriptional machinery and resulted in suppression of oncogenic programs. Furthermore, blocking the functionality of ENL sensitized leukaemia cells to inhibitors that target another distinct class of histone acetylation readers, the BET proteins, thus highlighting the crosstalk between epigenetic readers and potential benefit of combinatorial therapies. Our work established ENL as a missing molecular link between histone acetylation and gene activation critical for leukemia malignant state, and has inspired following studies investigating other YEATS domain-containing proteins as a new class of chromatin ‘readers’ in a broad range of human cancers. In addition to bringing novel insights into our basic understanding of chromatin regulation, this work also provides mechanistic guidance and structural basis for ongoing drug development to target chromatin reading activity of ENL in aggressive leukemias.

Wan L#, Wen H#, Li Y#, Lyu J, Xi Y, Hoshii T, Joseph JK, Wang X, Loh YE, Erb MA, Souza AL, Bradner JE, Shen L, Li W, Li H*, Allis CD*, Armstrong SA*, Shi X*. ENL Links Histone Acetylation to Oncogenic Gene Activation in Leukemias. Nature 2017 Mar 9;543(7644):265-269. (#Equal contribution). PMC5372383
Li Y*, Sabari BR*, Panchenko T*, Wen H, Zhao D, Guan H, Wan L, Huang H, Tang Z, Zhao Y, Roeder RG, Shi X, Allis CD, Li H. Molecular Coupling of Histone Crotonylation and Active Transcription by AF9 YEATS Domain. Mol Cell2016 Apr 21;62(2):181-93. (*Equal contribution) PMC4841940

New type of gain-of-function mutations in chromatin readers Recognition of modified histones by ‘reader’ proteins constitutes a key mechanism mediating the function of histone modifications, yet the mechanisms by which their dysregulation contributes to diseases remain poorly understood. Recurrent, hotspot mutations in the acetylation-reading domain (YEATS domain) of ENL were recently found in Wilms’ tumor, the most common type of pediatric kidney cancer. Whether and how these mutations cause the disease remained unknown. Our current work shows that these mutations confer ENL gain of function in driving abnormal gene expression implicated in cancer. Unexpectedly, these mutations promoted ENL self-association, resulting in the formation of discrete nuclear puncta that are characteristic of biomolecular condensates, a newly recognized form of protein assembly that often involves weak, multivalent molecular interactions and commonly underlies the formation of membrane-less organelles. We demonstrated that such a property drives ‘self-reinforced’ chromatin targeting of mutant ENL protein and associated transcriptional machinery, thus enforcing active transcription from target loci. Aberrant gene control driven by ENL mutations, in turn, perturbs developmental programs and derails normal cell fate to a path towards tumorigenesis. This work is a remarkable demonstration of how mistakes in chromatin reader-mediated process can act as a driving force for tumor formation. These mutations represent a new class of oncogenic mutations which impair cell fate through promoting self-association and reinforcing chromatin targeting.

Wan L#, Chong S, Fan X, Liang A, Cui X, Gates L, Carroll TS, Li Y, Feng L, Chen G, Wang S, Ortiz MV, Daley S, Wang X, Xuan H, Kentsis A, Muir TW, Roeder RG, Li H, Li W, Tjian R, Wen H#, Allis CD#. Impaired Cell Fate through Gain-of-function Mutations in a Chromatin Reader. Nature 2019 in press (#co-corresponding)

Targeting chromatin readers as cancer therapies My postdoctoral work suggested that the displacement of histone acetylation reader ENL from chromatin may be a promising epigenetic therapy, alone or in combination with BET inhibitors, for aggressive leukemia. I have contributed to the development of peptidomimetic and small molecule inhibitors targeting the YEATS domain protein family. The ultimate goal of these and other ongoing efforts is to develop chemical probes targeting the ‘reading’ activity of ENL and other family members as valuable research tools and potential therapeutic agents.

Li X*, Li XM*, Jiang Y, Liu Z, Cui Y, Fung K, van der Beelen S, Tian G, Wan L, Shi X, Allis CD, Li H, Li Y#, Li X#. Structure-guided Development of YEATS Domain Inhibitors by Targeting π-π-π Stacking. Nat Chem Biol. 2018 Dec;14(12):1140-1149. (*Equal contribution) PMC6503841

Molecular mechanisms of cancer metastasis Metastasis accounts for > 90% cancer-related deaths and yet is the most poorly understood aspect of cancer biology. I have contributed to studies in which we identified and characterized new molecular mechanisms for cancer metastasis. My graduate work focused on Metadherin (MTDH), a novel cancer gene identified by our group to be prevalently amplified in breast cancer and strongly associated with a high risk of metastasis and poor prognosis. What drives the strong selection of MTDH in primary tumors was unclear. By generating genetically engineered mouse models, we provided first evidence supporting an essential role of MTDH in the initiation and metastasis of diverse subtypes of breast cancer. We further showed that MTDH regulates the expansion and activity of cancer stem cells through working with its binding partner SND1. By determining the atomic structure of the complex via collaboration, we demonstrated that disrupting the complex impairs breast cancer development and metastasis in vivo. Our work establishes MTDH and SND1 as critical regulators of cancer development and provides mechanistic guidance for ongoing drug development efforts to target this complex as cancer therapy. More broadly, this work provides crucial experimental support for the emerging concept that metastatic potential could be conferred by early oncogenic events that possess additional metastasis-promoting function and advances our understanding of the origin of metastatic traits.

Wan L, Lu X, Yuan S, Wei Y, Guo F, Shen M, Yuan M, Chakrabarti R, Hua Y, Smith HA, Blanco MA, Chekmareva M, Wu H, Bronson RT, Haffty BG, Xing Y, Kang Y. MTDH-SND1 Interaction Is Crucial for Expansion and Activity of Tumor-Initiating Cells in Diverse Oncogene- and Carcinogen-Induced Mammary Tumors. Cancer Cell 2014 Jul 14;26(1):92-105. PMC4101059
Wan L, Pantel K, Kang Y. Tumor Metastasis: Moving New Biological Insights into the Clinic. Nature Medicine 2013 Nov;19(11):1450-64. PMID: 24202397
Guo F#, Wan L#, Zheng A, Stanevich V, Wei Y, Satyshur KA, Shen M, Lee W, Kang Y, Xing Y. Structural Insights into the Tumor-Promoting Function of the MTDH-SND1 Complex. Cell Reports 2014 Sep 25;8(6):1704-13.  (#Equal contribution). PMC4309369
Wan L, Hu G, Wei Y, Yuan M, Bronson RT, Yang Q, Siddiqui J, Pienta KJ, Kang Y. Genetic Ablation of Metadherin Inhibits Autochthonous Prostate Cancer Progression and Metastasis. Cancer Research 2014 Sep 15;74(18):5336-47. PMC4167565
Kang Y, Xing Y, Wan L, Guo F. Use of peptides that block metadherin-SND1 interaction as treatment for cancer. (U.S. Patent No. 10,357,539 B2).

Research Interest

The research interests in our laboratory lie in the intersection of cancer biology and epigenetics. We focus on chromatin – the complex of DNA and histone proteins – and its regulatory network. Cancer genome studies revealed that at least 50% of human cancers harbor mutations in genes encoding chromatin-associated factors, suggesting widespread roles of chromatin misregulation in cancer. We strive to understand chromatin function and its dysregulation in human cancer, with a focus on addressing how chromatin-based mechanisms regulate cellular fate transition and plasticity that endow cancer cells with tumor-promoting potentials. We use a host of different approaches in genetics, epigenetics, biochemistry, genome-wide sequencing, bioinformatics and functional genomics to address these questions. We are also interested in leveraging our basic mechanistic discoveries for therapeutics development.

Rajan Jain, Ph.D.

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.

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

Director, Center of Excellence in Type 1 Diabetes
Associate Director, Penn Diabetes Research Center
Associate Director, Penn Center for Molecular Studies in Digestive and Liver Diseases
Director, Next Generation Sequencing Center

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.

Rahul Kohli, M.D., Ph.D.

Resolving the enigmatic mechanisms of DNA demethylation. Despite the wealth of studies on the importance of 5-methylcytosine (mC) in mammalian genomes, the mechanism by which DNA can be demethylated has remained elusive. While high profile studies had suggested that deamination of mC or related analogs could be involved in DNA demethylation, the biochemical feasibility of this reaction had not been established. Further, with the discovery of TET family enzymes that can oxidize mC, new avenues for demethylation have been recently proposed. We were the first to show that enzymatic deamination of oxidized analogs of mC is a disfavored route for demethylation and also the first to show that TDG results in specific depletion of 5-formylcytosine from genomes. We have also recently demonstrated that TET2 shows catalytic processivity, providing a mechanism for the generation of highly oxidized mC species despite the relative dearth of their precursors. We have also found key active site determinants that control step-wise oxidation and fund TET enzymes that stall at hmC and provide a means to dissociate the different activities of hmC, fC and caC. We continue to probe the mechanism of TET family enzymes, aiming to unravel the many permutations of modifications with five cytosine states (C, mC, hmC, fC, cac), on two opposite strand (CpG pairs) that can be generated by three different TET enzymes (TET1, TET2, TET3).

Nabel CS, Jia H, Ye Y, Shen L, Goldschmidt HL, Stivers JT, Zhang Y, Kohli RM (2012) AID/APOBEC deaminases disfavor modified cytosines implicated in DNA demethylation, Nature Chem Biol 8:751-8. (PMC3427411)
Kohli RM, Zhang Y (2013) TET enzymes, TDG and the Dynamics of DNA Demethylation, Nature 502: 472-9. [Peer-reviewed Review] (PMC4046508)
Crawford DJ, Liu MY, Nabel CS, Cao XJ, Garcia BA, Kohli RM. (2016) Tet2 catalyzes stepwise 5-methylcytosine oxidation by an iterative and de novo mechanism. J Am Chem Soc 138:730-3.
Liu MY, Torabifard H, Crawford DJ, DeNizio JE, Cao XJ, Garcia BA, Cisneros GA, Kohli RM (2017) Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine, Nature Chem Biol, 13:181-187.

Explaining how targeted mutagenesis drives antibody maturation and genomic modification. Our lab has made great strides in understanding how targeted and purposeful mutation is used to improve our immune defenses. The enzyme Activation Induced Deaminase (AID) is the key driver of antibody maturation, catalyzing the targeted deamination of cytosine to generate uracil within the immunoglobulin locus. This targeted mutation is the initiating step in somatic hypermutation and class switch recombination. We have helped to decipher how targeting takes place at the molecular level, demonstrating how particular hotspots in the genome are targeted, and how the enzyme can discriminate DNA from RNA. These cytosine deaminase enzymes have been proposed to play a role in DNA demethylation. Furthermore, they offer new and unexploited tools to understand cytosine modification states in the genome.

Kohli RM, Abrams SR, Gajula KS, Maul RW, Gearhart PJ, Stivers JT (2009) A portable hotspot recognition loop transfers sequence preferences from APOBEC family members to activation-induced cytidine deaminase, J Biol Chem 284: 22898-22904 (PMC2755697)
Nabel CS, Lee JW ,Wang LC Kohli RM (2013) Nucleic acid determinants for selective deamination of DNA over RNA by activation-induced deaminase, Proc Natl Acad Sci USA 110: 14225–14230 (PMC3761612)
Gajula KS, Huwe PJ, Mo CY, Crawford DJ, Stivers JT, Radhakrishnan R, Kohli RM (2014) High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase, Nucleic Acids Res 42: 9964-75 (PMC4150791)
Schutsky EK, Nabel CS, Davis AKF, DeNizio JE, Kohli RM (2017) APOBEC3A efficiently deaminates methylated, but not TET-oxidized, cytosine bases in DNA, Nucleic Acids Res. doi: 10.1093/nar/gkx345 (PMID 28472485).

Research Interest

In mammalian cells, DNA modifications are centered to the largest extent around cytosine bases, which are targeted by three different DNA modifying processes: methylation, oxidation and deamination. Research in the Kohli laboratory focused on the biochemistry and chemical biology of the enzymes that make cytosine such a dynamic base in the genome.
Cytosine methylation by DNA Methyltransferases (DNMTs) generates 5-methylcytosine (5mC), an epigenetic modification associated with silencing, while TET family enzymes can catalyze step-wise oxidation of 5mC to generate three new oxidized 5mC bases (ox-mCs) – 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). These bases that are critical intermediates in the cycle of DNA demethylation and can also potentially serve as independent epigenetic marks. Deamination of either cytosine or modified cytosine bases by AID/APOBEC family enzymes yields targeted transition mutations in the genome. ‘Purposeful’ mutation by AID/APOBECs is used to garble foreign genomes, is exploited by the immune system to mature antibody responses, and has been posited to play roles in DNA demethylation. Such activity also carries risks and, accordingly, the deamination signatures of AID/APOBECs have been prominently left on cancer genomes.
In the Kohli laboratory, we utilize a broad array of approaches, which include: 1) biochemical characterization of enzyme mechanisms, 2) chemical synthesis of enzyme probes, and 3) biological assays spanning epigenetics and immunology to study DNA modifying enzymes.

Matt Weitzman, Ph.D.

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

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

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

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

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

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

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

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

Research Interests

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

Kavitha Sarma, Ph.D.

Research Interest

The Sarma laboratory is interested in the mechanisms of epigenetic gene regulation, or how the dynamic modifications of the architecture of chromatin, the complex of DNA, RNA, and proteins within the nucleus of our cells, impacts gene expression and cellular function. The lab investigates consequences of epigenetic alterations in neuronal cancers and neurodegenerative diseases using a combination of biochemistry, cell and molecular biology, and functional genomics approaches to gain mechanistic insight into how chromatin architecture is modified in disease. Our goal is to identify new pathways and interactions that can be targeted to correct these epigenetic perturbations.

Montserrat Anguera, Ph.D.

Mechanisms of X-chromosome Inactivation: We are investigating the molecular mechanisms of X-Chromosome Inactivation, and how altered dosage of X-linked genes affects early embryonic development and contributes to sex-biased disease. We focus on the autoimmune disorder lupus, which has a strong female-bias, exhibits overexpression of X-linked immune-related genes, and involves lymphocytes. We study the epigenetic status of the inactive X in female lymphocytes from humans and mice, and have 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 explain the female bias in autoimmune disorders such as lupus. We also study the dynamic mechanisms of Xist RNA localization and heterochromatin mark recruitment to the inactive X following lymphocyte activation.

Jianle Wang, Camille Syrett, Marianne Kramer, Arindam Basu, Michael Atchison, and Montserrat C. Anguera. (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
Anguera, Montserrat 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, Montserrat C., and Lee, J.T. (2013). “X Chromosome Inactivation and Epigenetic Responses to Cellular Reprogramming.” Annu Rev Genomics Hum Genet. 14:85-110. PMID: 23662665.
Anguera, Montserrat C., Sun, B.K., Xu, N., and Lee, J.T. (2006). “X-Chromosome Kiss and Tell: How the Xs Go Their Separate Ways.” Cold Spring Harb Symp Quant Biol. 71:429-37.

Long noncoding RNAs during early human development: We are also investigating sex-specific differences during human placental development using in vitro model systems. We discovered a novel X-linked long noncoding RNA specifically expressed in human placental progenitor cells that regulates the innate immune response.

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. PMCID: PMC4907097.
Jianle Wang, Montserrat C. Anguera. “In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells”. J Vis Exp. 2017 Mar 16;(121). doi: 10.3791/55268. PMID: 28362386

Research Interest

The research in the Anguera laboratory focuses on maintenance of X-chromosome Inactivation in the immune system and in stem cells.  They also study epigenetic mechanisms involving long noncoding RNAs during early human development and placental progenitors.

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