Posts classified under: University Of Pennsylvania

Penn Epigenetics > Program Members > University Of Pennsylvania
Eric-Joyce

Eric F. Joyce, Ph.D.

Contribution to Science

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

Research Interest

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

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

Contribution to Science

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

• Discovery of Nuclear Receptor Corepressor Complexes.

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

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

• Identification and characterization of PPAR g in Adipose Biology.

Research Interest

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

Mia Levine, PhD

Mia Levine, Ph.D.

Contribution to Science

The evolution of young genes via de novo- and duplication- based mechanisms
Evolutionary mechanisms of innovation at the molecular level are numerous. Codons diverge, regulatory elements arise and degenerate, and new genes are born. These signatures of adaptive evolutionary change are frequently species-restricted. My PhD research identified very young genes that harbor no homology to exons in related genomes. In contrast to classic mechanisms of novel gene formation like gene duplication, these de novo genes arise instead from fortuitous sequence evolution at noncoding DNA. These genes exhibited testis-biased expression and signatures of adaptive evolution, implicating male germline processes as potent agents of selection of these rare mutations. This publication was the first to describe such de novo genes that have since been documented in a wide array of taxa, including humans. My postdoctoral research focused instead on gene duplication as a potent mechanism of adaptive diversification. A shared domain structure between parent and daughter proteins facilitated my goal to identify lineage-specific innovations in proteins that package DNA. Prior to my research, the Heterochromatin Protein 1 (HP1) gene family was thought to encode between 2 and 5 members across eukaryotes. I discovered 22 new HP1 members in Drosophila. These 22 paralogs were all born less than 20 million years ago. Nevertheless, the number of HP1 genes per species remains relatively constant. This revolving door of gene replacement implicates conserved, currently undefined chromatin functions encoded by unconserved components recurrently generated by gene duplication.

Levine, M.T., C. D. Jones, A. D. Kern, H. A. Lindfors, and Begun, D.J. (2006) Novel genes derived from noncoding DNA in Drosophila melanogaster are frequently X-linked and exhibit testis-biased expression.Proceedings of the National Academy of Sciences 103: 9935-9939. PMCID: PMC1502557.
Levine, M.T., McCoy, C. Vermaak. D., LeeY.C.G, Hiatt, M.A., Matsen, F.A., and H.S. Malik (2012) Phylogenomic analysis reveals dynamic evolutionary history of the Drosophila Heterochromatin Protein 1 (HP1) gene family.  PLOS Genetics8(6): e1002729. PMCID: PMC3380853.

Adaption to novel environments at DNA packaging proteins

melanogaster, like humans, evolved in Africa but more recently invaded the New World and established populations from tropical to temperate climates. These geographically structured populations are panmictic (randomly mating), so any genotypic or phenotypic differences observed are likely the product of fitness variation across environments, i.e., natural selection. This spatially-structured system therefore presents a unique opportunity to elucidate the molecular basis of adaptation. I have taken both whole genome- and single locus- approaches. In one report, genomic DNA fromD. melanogastertropical and temperate populations from both Australia and the US were hybridized to whole-genome tiling arrays from which we inferred geographic sequence divergence (allele frequency variation) based on geographically structured differences in probe intensities. Our analysis demonstrated that a remarkably large fraction of the D. melanogaster genome has been targeted by spatially-varying positive selection. Our whole-genome analysis also uncovered many previously unsuspected biological functions associated with adaptation to novel environments. One of the most intriguing of these functions was chromatin binding. In light of the extensive data on the environment sensitivity of chromatin dynamics, I was especially interested in the unexplored role that chromatin-remodeling factors play in adaptation to novel habitat. Under one model, chromatin-remodeling factors evolve to maintain chromatin structure that is perturbed by environmental fluctuations. I focused primarily on the Polycomb Group genechameau. I discovered a linear relationship between latitude and allele frequency at several SNPs in both the US and Australian populations, which represent independent colonization events. Moreover, an amino acid-changing SNP predicted variation in tolerance to freezing temperatures. These data strongly implicated the action of natural selection and introduced chromatin-remodeling factors as a potentially rich source of adaptive genetic variation. Inspired by the observation that chromatin-based gene regulation can span more than one promoter, I also tested the hypothesis that adaptive expression variation across latitudinal gradients spans physically linked genes. I found that gene “neighborhoods” (of up to 15 genes), rather than single genes, exhibit adaptive transcriptional profiles, consistent with the notion that chromatin factors regulate adaptive expression variation across space.
Turner, L.T., Levine, M.T., and Begun, D.J. (2008). Genomic analysis of adaptive differentiation in Drosophila melanogaster.Genetics 179: 475-485. PMCID: PMC2390623.
Levine, M.T. and Begun, D.J. (2008). Evidence of spatially varying selection at four chromatin-remodeling loci inDrosophila melanogaster. Genetics 179: 455-473. PMCID: PMC2390624.
Levine, M.T., Eckert, M., and D.J. Begun (2011) Whole genome expression plasticity across tropical and temperateDrosophila melanogaster populations from eastern Australia. Molecular  Biology and Evolution 28: 249–256. PMCID: PMC3002243.

Evolutionary and functional diversification of essential DNA packaging proteins
Conserved nuclear proteins support conserved nuclear processes. Yeast and humans, for example, share essential, homologous chromatin proteins that package eukaryotic DNA and support shared, essential functions like chromosome segregation and telomere integrity. These cellular processes, however, also rely on unconserved molecular machinery. A surprisingly large fraction of essential genes that encode chromatin proteins evolve rapidly. My dissertation documented early evidence of this paradoxical phenomenon. The Dosage Compensation Complex (DCC) is responsible for equalizing X-linked gene dosage via chromatin remodeling of the single male X chromosome.  Loss of function at DCC genes is lethal. I discovered population genetic evidence of positive selection at four of the five DCC complex components. Continuing this theme during my postdoctoral research, I uncovered the essential function of the Heterochromatin Protein 1 paralog, HP1E. HP1E is required for faithful segregation of paternal DNA during the first embryonic mitosis. Nevertheless, a subset of Drosophila species apparently persists without HP1E. I discovered that in D. melanogaster not all paternal chromosomes are equally vulnerable to chromatin bridging during the first embryonic mitosis—the heterochromatin-rich sex chromosomes are more likely to mis-segregate than the large autosomes. Intriguingly, over evolutionary time major rearrangements of these same sex chromosomes co-occur with the pseudogenization of HP1E in the obscura group of Drosophila. These data support a model under which karyotype evolution rendered dispensable a once-essential gene. My findings thus provided a neat hypothesis to resolve the apparent paradox of HP1E’s essentiality in D. melanogaster together with its loss in related species.

Levine, M.T., Holloway, A.K., Arshad, U., and Begun, D.J. (2007) Pervasive and largely lineage-specific adaptive protein evolution in the dosage compensation complex of Drosophila melanogaster.Genetics 177: 1959–1962. PMCID: PMC2147993.
Levine, M.T. and H.S. Malik (2013) A rapidly evolving genomic toolkit of Drosophila heterochromatin.Fly 7: 137-141. PMCID: PMC4049844.
Levine, M.T., Vander Wende, H., and H.S. Malik (2015) Mitotic fidelity requires transgenerational action of a testis-restricted HP1.eLife 4: e07378. PMCID: PMC4491702

Research Interest

Chromatin proteins package our genomic DNA. Essential, highly conserved cellular processes rely on this genome compartmentalization, yet many chromatin proteins are wildly unconserved over evolutionary time. We study the biological forces that drive chromatin protein evolution and the functional consequences for chromosome segregation, telomere integrity, and genome defense.

Ronen Marmorstein, Ph.D.

Contribution to Science

1. My laboratory has pioneered the structure-function analysis of histone acetyltransferases (HATs) and continues to make seminal contributions in this area. Specifically, my laboratory determined the first crystal structure of a type A HAT and characterized its mechanism of catalysis, and the first to describe the mode of histone substrate binding by a HAT. My laboratory has extended our studies to the broader family of N- acetyltransferases including the non-histone lysine acetyltransferases (KATs) and the N-amino acetyltransferases (NATs). We have uncovered important molecular signatures that distinguish HATs, KATs and NATs. My laboratory has also contributed to the development of acetyltransferase inhibitors. The vast majority of the human proteome is acetylated in a functionally important manner and alterations occur in human diseases. This suggests that protein acetylation may rival protein phosphorylation as a biologically important protein modification and that KATs and NATs represent important therapeutic targets.
a. Deng, S., McTiernan, N., Wei, X., Arnesen, T. and Marmorstein, R. Molecular basis for N-terminal acetylation by human NatE and its modulation by HYPK, (2020) Nature Comm., 11: 14584-14587. PMID32042062: PMCID: PMC7010799
b. Deng, S., Pan, B., Gottlieb, L. and Marmorstein, R. Molecular basis for N-terminal alpha-synuclein acetylation by human NatB. (2020) eLife. 9: e57491. PMID32885784
Deng, S., Gottlieb, L., Pan, B., Supplee, J., Wei, Xuepeng, Petersson, E.J. and Marmorstein, R., Molecular mechanism of N-terminal acetylation by the ternary NatC complex. (2021) Structure, S0969-2126. PMID34019809
Gottlieb, L., Guo, L., Shorter, J. and Marmorstein, R. N-alpha-acetylation of Huntingtin protein increases its propensity to aggregate. (2021) J. Biol. Chem., 31: 101363-. PMID34732320

2. My laboratory is studying the molecular basis for how chromatin is assembled and maintain by histone chaperone complexes. We have focused on the binding and histone deposition of H3/H4 complexes by the ASF1 and VPS75 proteins and the multi-subunit HIRA complex, which specifically deposits the histone H3 variant, H3.3, in a replication independent manner. Histone H3.3 is deposited at active genes, after DNA repair and in certain forms of heterochromatin in non-proliferating senescent cells, and recurrent H3.3 mutations are found in pediatric glioblastoma and dysregulation of H3.3-specific activities in tumor growth and leukemia exemplifies the necessity for proper regulation of H3.3-specific deposition pathways. Together with the Peter Adams laboratory we have pioneered a molecular understanding of the HIRA complex highlighting the particular importance of the HIRA and Ubn1 subunits of H3.3-specific activities.
a. Ricketts, M.D., Frederick, B., Hoff. H., Tang, Y., Schultz, D.C. Rai, T.S., Vizioli, M.G. Adams, P.D. and Marmorstein, R. Ubinuclein-1 confers histone H3.3-specific binding specificity by the HIRA histone chaperone complex. (2015) Nature Commun. 6:7711-. PMID: 26159857: PMCID: PMC4509171
b. Haigney, A., Ricketts, M. D. and Marmorstein, R. Dissecting the Molecular Roles of Histone Chaperones in Histone Acetylation by Type B Histone Acetyltransferases (HAT-B), (2015) J. Biol. Chem., 290:30648-30657. PMID: 26522166: PMCID: PMC4683284
c. Ray-Gallet, D., Ricketts, M.D., Sato, Y., Gupta, K., Boyarchuk, E., Senda, T., Marmorstein, R., and Almouzni, G. Functional activity of the H3.3 histone chaperone complex HIRA requires trimerization of the HIRA subunit. (2018) Nat. Commun. 9:3103. PMID:30082790: PMCID: PMC6078998
d. Ricketts, M.D., Dasgupta, N., Fan, J., Han, J., Gerace, M., Tang, Y., Black, B.E., Adams, P.D. and Marmorstein, R. The HIRA histone chaperone complex subunit UBN1 harbors H3/H4 and DNA binding activity. (2019) J. Biol. Chem., 294: 9239-9259. PMID:31040182; PMCID: PMC6556585

3. My laboratory has leveraged our expertise in biochemistry and X-ray crystallography with small molecule screening for structure-based Inhibitor development for therapy of melanoma and other cancers. There is a particular interest in melanoma and the laboratory had developed inhibitors to several important oncogenic kinases in melanoma including BRAF, PI3K, PAK1 and S6K1. The laboratory has also targeted the oncoproteins E7 and E6 from human papillomavirus (HPV), the causative agent of a number of epithelial cancers, and a significant portion of head and neck cancers. These studies have important implications for therapy.
a. Qin, J., Rajaratnam, R., Feng, L., Salami, J., Barber-Rotenberg, J.S., Domsic, J., Reyes-Uribe, P., Liu, H., Dang, W., Berger, S.L., Villanueva, J., Meggers, E. and Marmorstein, R. Development of organometallic S6K1 inhibitors. (2015) J. Med. Chem. 58:305-314. PMID: 25356520; PMCID: PMC4289024
b. Grasso, M., Estrada, M.A., Ventocilla, C., Samanta, M., Maksimoska, J., Villanueva, J., Winkler, J.D. and Marmorstein, R. Chemically linked vemurafenib inhibitors promote an inactive BRAFV600E conformation. (2016) ACS Chem. Biol. 11: 2876-2888. PMID: 27571413: PMCID: PMC5108658
c. Emtage, R.P., Schoeberger, M.J. Fergusion, K.M., and Marmorstein, R. Intramolecular autoinhibition of Checkpoint Kinase 1 is mediated by conserved basic motifs of the C-terminal Kinase Associated-1 domain. (2017) J. Biol. Chem. 292:19024-19033. PMID:28972186: PMCID: PMC5704483
d. Grasso, M., Estrada, M.A., Berrios, K.N., Winkler, J.D. and Marmorstein, R. N-(7-Cyano-6-(4-fluro-3-(2-(3-(trifluoromethyl)phenyl)acetamido)phenoxy)benzo[d]thiazol-2-yl)cyclopropanecarboxamide (TAK632) promotes inhibition of BRAF through the induction of inhibited dimers. (2018) J. Med. Chem. 61:5034-5046. PMID: 29727562: PMCID: PMC6540792

4. My laboratory has more recently studied the connection between metabolism with cancer signaling and chromatin regulation, with a particular focus on the acetyl-CoA metabolism and metabolite acylation enzymes such as ATP citrate lyase (ACLY). Our studies uncovered the molecular mechanism of ACLY and provided a molecular scaffold for the structure-based development of ACLY inhibitors for therapy of cancer and metabolic and cardiovascular disorders.
a. Bazilevsky, G.A., Affronti, H.C., Wei, X., Campbell, S.L., Wellen, K.E. and Marmorstein. R. ATP-citrate lyase multimerization is required for coenzyme-A substrate binding and catalysis, (2019) J. Biol. Chem. 294:7529-7268. PMID: 30877197; PMCID: PMC6509486
b. Wei, X., Schultz, K., Bazilevsky, G.A., Vogt, A. and Marmorstein R. Molecular basis of acetyl-CoA production by ATP-citrate lyase. (2020) Nature Structural & Molecular Biology 27:33-41. PMID: 31873304
Wei, X. and Marmorstein, R., Reply to: Acetyl-CoA is produced by the citrate synthetase homology module of ATP-citrate lyase. (2021) Nat. Struct. Mol. Biol., 28: 639-641. PMID34294921
Wei, X., Kixmoeller, K., Baltrusaitis, E., Yang, X. and Marmorstein, R. Allosteric role of a structural NADP+ molecule in glucose-6-phosphate dehydrogenase activity, (2022) Proc. Nat. Acad. Sci. USA, 119(29): e2119695119

Research Interest

The Marmorstein laboratory studies the molecular mechanisms of (1) epigenetic regulation (2) protein post- and co-translational modification with a particular focus on protein acetylation, and (3) enzyme signaling in cancer and metabolism. The laboratory uses a broad range of biochemical, biophysical and structural research tools (X-ray crystallography and cryo-EM) to determine macromolecular structure and mechanism of action. The laboratory also uses high-throughput small molecule screening and structure-based design strategies to develop protein-specific small-molecule probes to interrogate protein function and for preclinical studies.

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