Posts classified under: University Of Pennsylvania

Penn Epigenetics > Program Members > University Of Pennsylvania

Rajan Jain, M.D.

Contributions to Science

Our work is broadly characterized by three major themes.

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

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

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

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

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

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

Research Interest

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

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

Research Interest

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

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

Contribution to Science

Elucidating and exploiting the mechanism of natural product macrocycle biosynthesis to make improved antibiotics. Many natural product antibiotics are brought to their bioactive conformations by cyclization. Macrocyclization rigidifies the small molecules to make them more effective at binding to their molecular targets. In my graduate work, I examined the natural product assembly lines of non-ribosomal peptide synthetases. In this work, I helped to establish that, rather than simply being responsible for unloading of the synthetase modules, the C-terminal domain of these assembly lines can catalyze release and macrocyclization of the natural products ref (a). After this initial discovery, I showed that this mechanism is broad ranging across a wide range of natural products spanning anti-infective and anti-cancer agents. We leveraged these advances to develop a chemoenzymatic approach to access alternative classes of related natural products (ref (b)), target new therapeutic areas (ref (c)), or improve antibiotic activity or specificity (ref (d)). This work demonstrates my abilities to decipher complex enzymatic reaction mechanisms.

Trauger JW, Kohli RM, Mootz HD, Marahiel MA, Walsh CT (2000) Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase, Nature 407: 215-218.
Kohli RM, Burke MD, Tao J, Walsh CT (2003) Chemoenzymatic route to macrocyclic hybrid peptide/polyketide-like molecules, J Am Chem Soc 125: 7160-7161.
Kohli RM, Takagi J, Walsh CT (2002) The thioesterase domain from a nonribosomal peptide synthetase as a cyclization catalyst for integrin binding peptides, Proc Natl Acad Sci USA 99: 1247-1252 (PMC122175).
Kohli RM, Walsh CT, Burkart MD (2002) Biomimetic synthesis and optimization of cyclic peptide antibiotics, Nature 418: 658-661.

Targeting the SOS Pathway to combat antibiotic resistance. My clinical training has exposed me to the ever-increasing impact of antibiotic resistance and made it evident that “next generation” antibiotics only stall resistance. My involvement in making “next generation” antibiotics (see (1) above) made me realized the need to tackle the root cause, i.e., how antibiotic resistance arises. In this vein, we have focused on understanding how the DNA damage response of bacteria (also known as the SOS response) allows them to adapt to antibiotics or evolve and acquire resistance. Our detailed biochemical analyses (ref (a)) have allowed us to devise strategies for targeting the SOS response to potentiate antibiotics (ref (b)) and we have isolated first-in-class inhibitors of LexA (ref (c)). This work represents my lab’s effort to advance a new paradigm (ref (d)) in a field with great need for innovation.

Mo CY, Birdwell LD, Kohli RM (2014) Specificity Determinants for Autoproteolysis of LexA, a Key Regulator of Bacterial SOS Mutagenesis, Biochemistry 53:3158-68 (PMC4030785).
Mo CY, Manning SA, Roggiani M, Culyba MJ, Samuels AN, Sniegowski PD, Goulian M, Kohli RM (2016) Systematically Altering Bacterial SOS Activity under Stress Reveals Therapeutic Strategies for Potentiating Antibiotics, mSphere 1:e00163-16 (PMC4980697).
Mo CY, Culyba MJ, Selwood T, Kubiak JM, Hostetler ZM, Jurewicz AJ, Keller PM, Pope AJ, Quinn A, Schneck J, Widdowson KL, Kohli RM (2018) Inhibitors of LexA Autoproteolysis and the Bacterial SOS Response Discovered by an Academic-Industry Partnership, ACS Infect Dis 4:349-359 (PMC5893282).
Merrikh H, Kohli RM (2020) Targeting Evolution to Inhibit Antibiotic Resistance, FEBS J, 287: 4341-4352 (PMC7578009).

Explaining the basis for targeted mutagenesis by DNA deaminases. My clinical experiences have highlighted the importance of understanding how diversity arises on both sides of the host-pathogen interface. As a clear example of our ability to integrate nucleic acid chemistry with enzyme mechanisms, my lab has made great strides in understanding how targeted and purposeful mutation is used to improve our immune defenses. Activation Induced Deaminase (AID) is the key driver of antibody maturation, catalyzing the targeted deamination of cytosine to generate uracil within the immunoglobulin locus. We have helped to decipher how targeting takes place at the molecular level, demonstrating how particular hotspots in the genome are targeted by AID and its APOBEC3 relatives (refs (a) and (c)) and how the enzyme can discriminate DNA from RNA (ref (b)). Most recently, we have leveraged this knowledge to develop small molecule controllable genomic base editors. This work demonstrates my success in manipulating enzymes to reveal mechanism and alter function.

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).
Berríos KN, Evitt NH, DeWeerd RA, Ren D, Luo M, Barka A, Wang T, Bartman CR, Lan Y, Green AM, Shi J, Kohli RM (2021) Controllable genome editing with split-engineered base editors. Nat Chem Biol. 17:1262-1270.

Addressing the enigmatic mechanism 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 that TDG can deplete 5-formylcytosine from genomes (ref (a)). We have also demonstrated that TET2 shows catalytic processivity (ref (b)), providing a mechanism for the generation of highly oxidized mC species despite the relative dearth of their precursors, uncovered new reactivities for TET enzymes (ref (c)), and helped to reveal new and unexpected DNA modifications which may mediate demethylation. Finally, we contributed to work led by Dr. Fraietta showing the clinical importance of TET2 in CAR-T therapy (ref (d)). This work demonstrates my role in helping to shape to the field’s current model for how methylation, deamination and oxidation collaborate to establish the epigenome.

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).
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 (PMC4762542).
Ghanty U, Wang T, Kohli RM. (2020) Nucleobase Modifiers Identify TET Enzymes as Bifunctional DNA Dioxygenases Capable of Direct N-Demethylation, Angew Chem Int Ed, 59: 11312-11315 (PMC7332413).
Fraietta JA, Nobles CL, Sammons MA, Lundh S, Carty SA, Reich TJ, Cogdill AP, Morrissette JJD, DeNizio JE, Reddy S, Hwang Y, Gohil M, Kulikovskaya I, Nazimuddin F, Gupta M, Chen F, Everett JK, Alexander KA, Lin-Shiao E, Gee MH, Liu X, Young RM, Ambrose D, Wang Y, Xu J, Jordan MS, Marcucci KT, Levine BL, Garcia KC, Zhao Y, Kalos M, Porter DL, Kohli RM, Lacey SF, Berger SL, Bushman FD, June CH, Melenhorst JJ. (2018) Disruption of TET2 promotes the therapeutic efficacy of CD19-targeted T cells, Nature, 558: 307-312 (PMC 29849141).

Rational engineering of DNA modifying enzymes for epigenetic analysis and sequencing. Utilizing a structurally guided approach, we evolved the first TET variants that selectively stall oxidation at 5-hydroxymethylcytosine (hmC) (ref (a)). These modified enzymes offer a novel tool that is being applied to understand the role of 5-hydroxymethylcytosine, as distinct from the highly oxidized bases 5-formylcytosine and 5-carboxycytosine. In a similar vein, we have exploited our knowledge of APOBEC selectivity (ref (b)) to develop an enzymatic method to localize 5-hydroxymethylcytosine in genomic DNA (ref (c)). These advances demonstrate our success in harnessing structure-function insights into DNA modifying enzymes to apply them to reveal new biology.
Liu MY, Torabifard H, Crawford DJ, DeNizio JE, Cao XJ, Garcia BA, Cisneros GA, Kohli RM (2016) Mutations along a TET2 active site scaffold stall oxidation at 5-hydroxymethylcytosine. Nature Chem Biol, 3:181-187 (PMC5370579).
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. 45:7655-7665 (PMC5570014).
Schutsky EK, DeNizio JE, Hu P, Liu MY, Nabel CS, Fabyanic EB, Hwang Y, Bushman FD, Wu H, Kohli RM. (2018) APOBEC-Coupled Epigenetic Sequencing permits low-input, bisulfite-free localization of 5-hydroxymethylcytosine at base resolution. Nature Biotech. 36: 1083–1090 (PMC6453757).
Caldwell BA, Liu MY, Prasasya RD, Wang T, DeNizio JE, Leu NA, Amoh NYA, Krapp C, Lan Y, Shields EJ, Bonasio R, Lengner CJ, Kohli RM, Bartolomei MS. (2021) Functionally distinct roles for TET-oxidized 5-methylcytosine bases in somatic reprogramming to pluripotency. Mol Cell. 81: 859-869 (PMC7897302).

Research Interest

While we conventionally think of genomic DNA as a simple polymer of A‘s, C‘s, G‘s, and T‘s, the chemistry of the genome is in fact far more interesting.

Our laboratory focuses on the DNA modifying enzymes that provide an added layer of complexity to the genome. These enzymes can be involved in the purposeful introduction of mutations or in the chemical modification of nucleobases, making DNA a remarkably dynamic entity. Many of these processes are at the heart of the battle between the immune system and pathogens or are central to epigenetics.

Our work can be broadly classified in two areas:

Enzymatic deamination, oxidation and methylation of cytosine bases, with a focus on AID/APOBEC DNA deaminases and TET oxygenases

Targeting Pathogen Pathways that Promote Evolution and Antibiotic Resistance, with a focus on the LexA/RecA axis governing the bacterial SOS response.

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 bacteriology, immunology, and virology to study DNA modifying enzymes and pro-mutagenic pathways.

Our research program aims to understand diversity generating enzymes and pathways in vitro, to perturb their function in physiological settings, and to harness the biotechnological potential of these diversity-generating pathways.

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.

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.

1 … 4 5 6 7 8
Previous Next
Close
Test Caption
Test Description goes like this