Shelley L. Berger - US grants

DESCRIPTION (provided by applicant): DNA-based transactions in the eukaryotic nucleus, such as transcriptional activation and repression, replication, rearrangements, and repair, occur in the context of chromatin. Long commonly thought to be an inert packaging scaffold, it is increasingly evident that signaling to nucleosomal histones is a controlling feature of genomic processes. One key class of histone alterations is post-translational modification (PTM), largely of their amino and carboxyl termini. Detailed evidence initially emerged for acetylation (ac), however histones are also phosphorylated (ph), methylated (me), ubiquitylated (ub), and sumoylated (su). The scale and intricacy of regulation via histone PTMs (hPTM) is only beginning to be appreciated and there are more complex layers of regulation via hPTMs yet to be elucidated. One key question regarding histone hPTMs is whether there is a predictive relationship linking individual PTMs and specific genomic processes. hPTMs are centrally important to regulate binding of key effector proteins. Our hypothesis is that regulatory information and effector protein binding is signaled by combinatorial hPTM patterns overtime and in 3- dimensional space. We will investigate several classes of hPTM cross-talk involved in transcriptional regulation in S. cerevisiae. The first is a temporal sequence of H2B ubiquitylation followed by its deubiquitylation during gene activation. The second is an alternative pattern of repressive H2B sumoylation vs activating histone acetylation/ubiquitylation. The third is a combinatorial pattern consisting of S10pi/K14ac on H3 during transcriptional initiation. Our specific goals in the proposed research are (1) to investigate mechanisms underlying the contrasting roles of histone ubiquitylation in transcriptional activation, compared to histone sumoylation in repression. Binding proteins to H2Bub and H2Bsu will be identified, and examine how these PTMs dictate chromatin structure. (2) To determine mechanisms involved in Ubp8-dependent deubiquitylation of H2B during transcriptional activation. We will test a model from our previous results, proposing that H2Bub functions as a barrier to phosphorylation on RNA polymerase II, and that a SAGA related complex containing Ubp8 proteolyzes ubiquitin from H2B within the ORF to promote transcript elongation. (3) To examine how the combinatorial pattern H3S10pi/K14ac regulates transcriptional activation. We believe that histone phosphorylation is a nexus to both inhibit binding of negative corepressors of transcription and, within the S10pi/K14ac pattern, promote binding of positive coactivators. It is now also known that aberrant histone PTMs underlie human disease. Indeed, histone deacetylase inhibitors constitute a promising class of anticancer medicines, and are already utilized in humans. Hence, elucidation of mechanisms and physiological roles of complex hPTMs may significantly impact our understanding and ultimate treatment of disease.

Description (provided by applicant) During latency, HSV-1 replication is blocked at the level of viral Immediate Early (IE) and Early (E) gene expression. This restriction of replication may be caused by the differentiated state of neurons, in which cellular factors required for viral gene expression are down-regulated, or transcriptional repressors are present. In addition, chromatin structure is generally repressive to gene activity. There is evidence that, in the latent state, the HSV-1 genome may be generally silenced because of nucleosomal association. Our previous work has shown that reactivation signals induce certain novel cellular genes, which may in turn up-regulate HSV IE and E genes, culminating in reactivation of HSV-4 from latency. The long-term goal of the proposed research is to define the functional role of the identified cellular factors, and their interplay with viral factors and alteration of chromatin structure, in causing reactivation of HSV-1 from latency in neurons. Our general methods will be to evaluate the role of cellular factors in the induction of the HSV-1 gene activity during viral infection, and during establishment of viral latency and reactivation. Our specific methods will be, first, to use quantitative RNA and protein detection analyses, as well as histology studies, to examine the correlation between up- or down-regulation of cellular genes, and reactivation of HSV1 in mouse trigeminal ganglia. Second, we will examine the functional interaction of the candidate factors with the viral genome, to obtain direct evidence that these factors influence HSV-1. Third, we will test the effect of the identified genes in HSV-1 reactivation using two model reactivation systems, murine trigeminal ganglia (TG) and NGF-differentiated PC12/HepG2 cell co-cultivation, to determine whether the candidate genes have a causal role in reactivating the viral genome. We will also determine whether mouse null mutants in these cellular genes alter HSV-1 latency or reactivation. Finally, we will use multiple approaches to study possible chromatin changes that occur during reactivation. We will focus on a transcriptional co-activator, called Bcl-3, that is up-regulated more than 5-fold in neurons during re-activation of HSV-1. Bcl-3 interacts with a DNA binding protein NFKB, and a histone modifying factor Tip60. In addition, there exist putative NFKB binding sites in the IE gene, IPCO, expression from which is required for HSV-1 reactivation from latency. We hypothesize that the protein complex NFKB/Bcl-3/Tip6O binds to the ICPO gene promoter to alter its chromatin structure and to initiate RNA synthesis, and then ICPO protein initiates further HSV-1 gene expression that is critical for reactivation from latency.

DESCRIPTION (provided by applicant): Epigenetics is defined as heritable changes in genomic function and phenotype that do not involve alteration to DNA sequence. This higher level control of genome function is embodied in chromatin, a composite of nucleosomes (DNA and histones), as well as other non-histone proteins. Human disease is increasingly being linked to epigenetic and chromatin changes. The central hypothesis of this Program Project is that chromatin, as an inherently dynamic structure, is prone to age-associated degeneration, but that this degeneration is also countered by protective processes. This Program Project studies these age-associated chcomatin changes as they occur in the context of cell senescence, an irreversible proliferation arrest of damaged cells that contributes to tissue aging. Our studies from the first cycle of funding confirmed the previously suspected role for epigenetics as a critical determinant of aging and longevity. As a cross-disciplinary and highly collaborative team (46 manuscripts to date [published or submitted] in the 2008-2013 funding cycle, of which 19 are collaborative), we will continue to employ biochemistry, structural biology, cell biology, yeas genetics, and state-of-the-art epigenomic technologies in yeast and human cells to elucidate the role of epigenetics in aging and senescence. In particular, we will define degenerative and protective changes to chromatin, and the molecular mechanisms underlying them. The relevance of these studies for aging will be tested by reference to young and old human tissues and in mouse models, assessing phenotypes of aging. Moreover, based on our findings from the first cycle of funding, we have already initiated efforts to leverage our mechanistic insights into lead compounds for novel therapies to promote healthy aging. Our ultimate goal is to understand the balance of processes that culminate in age-associated chromatin dysfunction, so that we can devise strategies to manipulate the balance to promote healthy aging.

Dynamic epigenetic alteration is central to differentiation of mammalian sperm, however the nature of these changes largely remains unknown. We propose that sequentially altered patterns of histone posttranslational modifications underlies chromatin restructuring during spermatogenesis and in mature sperm. We previously used sporulation in budding yeast S. cerevisiae, as a tractable model for gametogenesis, to uncover dynamic histone modifications, and then examined these in mouse spermatogenesis. Our data indicate that mouse sperm development involves temporal sequences of histone modifications, including multiple novel modifications, which are analogous in timing to the yeast. This conservation of the pattern of histone modifications during gametogenesis from yeast to mammals, strongly indicates that epigenetic regulation is key to the normal process of chromatin restructuring during gametogenesis. As part of the U54 Center, we will investigate novel epigenetic regulatory pathways in normal and abnormal mammalian spermatogenesis, in the mouse model and in human samples. Our hypothesis is that chromatin modulation is a highly evolutionarily conserved process in gametogenesis, is a key regulatory feature of spermatogenesis, and is altered in abnormal sperm, including in human infertility. We will investigate histone modifications during normal and abnormal spermatogenesis in the mouse model in collaboration with Project IV, and will examine sperm from human samples to determine whether modifications are altered, in collaboration with Project I. Our specific aims are: (1) to investigate histone post-translational modifications during mouse spermatogenesis, (2) to determine whether histone post-translation modifications are altered in mouse models having deregulated poly(ADP-ribose) (PAR) metabolism, and (3) to compare normal and abnormal human sperm from clinical IVF samples to discover potential disruptions of histone posttranslational modifications. Collaborations within our proposed Center provide unique synergistic approaches and research materials to uncover novel epigenetic pathways in normal and abnormal spermatogenesis, including in clinical human male infertility.

DESCRIPTION (provided by applicant): Many fundamental cellular processes are affected by epigenetic modulation, and in recent years it has become evident that chromatin-based epigenetic mechanisms underlie important aspects of the aging process. However, despite the fact that age is a prominent risk factor in neurodegenerative disease (ND), there is remarkably little information on the role of epigenetic alterations in mechanisms of ND such as Alzheimer's disease (AD), Parkinson's dementia (PD), frontotemporal degeneration (FTLD) or amyotrophic lateral sclerosis (ALS). We believe that a detailed biological, mechanistic and molecular understanding of the epigenetic factors that are altered in human ND holds promise for an improved understanding of disease pathogenesis and for the development of novel therapeutic interventions. The goals of this Project are to: (1) investigate whether major epigenetic modifications (histone post-translational modifications) change in the context of different NDs using an extensive bank of human samples available from the Penn Center for Neurodegenerative Disease Research (CNDR), (2) use our model systems to discover new epigenetic modifications that underlie ND disease, and (3) test the relevance of novel changes seen in human ND using models of ND. The proposed studies of this multiple-PI and co-Investigator effort will leverage complementary and intersecting interests from our laboratories concerning epigenetics and aging (Berger, Bonini, Johnson), ND (Bonini, Torres and Trojanowski), and the generation and bioinformatic analysis of genome-wide data obtained by chromatin immunoprecipitation followed by second generation sequencing (Gregory, Wang, Berger). The application of our combined expertise to the analysis of the rich collection of CNDR ND brain samples will launch a major new effort in the field of epigenetics in ND. In the broader scientific and medical communities, this effort will promote discoveries of epigenetic mechanisms of ND to provide the foundation for new insights and novel clinical approaches to treat ND.

DESCRIPTION (provided by applicant): Dynamic epigenetic alteration is central to differentiation of mammalian sperm, however the nature of these changes remains largely unknown. We are using the process of sporulation in the budding yeast S. cerevisiae, as a tractable model for mammalian spermatogenesis, to uncover dynamic chromatin and epigenetic regulation of transcription, meiosis and chromatin compaction. Our previous observations indicate that there are dramatic temporal changes in chromatin during sporulation, including histone modifications and other alterations. Further, our results indicate that mouse sperm differentiation involves similar temporal sequences of histone modifications, which are analogous in timing to the yeast. This conservation of the pattern of histone modifications during gametogenesis from yeast to mammals, strongly indicates that epigenetic regulation is fundamental to the normal process of chromatin restructuring during gametogenesis. Our hypothesis is that chromatin modulation is a highly evolutionarily conserved process in gametogenesis, and thus is a key regulatory feature of both yeast sporulation and mammalian spermatogenesis. We will address chromatin mechanisms during gametogenesis, through the following specific aims: (1) investigate chromatin mechanisms that operate through histones H3 and H4, including novel post-translational modifications and other regulatory features, identified via mutational screening in the previous funding period, (2) complete screening for histone substitution mutations in histone H2A and H2B that decrease or increase sporulation, and unravel their mechanisms through post-translational modifications, and other regulatory mechanisms, (3) examine linker histone Hho1-mediated mechanisms involved in meiotic gene transcriptional repression and post-meiotic chromatin compaction. As an important part of our proposal, we will determine whether these novel chromatin alterations are conserved during mouse spermatogenesis. Overall, results from these studies will provide novel views of dynamic changes in chromatin structure and function.

DESCRIPTION (provided by applicant): p53 is the most commonly mutated gene in human cancer. The p53 protein is a stress- and DNA damage-responsive transcriptional activator that binds to DNA to activate cell protective pathways, including cell cycle arrest, DNA repair, and apoptosis. The majority of p53 mutations in cancer inactivate the tumor suppressor via disruption of p53 DNA binding and hence transcriptional activation activity. The p53 protein is subject to numerous regulatory post-translational modifications, and additionally, the p53 transcription factor binds to target genes to recruit chromatin and histone modifiers as a key mechanism in gene activation. Given p53's normal role in chromatin regulation, p53 mutations and alterations in oncogenesis likely impair epigenetic pathways targeted by p53. Further elucidation of interactions between p53 and chromatin will therefore illuminate cancerous disruptions. Our recent published and preliminary data explore key mechanisms in the function and regulation of p53 as a transcriptional activator and regulating chromatin, and we will clarify these pathways in the proposed studies. First, we find that certain well-known p53 gain-of-function substitution mutations bind to and up-regulate an epigenetic gene signature, which, in turn, modifies histones to activate downstream ras/rho growth pathways. We plan to critically evaluate the mechanism and importance of this epigenetic signature in human tumor-derived cell lines bearing p53 gain-of-function substitutions and with related in vivo mouse models. Second, teratocarcinoma tumors paradoxically express high levels of wild type inactive p53; we show p53 in these cancers is decorated with elevated levels of repressive lysine methylation. In the proposed research, we will determine whether the methylation is crucial to inhibition of wild type p53 in teratocarcinomas. Third, we find that, beyond well-known p53 binding to promoters of target genes, surprisingly, the majority of stress- inducible p53 binding sites are distal to genes, appearing to be at enhancers. In the proposed studies we will address the interplay between p53 and certain histone modifiers in establishing enhancer function and gene activation. Fourth, by way of a high throughput si/shRNA screen for epigenetic regulators of p53-mediated gene activation, we observe an association of p53 with factors that mediate large-scale chromatin architecture. We will investigate direct roles of cohesion and CTCF in regulating the p53 transcriptional response via regulation of specific chromatin looping and architecture. Results from these studies will elucidate epigenetic mechanisms regulating p53 function, will explore involvement of these epigenetic pathways in p53 function in vivo and in cancer, and will launch investigation of tailored epigenetic therapies to ameliorate specific p53-driven cancer phenotypes.

ABSTRACT Ants exhibit highly evolved eusocial behaviors including stark division of labor among female castes, where the queen carries out all reproduction and worker castes forage for food and defend the colony. Interestingly, and of great relevance to aging research, the sterile workers are short-lived, while the reproductive queens are long-lived, with lifespans differing three to ten-fold between queen and worker. Remarkably, the genomes of these sterile and reproductive castes are nearly identical, and thus differences in lifespan (LS) and behavior likely result from epigenetic regulation. Furthermore, in the species Harpegnathos saltator, loss or removal of the queen leads to altered behavior in the workers, with antennal dueling and eventual ascendance of typically one or two workers into reproductive ?gamergate?, or pseudo-queen. From a longevity perspective, the gamergate exhibits longer LS and thus it appears that both behavior and lifespan are epigenetically determined during this switch. In addition, older workers reprogram much less efficiently into reproductive gamergate status. Our overall premise is that epigenetic regulation is at the heart of this caste-differentiated life span disparity, and that once we understand the basis of the epigenetic regulation, we can manipulate lifespan with epigenetic therapeutics and genetics in this relatively simple but socially complex organism. These results will provide fundamental knowledge that can be investigated in more sophisticated mammals. We propose to utilize H. saltator ants to investigate the epigenetic and physiological basis of the dramatic LS differences between reproductive and worker castes. We will carry out transcriptomic, proteomic, and epigenomic profiling of workers and queens of the same chronological age, and of young and old queens, to explore the basis of the plasticity in lifespan. We hypothesize that both known and novel mechanisms are lengthening LS in queens, which show such dramatic difference from worker LS. In addition, we will uncover the basis of the inefficient reprogramming of older workers into reproductive gamergates. Our recent published evidence (Science, 2016) supports the view that behavioral plasticity in ants is enhanced by epigenetic mechanisms during young adulthood, and that this plasticity is lost with age; however, the molecular mechanisms underlying this phenomenon remain unknown. Our preliminary data regarding chromatin marking show that regulatory loci near to active genes in gamergate queens bear activating histone H3K27 acetylation and these same loci in worker are marked with repressive H3K27 methylation. Intriguingly, these repressed loci in worker ants appear to be ?poised? for activation with H4K16 acetylation. We hypothesize that in young workers key loci are epigenetically poised to become activated and this poising becomes degraded as workers age, leading to inefficient reprogramming to reproductive status. In the proposed research we will test this proposal using epigenetic therapeutics and genetics. The ant model system provides an exceptional opportunity to integrate social behavior with aging, and to uncover key epigenetic processes underlying universal aging pathways.

The Administrative Core A of this Program Project is designed to provide scientific leadership and mentoring to trainees, effective communication, and an administrative support structure to ensure the coordination of all Program Project activities. The essential services provided by the Core include: administrative support for all project and core leaders; fiscal management and oversight for all components of the Program Project; and organization and communication of all Program Project meetings and activities. The overall goal of this Core is to provide effective and efficient leadership of the P01. The roles of the Core Leaders and administrative staff are to facilitate communication while stimulating scientific and technological interactions between the Projects, Cores and the associated staff and trainees. In order to achieve this goal, the Core has established three Aims: Aim 1: Establish and maintain an administrative structure to provide support for and management of all Program Project activities. Leadership will be provided by the Program PI (Dr. Berger) and Co-PI (Dr. Marmorstein) with input from the Executive Committee (Drs. Berger, Marmorstein, Adams, Zhang, Noma, Garcia, Lan and Schultz). Oversight of the program will also be provided by face-to-face Internal and External Scientific Advisory Board meetings on years 2 and 4 of the grant cycle. An Administrative Assistant (Sophia Castro, 50 % effort) will aid in communication among members of the projects, communication with Internal and External Scientific Advisory Boards and in coordinating fiscal management and timely grant reporting. Aim 2: Foster an environment to maximize collaborative research among Program Project investigators and between other P01 and NIA initiatives. This will be facilitated by Program Project Meetings every third week involving the Program Leaders, their trainees working on the project, and Core staff, with one group or Core presenting per meeting on a rotating basis. We will also have Program Project Retreats on years 1 and 3 of the grant cycle involving the Program Leaders and members of their respective groups and Cores to present talks and posters and interact at an off-site location. The PI will also advocate for space, personnel and resources for the Program Project and communicate with the NIA. Aim 3. Ensure compliance with all institutional, governmental and NIA regulations and policies. The PI will ensure compliance with animal research and ensure that the outlined Data Sharing Plan is followed.

Studies in animal models reveal that genetic differences and somatic mutations underlie longevity, but that non-genetic contributions also play a major role. Numerous observations, including our observations, suggest that epigenetic alterations occur as eukaryotes age. However, key questions remain, in particular, what are driving mechanisms and genomic changes that underlie the cellular phenotypes that characterize senescence and aging? Our hypothesis is that healthy aging involves homeostasis of the epigenomic landscape, which we refer to as chromostasis, and that chromostasis fails during aging, leading to tissue deterioration and to organismal death. Hence, genetic methods and pharmaco-therapeutics to enhance chromostasis are a prominent feature across all collaborative projects of this P01 application and in this Project 2. During the previous funding period we showed a key functional role of chromatin alterations in yeast replicative aging and massive chromatin alterations in mammalian senescence. In the next funding period we will explore and elucidate new chromatin regulatory pathways that alter genomic function in senescence and aging, leading to loss of chromostasis. In preliminary studies, we newly identified chromatin regulators whose reduction extends replicative lifespan, leading to new pathways that maintain epigenome and transcriptome fidelity during aging. We also discovered that nuclear disruption and shedding of LADs/chromatin into the cytoplasm during senescence and aging is perceived by a canonical cytoplasmic DNA sensing pathway, cGAS-STING, which in turn triggers cellular immunity pathways and the SASP (the senescence associated secretory phenotype) leading eventually to tissue damage during aging. To uncover the mechanisms and physiological importance of these new chromatin regulators and pathways in aging, we will carry out the following aims: 1. Investigate gene-internal cryptic transcriptional initiation during aging. We hypothesize that gene-internal transcriptional activation sites disrupt normal initiation at key longevity genes and lead to a global loss of transcriptional fidelity, contributing to reduction of chromostasis. (2) Investigate aging-associated upregulation of histone acetylation creating new enhancers. We hypothesize that dysregulated chromostasis licenses new enhancers during aging, leading to increased transcription of anti-longevity genes. (3) Investigate loss of chromatin integrity during aging triggering inflammation and autophagy of longevity chromatin regulators. Our preliminary findings show that LADs/chromatin in the cytoplasm triggers aging-promoting cellular immunity pathways via cGAS-STING. In the proposed studies, we will unravel the cGAS-STING pathway in promoting the SASP program in cellular senescence and the chronic inflammation associated with natural aging. This research will yield novel epigenetic mechanisms altering longevity, with potential for new therapeutic targets for intervention in age-related diseases and to extend healthy lifespan.

Abstract Understanding the molecular machinery underlying learning is critical to improve therapies for memory-related disorders that continue to burden our society. We recently identified a connection between cellular metabolism, epigenetic regulation, and memory-related neuronal plasticity. We found that ACSS2, a metabolic enzyme that generates acetyl-CoA is chromatin-bound in hippocampal neurons and required for long-term spatial memory, a cognitive process that relies on histone acetylation and gene expression. While these results established a strong functional link between nuclear acetyl-CoA generation by ACSS2, histone acetylation, transcription and memory, the exact molecular underpinnings of this metabolic- epigenetic axis remain to be elucidated. Here we propose to explore this phenomenon in further mechanistic detail. In particular, we aim to identify ACSS2-associated proteins, examine the mechanism of ACSS2 recruitment to specific genes, and identify higher-order structures that contribute to ACSS2-mediated transcriptional regulation via chromatin looping. Moreover, we will explore dorsal hippocampal transcriptional and epigenetic changes that accompany memory formation in a hippocampus-dependent mammalian learning model (spatial object recognition). We will assess genome-wide changes in transcript abundance and chromatin accessibility, study the enrichment of histone post-translational modifications, and the re-distribution of ACSS2 and select histone acetyl marks. Finally, using an array of pharmacological and genetic tools, we will assess the contribution of ACSS2 to the observed transcriptional, epigenetic and behavioral phenotypes. In addition, our preliminary data under Aim 3 indicate that ethanol-derived acetyl-groups are rapidly incorporated into neuronal chromatin in an ACSS2-dependent manner. This remarkably rapid epigenetic response might underlie alcohol-induced transcriptional and behavioral maladaptations in heavy drinkers. Indeed, we found that treating primary hippocampal neurons with acetate (the alcohol-derived metabolite and direct substrate of ACSS2) upregulates learning and memory-related genes and that ACSS2 reduction eliminates alcohol-related associative learning in conditioned place preference. We will explore hippocampal transcriptional and epigenetic changes associated with alcohol exposure in mice in vivo and assess the contribution of ACSS2 to molecular and cellular alterations induced by alcohol. Further, we will assess the effect of small molecule ACSS2 inhibitors on alcohol-related behaviors, as a basis for future therapeutic interventions. Overall, this work will significantly advance the field by characterizing the metabolic-epigenetic axis in the context of learning neurobiology. Furthermore, we expect our studies to identify efficacious novel therapeutic avenues for memory impairments and associated neurological and psychiatric conditions.