Alexander Meissner - US grants

DESCRIPTION (provided by applicant): The Reference Epigenome Mapping Centers (REMC) will aim to transform our understanding of human epigenetics through production and integrative analysis of comprehensive reference epigenomes for ES cells, differentiated cells and tissues. In pursuit of this goal, we have assembled a unique scientific team and infrastructure with broad expertise and capabilities in stem cell biology, epigenomics, technology, production research and computation. We recently demonstrated two complementary methods that leverage ultra high-throughput sequencing for epigenomic analysis. In the first method, genome-wide chromatin maps are acquired by deep sequencing chromatin IP DNA (ChlP-Seq). In the second, nucleotide-resolution DNA methylation maps are generated by high-throughput bisulfite-sequencing (HTBS). These methods represent major improvements over prior tools as they yield precise digital information, have high genome coverage, require fewer cells and are cost-effective. Multiple epigenomic maps have already been produced for stem cells and primary tissues, and pipelines have been assembled for efficient data collection, processing and analysis. For the REMC project, we propose to apply ChlP-Seq and HTBS pipelines to generate comprehensive high-resolution maps of chromatin state and DNA methylation for 100 diverse cell types. Cell types were selected for their biological and medical importance, and for their potential to maximize the comprehensiveness of acquired epigenomic data. They include human ES cells, ES-derived cells, mesenchymal stem cells, reprogrammed stem cells and primary tissues. ChlP-Seq will be used to map highly informative chromatin modifications and related chromatin proteins in each cell type. HTBS will be used to generate nucleotide-resolution DNA methylation maps. Reference epigenomes will reveal the locations and activation states of diverse functional genomic elements, inform on the developmental state and potential of studied cell populations, and provide a framework for understanding complex epigenetic regulatory mechanisms. All data will be made available to the scientific community upon verification. PUBLIC HEALTH RELEVANCE: Comprehensive characterization of epigenetic marks ('the epigenome') is a critical step towards a global understanding of the human genome in health and disease. The proposed mapping studies will provide unprecedented views of the human epigenetic landscape and its variation across cell states, offer fundamental insight into the functions and interrelationships of epigenetic marks, and provide a framework for future studies of normal and diseased epigenomes.

DESCRIPTION (provided by applicant): The grant being proposed "Mechanisms behind long-term and transgenerational effects of adolescent behavior" will address the Challenge Area (15) Translational Science, and the Specific Challenge Topic 15-AA-101* Determining If and How Adolescent Behaviors Affect Connections in the Developing Brain. A large body of both human and animal data support the idea that adolescent behaviors can have life-long effects on an individual. This sensitivity may derive, in part, from the dramatic developmental changes in brain function that are taking place at this time to generate skills for independence. Compounding this phenomenon is the adolescent-associated increase in risk-taking that predisposes them to initiate the use of addictive drugs. In addition, brain plasticity during adolescence may also contribute to heightened sensitivity to the long-term negative consequences of stress. Even more dramatic are new studies demonstrating that certain qualities acquired during youth in response to their environment may be passed on to future offspring, potentially magnifying the consequences of negative (or positive) adolescent behaviors. Many studies have begun to reveal how negative (and positive) experiences during adolescence, such as the ill-effects on adolescent exposure to nicotine or social stress, or the positive effects of an enriched environment, have long-term effects on life-long health and well being. However, they have focused on gene products involved in particular biochemical pathways and physiological processes of interest to individual investigators. In most cases molecular mechanisms underlying these effects have not been revealed, preventing rigorous test of whether they are physiologically relevant to long-lasting vulnerability to health problems that occurs in response to these specific adolescent behaviors. Thus, in this proposal, which is a joint effort by the Feig lab at Tufts University School of Medicine and the Meissner Lab at Harvard University and the Broad Institute, we will perform unbiased genome-wide screens to detect all genes in appropriate regions of the brain that display experience-induced, long-term changes in DNA methylation and/or histone modification that could that could account for long-term negative or positive consequences on health. Moreover, we will investigate whether any epigenetic-induced effects of these adolescent experiences are transmitted to the next generation It is anticipated that by identifying new long-lasting and/or transgenerational effectors of specific "negative" and "positive" adolescent behaviors, this study will generate new hypotheses that begin to answer the question proposed in this Challenge Area of how adolescent behavior "rewire(s) the developing brain thereby creating vulnerability for a number of persistent health problems including mental health disorders.....and addiction." This study will also suggest epigenetic mechanisms by which long-term and/or transgenerational changes in the expression of these effectors are regulated. As such, it will suggest strategies to specifically block their response to adolescent behaviors in mice to test their role in how "behaviors exhibited during this period can influence lifetime (and/or transgenerational) health and well-being. PUBLIC HEALTH RELEVANCE: Although a person's genetic blueprint strongly contributes to his/her susceptibility to disease, it is clear that the environment in which one lives influences how that blueprint is read. This appears to be particularly important during adolescent years where substantial evidence implies that some experiences during adolescence, such as exposure to nicotine or social stress have negative consequences on life-long health and well being. Our new data even suggest that these adolescent behaviors may influence future offspring when they are adolescents. This proposal will take advantage of state of the art tools to identify how these adolescent experiences have their effect through long-term structural modifications of genes that changes the way they are read. It is anticipated that this information will generate new hypotheses for how to reverse the long-lasting consequences of poor adolescent behaviors.

The purpose of Core A is to coordinate the administrative function of the Program Project and to foster a highly communicative, collaborative, and enthusiastic atmosphere to promote the science proposed in this application. Dr, Hanein, the PI of the core, will be responsible for the overall administration and supervision of this Program Project. Dr. Hanein will be assisted by an administrative assistant, Ms. Debbie Signer, provided by SBIMR, who will serve as the Administrator for the Program Project. Core A will serve as the interface of contact between the institutions (SBIMR, UCSD, UVA and Harvard), the 7 investigators, and all other personnel involved, as well as with the NIGMS. The six ether project leaders and co-investigators will receive funding via subcontracts issued by SBMRI, and each project leader and co-lnvestigator will be responsible for the day to day administration of the funds and the scientific oversight of the research. All Pis are highly motivated, successful scientists, so there is no anticipated need for a high level ef supervision. However, to track progress and, more importantly, to share new results and provide advice, all Pis will submit brief progress reports at monthly conference calls. Based on these reports and discussions, if there is any problem with productivity or management issues. Dr. Hanein, in consultation with the steering committee will work closely with any lab experiencing problems to put things back on the right track. Again, given the scientific experience and productivity of the individuals, this is highly unlikely. Core A will provide all coordination of collaborative meetings, organize regular meetings and reviews of the projects and cores (at least once a year), maintain contact with the Scientific Advisory Beard (SAB), organize the annual scientific retreat with the SAB and facilitate training of junior scientists. In addition to these responsibilities, the administrative core will oversee budget management for the Program Project, assist in the preparation of publications, and maintain the Program Project's website.

Abstract: We have proposed a comprehensive program project that will continue to address fundamental questions regarding the establishment and maintenance of the pluripotent state. Our four project components bring diverse expertise ranging from epigenetics, noncoding DNA, noncoding RNA and computational biology to understand the molecular circuits underlying reprogramming and pluripotent cells. There are however always many possible extensions of this program to further enhance its impact on regenerative biology and human health in general. To allow a continual expansion into these unknown or newly emerging areas we propose an administrative core with the objective to use a well-established pilot grant mechanism to attract new talent to the field and also respond rapidly to new trends by providing seed funds. We further integrate these pilot programs within and across our science through frequent interactions. Finally we aim to bring together scientists around our efforts with intra-lab retreats, local meetings, workshops and our international symposium. Given the interactions with the pilot recipients (including already several joint publications) we feel they are a significant enrichment of the program as a whole. The benefit is mutual and the interactions with the program PIs as well as the financial support is clearly helping the efforts of the pilot PIs, which are generally new to this field. Our inter labmeetings, retreats and also the main Symposium are effective ways the integrate all of the involved players and efforts into a high impact overall research program. The use of the administrative core in the ongoing project has been highly successful on all levels and we essentially propose to continue the program in the same structure in the current proposal. In summary, we are thrilled about the opportunity to have the administrative component and will continue to use it to the maximal benefit of the stem cell and larger scientific community.

The developmental biology and biomedical communities have had a long-standing interest in understanding how cells with identical genomes establish and maintain remarkably different behaviors and gene expression patterns. Attempts to define specific factors that are sufficient for reprogramming one cell type into another have identified single, or limited combinations of, transcription factors. For example, introduction of the ES cell- expressed transcription factors Oct4/Sox2/Klf4/c-Myc are sufficient to reprogram fibroblasts to a pluripotent state. Ectopic expression of transcription factors is likely to be a general and practical method for reprogramming between arbitrary cellular states. However, we also know from our preliminary studies that the factors themselves are unable to induce the full conversion and we find a great deal of heterogeneity and variability once the pluripotent state is established. Here we aim to dramatically advance our general understanding of reprogramming and pluripotency itself. To accomplish this we will first take advantage of the directionality of the reprogramming process and determine the cell context dependent remodeling capacity (aim 1), and then generate multidimensional data that describe the pluripotent population (aim 2), and finally built tools that enable us to further characterize subpopulations present within pluripotent cell cultures (aim 3). The proposed study presents an opportunity to dramatically alter and accelerate the utility of cellular reprogramming for basic, translational and clinical applications. RELEVANCE (See instructions): The proposed research effort aims to provide a detailed mechanistic understanding of the key protein factors that influence the cell state transitions (somatic to pluripotent) as well as the subsequent maintenance of the pluripotent state. A better understanding of the establishment and maintenance of pluripotency could transform biomedical research and health care delivery.

The developmental biology and biomedical communities have had a long-standing interest in understanding how cells with identical genomes establish and maintain remarkably different behaviors and gene expression patterns. Attempts to define specific factors that are sufficient for reprogramming one cell type into another have identified single, or limited combinations of, transcription factors. For example, introduction of the ES cell- expressed transcription factors Oct4/Sox2/Klf4/c-Myc are sufficient to reprogram fibroblasts to a pluripotent state. Ectopic expression of transcription factors is likely to be a general and practical method for reprogramming between arbitrary cellular states. However, we also know from our preliminary studies that the factors themselves are unable to induce the full conversion and we find a great deal of heterogeneity and variability once the pluripotent state is established. Here we aim to dramatically advance our general understanding of reprogramming and pluripotency itself. To accomplish this we will first take advantage of the directionality of the reprogramming process and determine the cell context dependent remodeling capacity (aim 1), and then generate multidimensional data that describe the pluripotent population (aim 2), and finally built tools that enable us to further characterize subpopulations present within pluripotent cell cultures (aim 3). The proposed study presents an opportunity to dramatically alter and accelerate the utility of cellular reprogramming for basic, translational and clinical applications. RELEVANCE (See instructions): The proposed research effort aims to provide a detailed mechanistic understanding of the key protein factors that influence the cell state transitions (somatic to pluripotent) as well as the subsequent maintenance of the pluripotent state. A better understanding of the establishment and maintenance of pluripotency could transform biomedical research and health care delivery.

DESCRIPTION (provided by applicant): The inability to manipulate DNA methylation or other epigenetic marks at will remains one of the biggest constrains in the field of epigenetics. Our goal is to overcome this limitation by designing an innovative approach for targeted manipulation of DNA methylation in a unique cellular system that also enables accurate measurements of such performance. Having the precise temporal and localized control over epigenetic marks will be essential for further dissecting their exact function(s) in genome regulation. The ability to write rather than just read epigenetic marks is the last missing piece t confidently and generally establish functional relationships, control the genome and will therefore have a wide impact on many fields. Over the past five years my lab has been one of the leading groups to map and manipulate DNA methylation at a genome-wide scale and we have accumulated likely the largest database of DNA methylation measurements at single base resolution. We have generated well over 2000 reduced representation bisulfite sequencing (RRBS) and more than 50 whole genome bisulfite sequencing (WGBS) datasets providing us with over a 100 billion CpG methylation measurements across more than a hundred mouse and human cell types. As a result we are confident to state that we know where in the genome DNA methylation can be found and how it is influenced by its genomic environment. Moreover we have created many mouse ES cells lines covering nearly every combination of loss and/or gain of function for the three catalytically active Dnmts (1, 3a and 3b) and their co-factor Dnmt3l. These in turn provide a unique foundation for this proposed study and enables us to most accurately determine efficiencies while controlling confounding factors. We strongly believe that simply engineering tools to write onto the genome without understanding the rules and principles on how this mark can be written in a genomic context will never provide a universal strategy.

DESCRIPTION (provided by applicant): This project is designed to obtain the first comprehensive assessment of epigenetic defects (epimutations) induced by the use of assisted reproductive technologies (ART) in the offspring produced. The genome-wide occurrence of abnormalities in DNA methylation patterns and gene expression patterns will be assessed in mice produced by intracytoplasmic sperm injection (ICSI) using cutting-edge, high-throughput epigenomic and genomic technologies. In addition, the etiology of ART-induced epimutations will be investigated by determining the precise timing of appearance of epimutations in mice produced by ART, as well as by discerning the relative contribution of each of three specific aspects of the ART process - gonadotropin stimulation of folliculogenesis, prolonged culture of preimplantation embryos, and embryo transfer - to the induction of epimutations. Finally, the fate of ART-induced epimutations will be examined to determine if these are consistently corrected by epigenetic reprogramming during either embryogenesis or gametogenesis. The significance of the research proposed in this application is that it will include high (single-base) resolution, comprehensive analyses of the disruptive effects of ART on epigenetic programming genome-wide, and it will facilitate controlled, prospective studies of the genesis, etiology, extent and fte of epimutations induced by ART, including parallel, simultaneous studies of the relative contribution(s) of multiple different aspects of the ART process to the induction of epimutations in the offspring produced. The proposed research is innovative and timely because we are now able to generate datasets of unprecedented scale and resolution describing abnormal epigenetic programming and gene expression in ART offspring, and we can use these to understand the genesis, consequences and fate of ART-induced epimutations in ways that will have significant implications for basic biology and human health. The resulting enhanced understanding of the etiology of ART-induced epimutations will inform future modifications of clinical ART protocols to minimize the induction of epimutations in ART-derived human offspring and thereby reduce the occurrence of epigenetic disorders in individuals produced by this technology.

Project Abstract Type 1 Diabetes (T1D) is an autoimmune disease in which the insulin-producing beta cells of the pancreas are destroyed. These beta cells are specifically targeted for destruction by autoreactive T cells, which manage to escape the normal elimination mechanisms in the thymus. Although it is clear that besides environmental factors a strong genetic component is involved, the underlying basis for autoimmune T1D is not well understood. GWAS for a number of diseases, including T1D, have recently highlighted regulatory DNA regions rather than protein coding sequences as hot spots where single nucleotide polymorphisms (SNPs) as causal variants can be found. Our long term goal is to decipher the molecular mechanism of genotype-phenotype causalities in T1D. Our overall objective is the in depth characterization of DNA regulatory elements and their risk variants that underlie T1D susceptibility on a molecular and functional level. Our central hypothesis is that T1D associated SNPs alter the function of cell type-specific DNA regulatory elements. Guided by strong preliminary data, we will approach this hypothesis from 3 complementary angles: We will use state-of-the-art and novel computational strategies to fine map T1D GWAS SNPs to candidate causal variants (Aim 1). We will characterize the mechanism of action of causal candidates SNPs by utilizing a combination of functional genomics and proteomics (Aim 2). Finally, we will determine the functional relevance of causal variants in T1D-relevant human(-ized) systems (Aim 3). This approach is highly innovative and will provide the first in depth functional characterization of DNA regulatory elements and their risk variants that underlie T1D susceptibility. Our studies will provide a significant shift from the correlative to the functional investigation of human T1D susceptibility. Ultimately, such knowledge has the potential to translate to novel approaches to predict, prevent and potentially to treat T1D.

Abstract Pluripotent stem cells can give rise to all cell types in the body and have therefore enormous potential for regenerative medicine, and provide a powerful tool for studies in developmental biology and pharmacology. Advances in transforming somatic cells directly into pluripotent (induced pluripotent stem: iPS) cells provide an attractive avenue for generating large numbers of customized stem cells. Notable progress has been made to efficiently generate iPS cells, including integration-free lines, however, detailed mechanistic insights regarding the establishment, subsequent maintenance and efficient exit from pluripotency are still lacking. Over the past four years our program project team has made significant contributions towards a better understanding of these (Smith et al. Nature 2012,14; Ziller et al. Nature 2013,14; Gifford et al. Cell 2013; Tsankov et al. Nature 2015; Liao et al. Nature Genetics 2015; Cacchiarelli et al Cell in 2015 and more). As would be expected the results have helped pinpoint areas that need further experimentation and also presented us with a new set of lingering questions that are central to our understanding of human pluripotency. Our aims in the framework of the larger program project (past and future) will provide crucial insights towards this end and as a result will have a broad impact on the fields of human reprogramming and pluripotency. Over the next years, we will apply a complex, recently engineered DNMT1 knockout/inducible rescue human ES cell line to address fundamental questions in human stem cell biology with a focus on the naïve versus primed states. We will apply the latest advances in single cell technology and analysis to gain unprecedented insights into the biology of human pluripotency. Lastly, we will use our recently developed and characterized human secondary reprogramming system to define and study one of the most critical points (the final transition) in the reprogramming process that is also the least understood.