Yousin Suh - US grants

DESCRIPTION (provided by applicant): With the completion of the human genome project, attention is shifting towards individual genetic variation. Such variation, most of which consists of single nucleotide polymorphisms (SNPs), may account for a substantial portion of human individual phenotypic variation, including differences in aging-related characteristics. Especially important are SNPs that affect the function of genes in candidate longevity assurance pathways, since they could provide novel targets for the early detection and possible prevention of aging-related functional decline. Optimal study populations in this respect are those in which aging related phenotypes can be defined in terms of their progression from middle age onwards, rather than as snapshots in time. We hypothesize that genetic variation at loci involved in genome maintenance can be related to individual differences in the rate and severity of aging. To address this hypothesis, a systematic multidisciplinary study is proposed in which SNP haplotypes of about 100 genes in 5 genome maintenance pathways will be determined in individuals of an ongoing longitudinal study of 586 Mexican American (MA) and 428 European American (EA) (SALSA; San Antonio Longitudinal Study of Aging). Subsequent association analysis in conjunction with functional assays of allelic gene variants will provide insight into their biological significance. The results should lead to increased understanding of the role of genome maintenance in healthy aging and provide genetic markers to identify individuals at risk for specific aging related phenotypes. This will open up the possibility of targeted and personalized intervention strategies, ultimately leading to improved quality of life of the elderly population.

With the entire human genome now almost completely decoded, attention is shifting towards individual genetic variation. Most of this variation consists of single nucleotide polymorphisms (SNPs), which can account for heritable inter-individual differences in, for example, disease susceptibility and response to medication. Aging is a major risk factor for most common human diseases. The identification of SNPs in candidate genes and the assessment of their potential functional impact on aging-related phenotypes will be important in assessing genetic components of aging, including exceptionally healthy aging. Optimal study populations in this respect are those in which aging-related phenotypes can be defined in terms of their progression from middle age onwards, rather than as snapshots in time. The objective of this proposal is to further optimize a previously developed SNP discovery method, Two-Dimensional Gene Scanning (TDGS), to comprehensively analyze SNPs in multiple candidate genes in large, aging populations. The validity of this approach will be assessed through association analysis, in a case control manner, of all possible SNPhaplotypes of a selection of nuclear and mitochondrial genes involved in musculoskeletal function in a population of 226 Mexican American individuals of an ongoing longitudinal study of aging (San Antonio Longitudinal Study of Aging; SALSA). The results are expected to enrich the ongoing study with a genetic component for this particular phenotype and to demonstrate the validity of TDGS as a high-throughput platform to screen aging populations for all possible SNPs in hundreds and ultimately thousands of candidate genes (comprehensive candidate approach).

DESCRIPTION (provided by applicant): With the completion of the human genome project, attention is shifting towards individual genetic variation. Such variation, most of which consists of single nucleotide polymorphisms (SNPs), may account for a substantial portion of human individual phenotypic variation, including differences in aging-related characteristics. Especially important are SNPs that affect the function of genes in candidate longevity assurance pathways, since they could provide novel targets for the early detection and possible prevention of aging-related functional decline. Optimal study populations in this respect are those in which aging related phenotypes can be defined in terms of their progression from middle age onwards, rather than as snapshots in time. We hypothesize that genetic variation at loci involved in genome maintenance can be related to individual differences in the rate and severity of aging. To address this hypothesis, a systematic multidisciplinary study is proposed in which SNP haplotypes of about 100 genes in 5 genome maintenance pathways will be determined in individuals of an ongoing longitudinal study of 586 Mexican American (MA) and 428 European American (EA) (SALSA; San Antonio Longitudinal Study of Aging). Subsequent association analysis in conjunction with functional assays of allelic gene variants will provide insight into their biological significance. The results should lead to increased understanding of the role of genome maintenance in healthy aging and provide genetic markers to identify individuals at risk for specific aging related phenotypes. This will open up the possibility of targeted and personalized intervention strategies, ultimately leading to improved quality of life of the elderly population.

DESCRIPTION (provided by applicant): With the completion of the human genome project, attention is shifting towards individual genetic variation. Such variation, most of which consists of single nucleotide polymorphisms (SNPs), may account for a substantial portion of human individual phenotypic variation, including differences in aging-related characteristics. Especially important are SNPs that affect the function of genes in candidate longevity assurance pathways, since they could provide novel targets for the early detection and possible prevention of aging-related functional decline. Optimal study populations in this respect are those in which aging related phenotypes can be defined in terms of their progression from middle age onwards, rather than as snapshots in time. We hypothesize that genetic variation at loci involved in genome maintenance can be related to individual differences in the rate and severity of aging. To address this hypothesis, a systematic multidisciplinary study is proposed in which SNP haplotypes of about 100 genes in 5 genome maintenance pathways will be determined in individuals of an ongoing longitudinal study of 586 Mexican American (MA) and 428 European American (EA) (SALSA; San Antonio Longitudinal Study of Aging). Subsequent association analysis in conjunction with functional assays of allelic gene variants will provide insight into their biological significance. The results should lead to increased understanding of the role of genome maintenance in healthy aging and provide genetic markers to identify individuals at risk for specific aging related phenotypes. This will open up the possibility of targeted and personalized intervention strategies, ultimately leading to improved quality of life of the elderly population.

PROJECT ABSTRACT Alzheimer?s disease (AD) is conventionally characterized by specific neuropathological features, including the appearance of extracellular amyloid deposits and the accumulation of intracellular neurofibrillary tangles. While several gene mutations are clearly associated with early onset Alzheimer?s disease, the large number of individuals exhibiting delayed onset, aging-associated AD, are likely to harbor many alterations in linked modifier genes that predispose to AD susceptibility. Genetic and genome wide association studies (GWAS) have identified numerous genes and risk alleles that indicate both cell autonomous and non-cell autonomous mechanisms contributing to loss of neurons and cognitive decline. In this regard, the majority of risk variants identified by GWAS reside in non-coding regions of the genome, implying that they act in part to alter gene expression. This proposal responds to the RFA indicating a particular need for approaches designed to delineate the transcriptional and cellular consequences of combinations of SNPs in the risk alleles by generating new cell line reagents to help unravel the question of the causative SNPs and their target genes in specific neurons derived from iPS cells of AD individuals. There are two features of sporadic AD that require molecular explanation- the potential role of aging in AD susceptibility, and the striking gender disparity, with the incidence of AD being exaggerated in females. These issues can only now be addressed based on new technologies and the availability of patient-derived samples. Our proposed research plan takes advantage of the invaluable samples stored at the brain bank of the Shiley-Marcos Alzheimer's Disease Research Center (ADRC) at UCSD, and the iPSC-derived neurons (Salk). This approach will interrogate the effects of different genetic variants with other risk factors (e.g. age, sex), and assess their effects on cell type-specific enhancer landscapes. By merging these data, we can begin to identify the potential causative SNPs that result in altered function of cell-type specific enhancers. We propose using a high throughput 4C screening approach (UMI-4C), and Hi-ChIP, to identify the most likely causative, enhancer-associated SNPs for functionally-implicated coding target genes. Exploiting the power of contemporary gene editing approaches in control or patient-derived iPS cells to specific neuronal cell types, and to astroglia, we can assess the transcriptional phenotypes and functional behaviors of neurons harboring different combinations of risk alleles, both in the isolated cell lines alone and in combination with coculture experiments with astroglia and microglia, as effects of these SNPs may be manifest only with astroglial:neuronal interactions. Together these studies will use powerful contemporary global genomic approaches to determine the coding transcriptional targets of several of the most significant SNPs in enhancers, and the link to roles of estrogen receptor in the gender disparity for AD.

Contact PD/PI: ROSENFELD, MICHAEL G PROJECT ABSTRACT Alzheimer?s disease (AD) is conventionally characterized by specific neuropathological features, including the appearance of extracellular amyloid deposits and the accumulation of intracellular neurofibrillary tangles. While several gene mutations are clearly associated with early onset Alzheimer?s disease, the large number of individuals exhibiting delayed onset, aging-associated AD, are likely to harbor many alterations in linked modifier genes that predispose to AD susceptibility. Genetic and genome wide association studies (GWAS) have identified numerous genes and risk alleles that indicate both cell autonomous and non-cell autonomous mechanisms contributing to loss of neurons and cognitive decline. In this regard, the majority of risk variants identified by GWAS reside in non-coding regions of the genome, implying that they act in part to alter gene expression. This proposal responds to the RFA indicating a particular need for approaches designed to delineate the transcriptional and cellular consequences of combinations of SNPs in the risk alleles by generating new cell line reagents to help unravel the question of the causative SNPs and their target genes in specific neurons derived from iPS cells of AD individuals. There are two features of sporadic AD that require molecular explanation- the potential role of aging in AD susceptibility, and the striking gender disparity, with the incidence of AD being exaggerated in females. These issues can only now be addressed based on new technologies and the availability of patient-derived samples. Our proposed research plan takes advantage of the invaluable samples stored at the brain bank of the Shiley-Marcos Alzheimer's Disease Research Center (ADRC) at UCSD, and the iPSC-derived neurons (Salk). This approach will interrogate the effects of different genetic variants with other risk factors (e.g. age, sex), and assess their effects on cell type-specific enhancer landscapes. By merging these data, we can begin to identify the potential causative SNPs that result in altered function of cell-type specific enhancers. We propose using a high throughput 4C screening approach (UMI-4C), and Hi-ChIP, to identify the most likely causative, enhancer-associated SNPs for functionally-implicated coding target genes. Exploiting the power of contemporary gene editing approaches in control or patient-derived iPS cells to specific neuronal cell types, and to astroglia, we can assess the transcriptional phenotypes and functional behaviors of neurons harboring different combinations of risk alleles, both in the isolated cell lines alone and in combination with coculture experiments with astroglia and microglia, as effects of these SNPs may be manifest only with astroglial:neuronal interactions. Together these studies will use powerful contemporary global genomic approaches to determine the coding transcriptional targets of several of the most significant SNPs in enhancers, and the link to roles of estrogen receptor in the gender disparity for AD. Project Summary/Abstract Page 7

Abstract Based on the importance of defining new insights into cellular senescence, we initiated studies to investigate whether there might be a specific enhancer activation ?code? that underlies cellular senescence for identifying the responsible DNA binding transcription factors. While there is rapidly-emerging, and now unassailable evidence, on the role of the 40-70,000 enhancers in each cell type in development, homeostasis and, often, pathological events, their role in cellular senescence remains undefined. Furthermore, while cellular senescence represents a fundamental process of aging and a known driver of pathologies, the causative role of newly activated enhancer cohorts underlying progression of senescence remain poorly understood. Therefore, the goal of this proposal, supported by extensive preliminary data, is to test a novel hypothesis that the de novo appearance of two specific cohorts of enhancers sets into motion a progressive, functionally- important, alteration in gene transcription programs. Based on our study of the altered enhancer and chromosomal landscape during replicative senescence, we have begun to establish that the geroprotective mTOR inhibitor, Rapamycin, markedly delays all aspects of cellular senescence, including the appearance of new, functional, enhancers. Our focus is to elucidate the functional importance of a gained enhancer program underlying cellular senescence, and identify the critical DNA binding transcription factors underlying the transcriptional programs that are determinants of replicative senescence, based on the complementary expertise of the Suh and Rosenfeld laboratories. Specifically: i) We will use unbiased screens to document that at least two distinct activated enhancer networks independently regulate the proliferation arrest and SASP aspects of replicative senescence, respectively. ii) We will identify combinatorial factors synergizing with the previously-unrecognized transcription factors, NFI-A, NFI-C, to regulate the gained enhancers underling proliferation arrest, and those that, with SMAD2/3 and NFkB, to regulate the SASP program. In parallel, we can implicate the underlying signaling pathways. iii) We will identify previously unrecognized histone modification signatures of, and their functional importance in replicative senescence . iv) We will Identify Activin and Tgf?2 as inhibitors of the proliferation and SASP enhancer programs, respectively. Our proposal promises to provide transformative insights into molecular events that initiate and perpetuate the senescent cell phenotypes, and help elucidate potential novel therapeutic modalities against the deleterious SASP program.

ABSTRACT Here we propose a Supplement to the research proposed in the parent grant Combinatorial regulation of the enhancer codes in senescence to perform a logical extension of our initial Specific Aims regarding cellular aging/senescence to achieve initial insights into the distinction between these events and distinct pathological causal features in each CNS cell type that may represent the underlying mechanisms underlying sporadic Alzheimer?s Disease (AD). The Specific Aims of the initial grant employed and developed biostatistical tools relevant to cellular aging and replicative senescence, including an examination of underlying epigenomic alterations and enhancer activation codes ultimately leading to cellular senescence approaches and proposed application of single cells approaches. Appling these specific aims/approaches is particularly suitable for enhancing our understanding of the potential initial causal events that eventuate in clinical sporadic AD, an aging- associated disease affecting both men and, to a greater extent, women. In concert with the original Aim of understanding the molecular basis for enhancer-mediated programs of cellular aging and senescence, in this Supplement, we propose to extend our original Specific Aims to uncover the enhancer program underlying the aging events in each CNS cell type, to permit examination of the central question whether the altered enhancer and transcriptome changes in AD in each cell type represent a trajectory distinct from the normal aging-related alterations in these cell types. We hypothesize that, while each cell type will, of course, exhibit specific features of enhancer activation characteristic of aging and even cellular senescence, as we have uncovered in the parent grant, AD represents a distinct trajectory for these cell types. This Supplement is licensed by our ability by our development of the technology to perform simultaneous quantitation of single nucleus (sn) RNA-seq and snATAC-seq using archival samples stored at the brain bank of the Shiley-Marcos Alzheimer's Disease Research Center (ADRC) at UCSD. We have carefully piloted this approach to ensure that we are technically able to obtain high quality data and that all of the proposed informatic pipelines and our ability to successfully aggregate such massive data sets is fully established. The Supplement would license our ability to scale the analysis to obtain data sets capable of generating statistically significant results and, therefore, informative conclusions, which can be ultimately be further validated by imputation from available data bases and using hiPSCs to generate specific cell types for validating transcriptional analyses. Our overarching goal in this Supplement is to apply the described technologies and new informatic approaches to a sufficient number of archived specimens to permit formulating the actual transcription factors and pathways that distinguish initiation of the AD process in specific CNS cell types.

PROJECT SUMMARY Alzheimer?s disease (AD) is a complex neurodegenerative condition that affects millions of individuals worldwide and currently has no preventative or disease-modifying therapies. Exciting results from heterochronic parabiosis studies indicate the presence of rejuvenating factors in the circulation that can restore youthful characteristics to aged cells and tissues, including the restoration of aged neural stem activity and cognitive function. Intriguingly, heterochronic parabiosis studies have also shown that exposure of transgenic AD model mice to a young systemic environment results in a marked amelioration of the cognitive deficits and AD-like neuropathology observed in these animals. The beneficial effects of young blood have been observed in multiple transgenic AD models. However, the identities of the blood-borne factors that mediate these effects are still unknown. This proposal is built upon the exciting results emerged from studies funded by R01AG057433, with the goals of identifying and characterizing circulating anti-geronic factors conserved in mammals, including human, bovine, and mouse. We found that systemic administration of one of the top candidate anti-geronic factors, PEDF, significantly improves deficits in cognitive function in aged wild type mice. We hypothesize that conserved circulating anti-geronic factors such as PEDF mediate the rejuvenation effects of young blood in both aged wild type animals and transgenic animal models of AD, and that systemic treatment of these factors to an AD mouse model will improve cognitive function and ameliorate AD-like neuropathology. To test this hypothesis, we will employ the well-studied 5XFAD transgenic mouse model of AD and will investigate whether and to what extent top conserved anti-geronic factors exert beneficial effects on cognitive function and AD-like neuropathology in vivo by measuring changes in amyloid plaque formation, gliosis, synaptic density, cerebrovascular integrity, and cognitive behavior, as compared to vehicle-treated controls. To understand the mechanisms through which these factors exerts protective and restorative effects, we will perform single cell RNA-seq analysis to identify the biological processes and specific cell types mediating their beneficial effects in the brain. Defining the cell type- specific transcriptional programs that are altered in the brain of the 5XFAD mouse model during the progression of AD pathology and how these are affected by treatment with anti-geronic factors will provide mechanistic insights into the protective and restorative effects of youthful blood-borne factors and may identify new therapeutic targets for AD.

Abstract Type 1 diabetes (T1D) is an organ-specific autoimmune disease, whereby immune cell-mediated and inflammatory cytokines lead to loss of the insulin-producing ? cells in the pancreas. Genome-wide association studies (GWAS) have identified ~60 genomic regions associated with T1D risk. The vast majority of GWAS risk variants associated with T1D reside in non-coding regions, particularly enhancers, suggesting that gene regulatory changes substantially contribute to inter-individual differences in susceptibility to T1D. Driven by our T1D GWAS annotation data, highlighting the importance of a systematic analysis of both immune and ? cell systems of both T1D patients and controls, we propose to identify causal enhancer variants and causal target genes using contemporary computational methods and cutting-edge global genomics. We will perform our PRO-cap and PRO-seq assays to comprehensively identify active enhancers harboring T1D-associated variants in T cells, monocytes and stem cell derived ? cells (sc-? cells) from T1D patients and controls, followed by validation of active enhancers harboring T1D-associated variants using our eSTARR-seq assays (Aim 1). We will perform our Tri-HiC assays to profile the enhancer-promoter interactomes at unprecedented high resolution in primary T cells, primary monocytes and sc-? cells from T1D patients and controls as well as pancreatic islets to comprehensively identify target genes of T1D-associated variants, and refine targets of T1D-associated variants with T1D-sepcific alteration in target gene expression in each cell type using our coupled single cell nucleus (sn) RNA-seq and snATAC-seq assays (Aim 2). We will validate targets of T1D-associated variants in T cells, monocytes and ? cells using CRISPR/Cas9 endogenous enhancer mutational strategies in the context of the endogenous chromatin landscape in each cell type. Our analytical and experimental framework represents an exciting new paradigm for studying T1D, and the subsequent clinical research based on our results has the potential to develop preventive and therapeutic strategies against T1D. The data sets generated by these studies will represent important, but currently lacking, resources for the T1D research community.

Reproductive aging is a major health, personal and societal issue, but ovarian aging has received limited scientific attention, even in large genomic survey projects. Ovarian aging influences diverse health outcomes in women including lifespan, cardiovascular disease, metabolic syndromes, neurodegenerative disorders and various types of cancer. Yet the molecular mechanisms underlying ovarian aging, timing of menopause and inter-organ feedback loops remain elusive. As one of the most dynamic organs in the human body, the ovary undergoes significant remodeling across the entire reproductive period. The dynamic transcriptional regulation of and interactions between oocytes and their surrounding cells during aging remain unknown. The objective of this proposal is to understand the regulatory landscapes underlying the complex interplay among the different cell types in the ovary and to investigate the molecular mechanisms that regulate the remarkably complex processes of reproductive aging. We will apply powerful single-cell (sc) RNA-seq and scATAC-seq analysis to define specific transcriptional programs and regulated enhancer networks that are altered in distinct ovarian cell types or subtypes during aging. By defining the roles of specific enhancers in specific cell types, and how these change with aging, we aim to understand the identities of the regulatory factors and environmental signals that impact aging in each ovarian cell type. Genetic variation affecting enhancer selection and function is a major determinant of differences in cell-specific gene expression between individuals. To investigate the mechanisms by which altered regulatory enhancer landscapes contribute to ovarian aging by licensing changes in transcriptional programs, we will investigate the roles of genetic variants associated with age at menopause, detected by genome-wide association studies (GWAS), in modulating transcription programs during ovarian aging. In particular, we hope to provide mechanistic insights into genetic modulation of transcriptional regulation of critical homeostatic and inflammatory pathological functions in the granulosa cells (GC), the supporting cell type immediately surrounding the oocyte, by modeling the causal regulatory variants in human GC models that are differentiated from human ESCs engineered to carry causal variants by CRISPR gene editing.

OVERALL: PROJECT SUMMARY Defining the molecular and cellular heterogeneity underlying senescent cell states is a critical knowledge gap in the field. The Columbia University Senescence Tissue Mapping (CUSTMAP) Center is uniquely poised to address this gap by creating a multi-scale atlas of senescence in diverse tissue types across the adult human lifespan. CUSTMAP is a highly collaborative effort that builds upon long-standing and established collaborations between Columbia University Irving Medical Center (CUIMC), The University of Edinburgh, New York University (NYU), and the New York Genome Center (NYGC). Using state-of-the-art spatial genomics technologies and leveraging our established experimental workflows and analytical pipelines, CUSTMAP will generate three- dimensional maps of senescent cells in tissues with vulnerability to age-related degenerative processes: the central nervous system (brain and spinal cord) and the skin. We will perform spatially resolved transcriptomics (ST), single-nucleus RNA-sequencing, and multiplexed proteomics using iterative indirect immunofluorescence imaging (4i) experiments in central nervous system (CNS) and skin tissues across the human lifespan, allowing for unprecedented genome-wide molecular characterization at single-cell resolution in space. CUSTMAP is structured around three scientific Cores, as well as an Administrative Core to support these integrated efforts. Human tissue samples characterized and collected through the Biospecimen Core (BIO) will be analyzed using the tools and techniques developed through the Biological Analysis Core (BAC) and Data Analysis Core (DAC), leading to detailed maps of senescent cells and their effects in human tissues at single-cell resolution. The success of CUSTMAP is predicated on access to a continuous supply of human tissues from healthy individuals across the lifespan. CUSTMAP investigators have access to post-mortem samples and prospective tissue collection from local resources at CUIMC as well as through established collaborations. Our unique geographic location in Upper Manhattan enables tissue acquisition from a local population of patients that is rich in racial and ethnic diversity. Thus, CUSTMAP is uniquely poised to obtain high-quality tissue from these diverse populations and apply cutting-edge multiomic approaches to build integrated 3D molecular atlases of senescent cells in CNS and skin tissues. Our approach provides a unique platform for driving transformative discoveries of novel molecular, cellular and regional correlates of age-related changes in cellular senescence in human tissues. The CUSTMAP workflow and computational tools are readily generalizable to other tissue types and can be efficiently shared with and deployed across the SenNet Consortium.