Kathrin Plath - US grants

NIH Roadmap Initiative tag

Analysis, Data; Binding; Binding (Molecular Function); Bio-Informatics; Bioinformatics; Cells; Chromatin; Chromatin Structure; DNA Methylation; Data; Data Analyses; Data Analysis, Statistical; Data Banks; Data Bases; Data Interpretation, Statistical; Data Set; Databank, Electronic; Databanks; Database, Electronic; Databases; Dataset; ES Cell Line; ES cell; Educational process of instructing; Embryonic Stem Cell Line; Epigenetic; Epigenetic Change; Epigenetic Mechanism; Epigenetic Process; Experimental Designs; Frequencies (time pattern); Frequency; Genes; Genetic; Genomics; Germ Lines; Head; Hematopoietic; Histones; In Vitro; Investigators; Laboratories; Methylation; Microarray Analysis; Microarray-Based Analysis; Modeling; Modification; Molecular Interaction; Mother Cells; Nervous; Pathway interactions; Progenitor Cells; Programs (PT); Programs [Publication Type]; Protein Methylation; Publications; Publishing; Quality Control; Research Design; Research Personnel; Researchers; Role; Sample Size; Science of Statistics; Scientific Publication; Services; Somatic Cell; Standards; Standards of Weights and Measures; Statistical Data Analyses; Statistical Data Interpretation; Statistics; Stem cells; Study Type; T-Cells; T-Lymphocyte; Teaching; Testing; Thymus-Dependent Lymphocytes; Work; cell type; clinical data repository; clinical data warehouse; data mining; data repository; datamining; density; embryonic stem cell; experience; experiment; experimental research; experimental study; hESC; human ES cell; human ES cell lines; human ESC; human embryonic stem cell; human embryonic stem cell line; in vivo; microarray technology; neural; pathway; programs; reconstitute; reconstitution; relating to nervous system; relational database; research study; response; self-renewal; social role; statistics; stem cell fate; stem cell of embryonic origin; study design; thymus derived lymphocyte; transcription factor

Description The Administrative Core will support all of the Projects and the Bioinformatics Core of this Program. Kathrin Plath, Program PI, will be the Leader of the Administrative Core, with support from Drs. Smale and Zaret as Co-Investigators. This Core will enable all of the scientific components of the Program to interact by providing the structure and financial support for regular planning, sharing, and evaluation of the research. The Core will be responsible for ensuring that the investigators act as a cohesive and interactive group by providing the planning and leadership required for regular and frequent interactions among the Projects and the Bioinformatics Core. A shared Web site will support the projects with integrated services for optimal quality, efficiency, and data-sharing. In addition, the core will ensure efficient collaborations with the existing stem cell research centers at UCLA and UPenn to provide optimal access to basic resources and infrastructure and leverage existing high-throughput sequencing, microarray, and stem cell resources at the respective institutions. The Core will recruit additional investigators to the study of human cell pluripotency and reprogramming through the distribution of pilot projects. The Administrative Core will also provide centralized grant administration, including coordinating interactions of the investigators. Core usage, reporting to the NIGMS, budget oversight and management, and ensuring compliance with all governing IBC and ESCRO regulations and policies. Program interactions and oversight will be facilitated by a Scientific Advisory Board composed of both internal and external experts in stem cell biology and epigenetics.

DESCRIPTION (provided by applicant): The long-term objective of this Program is to determine the fundamental mechanisms underlying the interplay between transcription factors, chromatin structure, and higher-order genomic organization during the cellular conversion to and maintenance of pluripotency. Despite the remarkable ability of transcription factors and miRNAs to convert cells to pluripotency, undefined stochastic parameters presently limit the efficiency of the process. In addition, different pluripotent lines have different capacities for terminal differentiation and we poorly understand parameters that determine how well in vitro differentiation compares to in vivo differentiation. Understanding the molecular mechanisms in pluripotency induction and maintenance as well as those limiting differentiation will allow enhancements of the process that, in turn, will facilitate the use of small human biopsy samples much more efficiently than present techniques allow. To this end, the four projects of the Program ask: 1) How is the differentiated cell genome reorganized within the nucleus, during reprogramming to pluripotency, what aspects of reorganization are important, and what controls genome organization in pluripotent cells? 2) How do ectopic pluripotency transcription factors gain access to silent, chromatinized target sites to activate the endogenous pluripotency network, and how can the process be enhanced? 3) What regulatory circuits need to be properly established within pluripotent cells to allow their subsequent differentiation to fully mature progeny? 4) What marks of the competence to differentiate exist in pluripotent cells and how do they get established? By seeking answers to these questions in a single Program, we can obtain a time-resolved, integrated view of the mechanisms by which different aspects of the nuclear genome change coordinately to properly convert a cell to pluripotency and the process by which cells return to the somatic state. We also anticipate that the coordinate mechanisms unveiled by our studies will provide insights into direct cell reprogramming, independent of pluripotency. Administrative and Bioinformatics Cores and a shared Web site will support the projects with integrated services for optimal quality, efficiency, and data-sharing. The Administrative Core leverages existing high throughput sequencing, microarray, and stem cell cores at the respective institutions. The Project and Core leaders have complementary expertise in the relevant areas of stem cell biology, differentiation, transcription and chromatin/ epigenetics and have a long-standing record of interactive collaborations and publications. The plan provides unique experimental synergies that address the objectives of the funding announcement. PUBLIC HEALTH RELEVANCE: The information generated by these investigators will provide valuable knowledge to increase the efficiency and effectiveness of generating properly reprogrammed pluripotent stem cells from differentiated cells. Such increases will greatly facilitate the ability to generate reprogrammed cells to make autologous cells for transplantation and modeling of diseases, provide insights into other forms of cellular reprogramming, and further our understanding of basic mechanisms that control self-renewal and differentiation of stem cells.

Description The goal of this proposal is to reveal the mechanisms that govern the three-dimensional (3D) architecture of the genome in pluripotent cells and during reprogramming to the IPS cell state. The spatial organization of chromosomes is a fascinating problem of metazoan biology, but leaves many unanswered questions, particularly the challenge to decipher the mechanisms driving the co-localization of genetic loci. Based on differentiated cell data, the 3D network of chromosomal interactions in the nucleus is thought to be important for the maintenance of cell identity and affect gene expression by spatial clustering of genes and their regulatory regions and by congregating groups of genes at the same sub-nuclear structure allowing their coordinated expression and modulation of epigenetic states. The 3D structure of the pluripotent cell genome is basically unstudied. However, a recent study of DNA replication timing, to which my lab contributed, suggested that a large-scale reorganization of the genome coincides with the commitment of pluripotent cells to differentiation, prior to germ layer specification. The reversal of this process appears to be one of the final steps of reprogramming, linked to the binding of the reprogramming factors to pluripotency gene targets and changes in global chromatin structure and DNA replication patterns. Based on these findings, we hypothesize that a true understanding of genome regulation in pluripotent cells and during reprogramming can only be obtained by revealing the structural and functional relationships between the spatial organization of the genome and linear genomic features such as chromatin and expression states and association with transcription factors; and that the establishment of the pluripotent nuclear architecture represents a road block to reprogramming. In an effort to begin to address 3D genomic interactions in pluripotent cells, my laboratory has successfully established the 4C-seq method to identify regions throughout the genome that are physically close to the Oct4 locus, which revealed that this locus interacts with early replicating, highly expressed genes that are bound by pluripotency transcription factors that themselves are enriched at the Oct4 locus. Based on this extensive work, our expertise in reprogramming and pluripotent cell chromatin, along with specific collaborations within the P01 and strong ties to the P01 Bioinformatics Core, we are well positioned to unveil molecular mechanisms governing the 3D genomic interactions in human and mouse embryonic stem (ES) cells and to study how the differentiated cell genome is reorganized during human cell reprogramming; with these Aims: Aims: Aim 1. We will assess and compare genomic interactions of key loci in mouse and human ES cells; discern the relationship with linear genomic features; and unveil mechanisms that control 3D organization. Aim 2. We will systematically test how expression level, pluripotency transcription factors, and chromatin proteins regulate 4C interactions at an isolated locus in mouse ES cells. Aim 3. We will define how genomic interactions are reorganized during human cell reprogramming and discover how chromatin limits this process. Each of the aims will be performed in conjunction with other members of the P01 proposal and build on the work of the P01 Bioinformatics Core. We do not propose human and animal experimentation.

? DESCRIPTION (provided by applicant): The goal of this proposal is to reveal mechanisms by which the long non-coding (lnc) RNAs Xist and Tsix find their genomic target sites and control gene expression at these sites during the process of X chromosome inactivation (XCI). XCI is a remarkable paradigm of lncRNA-mediated chromatin regulation, controlled by the oppositely transcribed RNAs Xist and Tsix, that transcriptionally silence one of the two X chromosomes in female mammalian cells. While Xist induces silencing of the X chromosome, Tsix negatively regulates Xist early in development to ensure that Xist only becomes active on one X chromosome. We already know several functions of Xist, including spreading in cis from its site of transcription to cover the entire X chromosome, excluding RNA Polymerase II, triggering the accumulation of repressive chromatin marks, and inducing a chromosome-wide compaction, demonstrating the remarkable versatility of this lncRNA. The mechanisms by which Xist carries out these functions remain poorly understood. In particular, with few exceptions, we still do not know domains of Xist or interacting proteins that mediate these various roles. However, our preliminary data yield new insight into the roles of Xist and Tsix. Specifically, we have been able to define three domains of Xist as mediators of distinct functions, consistent with the idea that distinct RNA domains carry out different functions. We hypothesize that these domains are required for the efficient association of Xist with the X, ensuring its function in cis; the targetng of Xist to active genes, which is a prerequisite for their subsequent silencing; and for changes in chromosome conformation, respectively. To further define the mechanisms by which these domains act, we have also identified a critical protein interaction partner of one of these domains. Moreover, our recent work on Xist spreading suggests an intriguing link between Xist and genome organization, that led to the hypothesis that Xist utilizes long-range chromatin interactions to spread across the X chromosome, and, upon tethering to distal sites acts as a nuclear organization factor that creates a new 3D topology of the chromosome to induce gene silencing. Finally, by comparing the RNA localization of Tsix and Xist on the X chromosome, we found that Tsix can spread along the X, but in contrast to Xist, only spreads locally around its site of transcription. We speculate that specific RNA sequences in Xist and Tsix direct these distinct localization patterns. Based on our findings, we are well positioned to unveil regulatory mechanisms by which Xist and Tsix control XCI, with these Aims: 1) To understand how Xist RNA mediates its various functions through specific RNA domains. 2) To investigate the link between 3D chromosome structure and Xist. 3) To characterize the function and localization of Tsix. Each aim will be supported by outstanding collaborators. Together, our proposed studies will provide novel insights into XCI, and should significantly advance our understanding of the role of nuclear architecture on nuclear function. Exploring the differences between Xist and Tsix will also yield a better understanding of how lncRNAs locate to their genomic targets.

PROJECT SUMMARY Women and men are well known to have different propensities to neuropsychiatric illness, but the source of these differences is not understood. In particular, the molecular events that determine functional dimorphism between the female and male brain need to be defined. We will examine how the female-specific long noncoding RNA Xist and its newly identified interaction with the pre-mRNA splicing regulators PTBP1 and 2 affect gene expression and alternative splicing in the female brain. The project will use expertise and tools developed in three labs for the study of Xist RNA, of neuronal splicing regulation by the PTB proteins, and of gene expression and alternative splicing using computational methods. RNA-seq data from defined regions of both human and mouse brain will be analyzed using the new rMATS analysis tool to create a large statistically robust database of differential gene expression and alternative pre-mRNA splicing between males and females. Expression and splicing changes will be correlated with changes in PTBP1/2 mRNA and Xist across the same datasets to define genes potentially regulated by these molecules at the transcriptional and post- transcriptional levels. Female specific patterns of expression and splicing caused by the XX genotype will be distinguished from events driven by female hormones using four core genotype mice. Xist targeting will be confirmed using conditional Xist alleles that allow either removal or activation of Xist during brain development and measurement of the resulting changes in splicing. The expression of Xist relative to PTBP2 will be quantified over neuronal differentiation in culture. The PTBP targeting of Xist-dependent changes in splicing will be confirmed in PTBP2 knockout and PTBP1 transgenic mice, and by transcriptome-wide binding analyses by iCLIP. Alternative splicing is a widespread mechanism of gene regulation, but has been only minimally examined in relation to the XX genotype of female cells. Using sophisticated new genome-wide methods and molecular tools, these studies promise to identify new genetic determinants of sexual dimorphism in the mammalian brain and to elucidate their underlying molecular mechanisms. In the longer term, the identified molecular changes driven by Xist and PTBP will provide entrée to the examination of the functional consequences of these dimorphisms.

Administrative Core Program Lead: Kathrin Plath, PhD. The Reprogramming P01 Administrative Core will support all of the Projects of this Program. Kathrin Plath, Program PI, will be the Leader of the Administrative Core, with support from Drs. Zaret and Hochedlinger as Co-Investigators. This Core will enable the scientific components of the Program to interact by providing the structure and financial support for regular planning, data sharing, and evaluation of the research. The Core will be responsible for ensuring that the investigators act as a cohesive and interactive group by providing the planning and leadership required for regular and frequent interactions among the Projects. In addition, the Core will ensure efficient collaborations with the existing genomics, computational, and stem cell research centers at UCLA, UPenn, and MGH to provide optimal access to basic resources and infrastructure and leverage existing high-throughput sequencing, bioinformatics, and stem cell resources at the respective institutions. The Core will recruit additional investigators to the study of pluripotency and reprogramming through the distribution of pilot projects. The Administrative Core will also provide centralized grant administration, reporting to the NIGMS, budget oversight and management, and will ensure compliance with all governing IBC and ESCRO regulations and policies. Program interactions and oversight will be facilitated and evaluated by a Scientific Advisory Board composed of both internal and external experts in stem cell biology, reprogramming, and epigenetics. Through all of its activities, the Administrative Core will provide the infrastructure for the Program Project mechanism to enhance synergism and productivity in our thematically linked proposals.

PROJECT SUMMARY The overall goal of this proposal is to reveal the molecular mechanisms by which the reprogramming factors Oct4 (O), Sox2 (S), Klf4 (K), and cMyc (M) revert somatic cells to iPSCs. Conversion of somatic cells to pluripotency is the most robust example of cellular reprogramming. In many other cases of transcription factor (TF)-induced cell fate conversion, for example the direct conversion of fibroblasts to neurons, cells fail to completely silence the starting cell transcriptome. Thus, uncovering mechanisms by which OSKM inactivate the somatic program and activate the pluripotency program during reprogramming to iPSCs will reveal important principles of how cell identity is maintained and effectively converted, and enable novel approaches to make cellular reprogramming, including lineage conversions, more efficient and faithful. In preliminary studies, we determined the genomic locations of OSKM early in reprogramming of mouse fibroblasts and in the pluripotent state, which revealed a dramatic change of OSKM targets during reprogramming and engendered new hypotheses about the action of OSKM. Early in reprogramming, the essential reprogramming factors OSK only engage a small set of pluripotency enhancers and, unexpectedly, bind extensively to somatically active enhancers. At this early time point, when very few transcriptional changes occur, we observed that almost all somatic enhancers lose active chromatin marks. Explaining how somatic enhancer decommissioning may occur, we found that somatic TFs are redistributed away from somatic enhancers early in reprogramming, to other genomic sites bound by OSK. Our findings lead to the unique hypothesis that OSK orchestrate reprogramming by mediating the activation of pluripotency enhancers as well as the silencing of somatic enhancers, and we speculate that the effect of OSK on somatic TFs is similarly important for reprogramming as step-wise pluripotency enhancer activation. Another crucial step that we defined in the reprogramming process is the reactivation of the inactive X chromosome (Xi). We discovered that Xi-reactivation is associated with DNA demethylation and is one of the final steps of reprogramming. How the disassembly of this heterochromatic structure is achieved is unknown. Based on our studies and collaborations with the Zaret lab on silencing via heterochromatin and the Hochedlinger lab on post-transcriptional control, we are well positioned to unveil the mechanisms underlying OSK's step-wise engagement of pluripotency enhancers, somatic enhancer decommissioning by OSK, and Xi-reactivation, with these Aims: 1) To uncover the mechanisms by which OSK target and modulate somatic and pluripotency enhancers early in reprogramming. 2) To define the co-factors required for the step-wise selection and activation of pluripotency enhancers by OSK. 3) To delineate mechanisms of Xi-reactivation and heterochromatin disassembly. While we will perform most experiments on fibroblast reprogramming, we will also investigate how OSKM can induce reprogramming across different cell types to define common pathways by which OSKM re-wire genetic networks.

PROJECT SUMMARY The goal of our Program renewal is to build upon the novel and unexpected discoveries of our last grant period to dissect, perturb, and control molecules and networks that enable or restrict the conversion of a somatic cell to pluripotency. Our last grant period revealed that, during conversion to pluripotency, resident enhancers in somatic cells that maintain cell differentiation must be decommissioned and pluripotency enhancers must be gradually activated, heterochromatic domains must be disassembled, and the post- transcriptional processes of polyadenylation and protein sumoylation must be altered. By investigating these parameters collectively in the next grant period, the Plath, Zaret, and Hochedlinger labs will determine how gene-regulatory networks integrate with cell biological transitions that are crucial for timely and efficient cellular reprogramming. Our three specific projects address the following fundamental questions: 1) How are enhancer patterns reorganized during the conversion of somatic cells to pluripotency, which genomic and epigenomic features are important, and how can heterochromatin be disassembled? 2) What comprises heterochromatin and how can its constituents be antagonized to facilitate reprogramming? 3) How do post-transcriptional regulation of mRNA polyadenylation and protein sumoylation suppress the pluripotency program? Answers to these mechanistic questions will provide insights to facilitate specific avenues to enhance cellular reprogramming to pluripotency as well as direct reprogramming from one somatic fate to another. The principles we uncover will also have direct applications for understanding cell type conversions in development, disease, and regeneration. Notably, new discoveries and scientific overlap between the three P.I.'s makes this a far more synergistic and interdependent proposal than the previous submission. The Program also takes advantage of extensive collaborations between our three groups, all leaders in the field of reprogramming, with a track record of working and publishing together. Our plan to evaluate chromatin and transcriptional features and post-transcriptional mechanisms within one Program will greatly enhance a sophisticated and cohesive view of how somatic cells can change fate to become pluripotent, identify commonalities and potential differences between human and mouse models, and allow us to determine how diverse features can be modulated coordinately to boost the efficiency and faithfulness of reprogramming to pluripotency and of one somatic fate to another. An Administrative Core will ensure efficiency and data sharing, leverages existing cores at our respective stem cell institutions, offer a pilot program to bring in additional investigators supporting our goals, and integrates a Scientific Advisory Board, and make sure that our goals are achieved collaboratively.

PROJECT SUMMARY The goal of this proposal is to investigate the role of ubiquitin-dependent protein degradation in initiating and maintaining X-chromosome inactivation (XCI), which produces the inactive X chromosome (Xi) to equalize the dose of X-linked genes between male and female mammals. XCI is necessary for proper development and depends on the long non-coding (lnc) RNA Xist, which implements silencing early in development. During initiation of XCI, Xist spreads in cis from its site of transcription on the X-chromosome to cover the entire chromosome, recruits repressive chromatin regulators, induces gene silencing, leads to the depletion of active chromatin and transcriptional regulators, and mediates chromosome-wide compaction. The molecular mechanisms underlying all these changes on the Xi during initiation of XCI and how they contribute to XCI maintenance are still poorly understood. Interestingly, studies of Xist deletion support a model where Xist promotes removal of active transcriptional and chromatin regulators from the Xi and continued removal is important to prevent reactivation of genes on the Xi. The mechanism by which Xist carries out this function is not understood. We recently discovered that a subunit of the anaphase-promoting complex/cyclosome (APC/C) accumulates on the Xi and that this localization depends on Xist. APC/C is a multi-subunit ubiquitin ligase that catalyzes the attachment of ubiquitin to target proteins to trigger degradation by the proteasome. APC/C functions in various cellular processes including regulating progression through different phases of the cell cycle and also has cell cycle-independent functions. Based on our preliminary findings, we hypothesize that Xist recruits APC/C to the Xi where it ubiquitinates regulators of active chromatin and transcriptional regulators, such as chromatin modifying enzymes or transcription factors, to promote their degradation by the ubiquitin- proteasome system. Here, we will test this model by defining the localization of APC/C and components of the ubiquitin-proteasome system on the Xi, and by examining how blocking APC/C and the ubiquitin-proteasome system affects the Xi. This work is a multi-PI application combining the expertise of two labs: Kathrin Plath in XCI and genomics approaches, and Lars Dreier in cell biological studies of the ubiquitin-proteasome system, allowing us to effectively integrate cell biological and genomic approaches. Our complementary strengths, together with promising preliminary data and expert support, position us well to explore a role for ubiquitin- dependent protein degradation on the Xi. The proposed experiments will provide a starting point for defining critical proteins that need to be continuously degraded to prevent a transcriptionally permissive environment on the Xi. Our work will also provide novel insights into the regulation of chromatin states by lncRNAs and may link the function of two fundamental processes occurring in the cell.

PROJECT SUMMARY The overall goal of this proposal is to study the regulation of X-chromosome dosage compensation by long- noncoding (lnc) RNAs in human embryonic stem cells (hESCs) to fundamentally advance the understanding of early human development, the regulation of pluripotency states, and mechanisms of gene regulation by lncRNAs. X-chromosome dosage compensation is an essential process that equalizes X-linked gene expression between females and males and is initiated early in development. In mammals, it is mediated by X-chromosome inactivation (XCI), the transcriptional silencing of genes on one of the two X-chromosomes in females. Mechanistic studies of XCI have been carried out in mice and established that the lncRNA Xist, encoded on the X chromosome, spreads along the X chromosome to mediate XCI. Although XCI occurs in both mouse and human post-implantation development, the regulation of X-chromosome dosage in pre-implantation embryos has evolved differently between the two species. In pre-implantation development, mouse embryos undergo an imprinted form of XCI, while human embryos lack imprinted XCI and instead regulate gene expression by dampening transcription on both X-chromosomes. X-chromosome dampening (XCD) is different from XCI as it occurs on both X chromosomes in contrast to the complete transcriptional silencing of one X. Additional remarkable differences between human and mouse include the unprecedented expression and accumulation of XIST on the active (dampened) X-chromosomes and the expression of the primate- and pluripotency-specific lncRNA XACT from the X in human pre-implantation embryos. Thus, X-linked gene dosage in humans is regulated first by XCD and upon implantation by XCI, and remarkably, XIST expression is uncoupled from silencing when XCD takes place. A mechanistic understanding of XCD, lack of silencing by XIST in pre- implantation embryos, XIST's transition to mediating XCI, and the role of the lncRNA XACT is lacking, and it is not known if XIST has any role in human pre-implantation cells or XCD. We discovered that naïve hESCs recapitulate in vitro many of the unique features of human X-chromosome dosage compensation, including XCD, expression of XIST on active X-chromosomes, XACT expression, and initiation of XCI upon differentiation. Here, we will take advantage of naïve hESCs to investigate how XIST expression does not induce silencing early in development and later acquires the ability to silence, to reveal regulatory mechanisms of human XCD and XCI, and if XACT controls XIST functions. We have the following Specific Aims: 1) We will define the function of XIST in naïve hESCs, its requirement for XCD, and mechanisms of XCD. 2) We will define chromatin targets and protein interactors of XIST to understand its differential silencing ability in naïve and primed hESCs. 3) We will characterize the function and localization of the lncRNA XACT. Understanding the mechanisms of human X- chromosome dosage compensation will allow us to advance our understanding of epigenetic features unique to human development and reveal how key epigenetic processes are changing in evolution.

PROJECT SUMMARY The reprogramming of somatic human cells to induced pluripotent stem cells (iPSCs) by only four transcription factors (TFs) Oct4, Sox2, Klf4, and cMyc (OSKM) is one of the most striking remodelings of gene regulatory networks. The remarkable ability of OSKM to reprogram diverse somatic cell types into iPSCs that are functionally indistinguishable from embryonic stem cells indicates that OSKM leverages a fundamental mechanism for network remodeling that may be generally applicable to all cell fate transitions. Previous studies of reprogramming have identified the crucial role of cooperative TF binding in repressing somatic programs and activating pluripotent ones. However, associating TF binding dynamics and epigenomic remodeling with key bifurcation events during reprogramming is confounded by the highly heterogeneous nature of the reprogramming process and the lack of knowledge regarding how the transition from somatic to pluripotent regulatory programs occurs in individual cells. In this project, we aim to model the regulatory network underlying the cell fate change of reprogramming using three types of single-cell multi-omic profiles generated from critical time points during reprogramming. We will interrogate the network leveraging natural perturbation of reprogramming and pluripotency by genetic variants. Genetic variation is well known to modulate the regulatory network of pluripotency and contributes to the variability of cellular phenotypes and differentiation capacity of iPSC lines. We will generate population-scale single-cell joint profiling of RNA and DNA methylation (snmCT- seq), joint profiling of RNA and chromatin accessibility (scRNA + ATAC-seq) and single-nucleus joint profiling of chromatin conformation and DNA methylation (sn-m3C-seq), allowing the cell-type-specific determination of transcriptome, chromatin accessibility and methylation states at regulatory elements, as well as enhancer-gene looping to connect non-coding variants to their regulatory target. To integrate OSKM binding with the single-cell transcriptomic and epigenomic dynamics, we will determine the allele-specific binding of TFs and histone modifications using a pooled-alleles ChIP-seq strategy. We will use Dynamic Regulatory Events Miner (DREM) to construct predictive models by integrating transcription factor-gene interaction information with time- and pseudotime-series genomics data. To determine the genetic regulation of the reprogramming network, we will apply the novel statistical method FastGxE to distinguish cell-type-specific from the shared genetic component of gene expression regulation, to enhance the sensitivity for identifying cell-type-specific quantitative trait loci (QTLs). To test the regulatory network, we will experimentally determine the function of network hub genes and non-coding variants using high-throughput CRISPR interference and precise variant replacement experiments. Our proposed project integrates diverse approaches including single-cell multi-omics, computational modeling, and genetic engineering, and will likely provide new insights into the mechanism by which TFs remodel regulatory networks of cell type identity and serve as a model for similar analyses in other systems.