U01HG012079

Leveraging Genetic Variation to Dissect Gene Regulatory Networks of Reprogramming to Pluripotency - 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.

Los Angeles University Of California

NHGRI SUPPORTS THE DEVELOPMENT OF RESOURCES AND TECHNOLOGIES THAT WILL ACCELERATE GENOME RESEARCH AND ITS APPLICATION TO HUMAN HEALTH AND GENOMIC MEDICINE. A CRITICAL PART OF THE NHGRI MISSION CONTINUES TO BE THE STUDY OF THE ETHICAL, LEGAL AND SOCIAL IMPLICATIONS (ELSI) OF GENOME RESEARCH. NHGRI ALSO SUPPORTS THE TRAINING AND CAREER DEVELOPMENT OF INVESTIGATORS AND THE DISSEMINATION OF GENOME INFORMATION TO THE PUBLIC AND TO HEALTH PROFESSIONALS. THE SMALL BUSINESS INNOVATION RESEARCH (SBIR) PROGRAM IS USED TO INCREASE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, TO INCREASE SMALL BUSINESS PARTICIPATION IN FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION. THE SMALL BUSINESS TECHNOLOGY TRANSFER (STTR) PROGRAM IS USED TO FOSTER SCIENTIFIC AND TECHNOLOGICAL INNOVATION THROUGH COOPERATIVE RESEARCH AND DEVELOPMENT CARRIED OUT BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO FOSTER TECHNOLOGY TRANSFER BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO INCREASE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION.

93.172 - Human Genome Research

National Human Genome Research Institute (NHGRI) [HHS - NIH]

Los Angeles, California 900958348 United States

Single Zip Code

Defining Genomic Influence on Gene Network Regulation (U01 Clinical Trial Not Allowed) (RFA-HG-20-044)

Amendment Since initial award the total obligations have increased 533% from $950,000 to $6,016,057.