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:
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.
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