DNA replication is central to the structural and functional integrity of the genome and intimately tied to large- scale 3D chromosome organization and cell lineage specification, but we understand little about its regulation. We have identified specific cis-elements, termed Early Replication Control Element (ERCEs), that regulate replication timing (RT), chromosome architecture, and transcription in murine embryonic stem cell (mESCs). ERCEs harbor acetylated histones, form CTCF/cohesin-independent 3D interactions and are co-occupied by pluripotency transcription factors Oct4, Sox2 and Nanog (OSN). What is not known is how these properties control chromosome structure and function and whether their activities are separable. Our long-term goal is to understand the relationship of RT to chromosome architecture, epigenetic states and disease. Our immediate goal is to identify mechanisms by which ERCEs co-regulate RT, chromatin architecture and transcription. Our central hypothesis is that ERCEs interact to create 3D hubs of histone acetylation that recruit replication initiation factors while independently regulating transcription. Our rationale is that elucidating mechanisms by which ERCEs co-regulate RT, transcription and genome architecture will open new horizons for studies of chromosome structure and function and, ultimately, its mis-regulation in disease states.
Aim1 will genetically dissect ERCEs to identify minimal sequences necessary and sufficient for their associated activities.
Aim2 tests the hypothesis that Rif1, which resides in late replicating chromatin but is necessary RT genome-wide, focuses histone acetylation to ERCEs to recruit the replication initiation protein Treslin through its interaction with histone acetylation binding proteins Brd2 and Brd4.
Aim3 will address the longstanding relationship of RT to transcription. We propose that cell type specific transcription factors create hubs of histone acetylation independent of their roles in RT and architecture. This contribution will be significant because it will lift a major barrier to the study of mechanisms regulating chromosome structure and function, how they are regulated during cell fate transitions and, ultimately, how they are mis-regulated in human disease. This work is innovative because the breakthrough discovery of ERCEs introduces novel hypotheses, concepts and approaches to the genome-architecture field.
The proposed project is important for public health because abnormalities in the temporal order in which chromosome segments are duplicated have been detected in many diseases, yet this replication timing process remains one of the most poorly understood chromosomal functions. The proposed experiments build on a breakthrough discovery that makes possible for the first time a genetic analysis of replication timing that is certain to have major impact on our understanding of how replication timing is regulated. Thus, the proposed experiments are relevant to the part of NIH?s mission that pertains to developing fundamental knowledge that will increase our understanding of the pathogenesis of disease.
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