cis-Acting Elements Regulating Developmental Control of Replication Timing

San Diego Biomedical Research Institute, San Diego CA

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Abstract

PROJECT SUMMARY / ABSTRACT DNA replication is central to the structural and functional integrity of the genome and intimately tied to large- scale 3D chromosome organization, cell lineage specification and disease, but we understand little about its regulation. We have identified specific cis-elements, termed Early Replication Control Element (ERCEs), that are essential for replication timing (RT), chromosome architecture, and transcription in murine embryonic stem cell (mESCs). These novel elements likely coordinate the major correlated features of large-scale chromosome structure and function, underscoring an urgent need to understand how they function and whether they can be exploited in human health and disease. Our long-term goal is to understand the relationship of RT to chromo- some architecture, epigenetic states and disease. Our immediate goal is to identify mechanisms by which ERCEs co-regulate RT, chromatin architecture and transcription. ERCEs harbor acetylated histones, form CTCF/cohesin-independent 3D interactions and are co-occupied by pluripotency transcription factors Oct4, Sox2 and Nanog (OSN) in mESCs. Preliminary data show that their activity is DNA sequence encoded and can be transferred to ectopic sites to manipulate RT. Our central hypothesis is that lineage-specific transcrip- tion factors bind to ERCEs and seed a sub-nuclear microenvironment that potentiates early RT, which in turn drive modifications in chromatin and genome architecture. Our rationale is that elucidating mechanisms by which ERCEs co-regulate RT with transcription and genome architecture will open new horizons for studies of chromosome structure and function, ultimately providing tools to correct RT aberrations in disease states and to develop more robust cellular therapeutics. Aim1 will identify sequence motifs and trans-acting factors under- pinning ERCE activity, a critical first step to probe mechanisms regulating RT and to develop tools to manipu- late RT. Aim2 will examine mechanisms by which multiple ERCEs cooperate to elicit large changes in RT and the structural and functional consequences of altering RT to the engineered chromosome domain. Aim3 will clarify the longstanding enigmatic relationship between RT and transcription during cell fate transitions. This contribution will be significant because it will lift a major barrier to the study of mechanisms regulating chromo- some 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 intro- duces novel hypotheses, concepts and approaches to the genome-architecture field.