PROJECT SUMMARY: Comprehensive regulation of transcription is a major mechanism by which immature cells functionally determine their identity. The epigenetic factors responsible for this transcriptional regulatory activity are some of the most potent, and most commonly mutated, factors implicated in tissue homeostasis and disease. We understand the role of many epigenetic factors responsible for activating transcription, but we have a limited understanding of the factors that repress transcription associated with differentiation. Additionally, we know many of the epigenetic transcriptional regulators present on the chromatin before and after replication, but we have a limited understanding of how these factors interact during replication. The relative lack of understanding of how the replication machinery regulates transcription associated with cell identity is a key knowledge gap in the field of epigenetics. Our studies suggest that the Chromatin Assembly Factor 1 (CAF1) complex, functioning in its canonical role of replication-linked chromatin assembly, is the critical factor responsible for directly regulating fate-specific transcription during replication. CAF1 is a heterotrimeric protein complex responsible for facilitating histone H3/H4 heterodimer assembly at the replication fork during the S phase of cell cycle. Our lab focuses on the functional contributions of CAF1 through studying its p60 subunit: Chromatin Assembly Factor 1B (CHAF1B). We are using CHAF1B as a model for CAF1 function because of the abundance of tools and expertise we have generated to study CHAF1B and the overwhelming evidence that readouts of CHAF1B and the CAF1 complex are functionally identical. CHAF1B is highly expressed in uncommitted stem cells, and its expression decreases as cells differentiate. We recently reported that CHAF1B directly binds chromatin at promoters and enhancers of differentiation genes, repressing their expression by blocking transcription factor binding. Depletion of CHAF1B in independent models led to massive upregulation of gene activation caused by transcription factors flooding regions of chromatin previously occupied by CHAF1B. This led to our hypothesis: CHAF1B is a master regulator of cell identity through a novel role as transcriptional repressor of fate genes in immature cells. Our long-term goal is to understand how cells regulate transcription associated with cell fate throughout differentiation. Our short-term goal is to understand how the chromatin assembly machinery affects transcriptional repression of fate genes in immature cells. In this proposal we will study the functional roles of the factors responsible for CHAF1B direct binding to chromatin on transcription and phenotype (Focus 1), and the contributions of the DNA/protein complexes recruited by CHAF1B to the chromatin on transcription and phenotype (Focus 2). Discoveries about CHAF1B, the CAF1 complex, and the larger DNA/protein chromatin landscape it promotes, have t...