Interrogating YY1's role in chromatin organization during erythroid cell state transitions

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Project Summary Chromatin structure plays an important role in instructing gene expression and maintaining cellular identity. However, much remains unknown regarding how the genome becomes reorganized during crucial transitions, such as cell cycle progression and cellular differentiation. Mitosis is marked by a global cessation of transcription, eviction of transcription factors, and the dissolution of most chromatin structure. During the mitosis to G1 phase transition, newly born cells must therefore address the challenge of rapidly re-establishing 3D genome organization that faithfully reflects that of the mother cell. While CTCF and cohesin-mediated loop extrusion has been shown to forge some chromatin loops, many observed architectural features cannot be explained by this mechanism. Another important architectural factor, YY1, has been implicated in enhancer-promoter loops in studies in interphase cells. However, its dynamics and role in chromatin loop formation has not been explored at the critical juncture between mitosis and G1 phase. We aim to characterize YY1 chromatin occupancy kinetics as it relates to the emergence of chromatin loops. We will test its necessity by interrogating effects of specifically timed depletion during metaphase and during G1 phase entry. We also propose to study the functional importance of YY1- mediated loop formation by characterizing transcriptional changes caused by mitotic depletion. Similarly, the role of YY1 in chromatin organization during hematopoiesis has yet to be clearly defined. YY1 has been proposed to be a regulator of loops essential for development, but previous work has only focused on select loci in limited cell types. To elucidate YY1’s involvement in genome reorganization during developmental transitions, we will characterize YY1 binding and corresponding architectural changes by generating high-resolution Micro-C maps before and after erythroblast maturation. We will also acutely deplete YY1 during differentiation to test its necessity in orchestrating looping reconfiguration in erythroblasts. By utilizing two natural state transitions – cell cycle and erythroid differentiation – as well as an acute degradation system, we aim to gain new insights into the fundamental mechanisms underlying genome organization.