Quiescence to Activation: An Epigenetic Study of the Co-activators of the Homeoprotein Six1 in Muscle Stem Cells

Abstract

Adult skeletal muscle regeneration depends on muscle stem cells, which are, in non-injury conditions, quiescent but activate in response to injury. Within the earliest hours after disruption of their niche, MuSCs begin to reorganize their transcriptional and chromatin states. Although chromatin accessibility changes rapidly during this period, the enzymatic activities that establish and maintain this epigenetic landscape remain incompletely understood. This thesis focuses on three chromatin-linked regulatory systems with the potential to shape early MuSC activation: p300/CBP-mediated acetylation, BRG1/BRM-dependent SWI/SNF remodelling, and CDK9/P-TEFb-associated kinase activity. To investigate these pathways, primary MuSCs were isolated from adult mice by fluorescence-activated cell sorting and, during the isolation, were exposed to selective inhibitors. A-485 was used to inhibit p300/CBP acetyltransferase activity, BRM014 was used to inhibit BRG1/BRM ATPase activity, and NVP-2 was used to inhibit CDK9 kinase activity. OMNI-ATAC-seq was then used to profile genome-wide chromatin accessibility, with ATAC-qPCR and fragment analysis as quality control measures for library evaluation. In parallel, proximity ligation assays were used to assess whether Six1, a homeodomain transcription factor implicated in adult MuSC regulation, is detectably proximate to candidate co-regulators including p300, BRG1, and CDK9. Inhibitor-based accessibility profiling showed that early MuSC chromatin is not uniformly dependent on a single co-regulatory pathway. BRG1/BRM ATPase inhibition caused broad reductions in accessibility. p300/CBP inhibition produced a more restricted accessibility response which may suggest that the contribution of acetyltransferase activity is limited to a specific subset of regulatory elements as it relates to chromatin accessibility. CDK9 inhibition suggested transcription-linked accessibility dependencies which may support the idea that productive transcriptional elongation and chromatin accessibility are coordinated during early activation. Comparative analysis revealed some shared responses and highlighted inhibitor-specific responses. Because Six1 has been implicated in myogenic transcriptional control and chromatin regulation, this thesis also considered whether inhibitor-sensitive regions may intersect with Six1-associated regulatory logic. Integration of accessibility changes with Six1-related features suggested that subsets of differentially accessible regions overlap with regulatory environments relevant to Six1-dependent myogenesis. PLA analysis provided complementary evidence that Six1 can be detected in proximity to p300 and BRG1 in MuSCs. This supports the possibility that Six1 participates in multiple regulatory contexts, where different chromatin-modifying, remodelling, or elongation-associated activities may contribute at distinct loci. Together, these findings show that early MuSC activation depends on multiple chromatin-regulatory activities with distinct effects on accessibility. SWI/SNF remodelling, p300/CBP-mediated acetylation, and CDK9-dependent transcriptional regulation each contribute to different aspects of the accessible chromatin landscape during the early activation window. By integrating inhibitor-based chromatin profiles with Six1-centered DNA-binding data and proximity analysis, this thesis connects Six1-associated regulatory regions to distinct co-activator-dependent chromatin states during early adult MuSC activation.