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Muscle progenitor specification and myogenic differentiation are associated with changes in chromatin topology.

ABSTRACT: Using Hi-C, promoter-capture Hi-C (pCHi-C), and other genome-wide approaches in inducible Pax7-expressing skeletal muscle progenitors that inducibly express a master transcription factor, Pax7, we systematically characterized at high-resolution the spatio-temporal re-organization of compartments and promoter-anchored interactions as a consequence of myogenic commitment and differentiation. We identified key promoter-enhancer interaction motifs, namely, cliques and networks, and interactions that were dependent on Pax7 binding. Remarkably, we found that the majority of super-enhancers were bound by Pax7 and a cadre of associated factors that maintained an epigenetic memory of active enhancers in the absence of Pax7. Lastly, we identified a previously uncharacterized Pax7-bound enhancer hub that simultaneously regulates the essential myosin heavy chain cluster during skeletal muscle cell differentiation. Our studies lay the groundwork for understanding the three-dimensional conformation of chromatin in muscle stem cells.

ORGANISM(S): Mus musculus

PROVIDER: GSE150638 | GEO | 2020/10/31

REPOSITORIES: GEO

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Muscle progenitor specification and myogenic differentiation are associated with changes in chromatin topology

Project description:Using Hi-C, promoter-capture Hi-C (pCHi-C), and other genome-wide approaches in skeletal muscle progenitors that inducibly express a master transcription factor, Pax7, we systematically characterized at high-resolution the spatio-temporal re-organization of compartments and promoter-anchored interactions as a consequence of myogenic commitment and differentiation. To further investigate transcriptional regulatory mechanisms governed by Pax7, we used immuno-affinity purification and mass spectrometric sequencing to identify the compendium of Pax7-associated proteins in muscle progenitors. Although Pax7-associated proteins have been identified in myoblasts, it is likely that such an approach would fail to uncover progenitor specific interactions with this protein. We therefore isolated chromatin from iPax7 cells engineered with a single, inducible copy of the Flag-tagged Pax7 transgene integrated next to the Hprt locus, which is expected to maintain expression levels comparable to those of satellite cells in the presence of Dox. Through purification of Flag-Pax7, we identified a cohort of factors and complexes involved in gene activation or repression and remodeling of chromatin and genome architecture that were substantially enriched compared to the uninduced control. We also identified a large cohort of sequence-specific TFs, including several that were shown to play an essential role in muscle stem cells (e.g., Foxk1, Six1, Runx1, Tead1, Nfix) and that are recruited to Pax7-bound enhancers in muscle cells. Importantly, we also confirmed multiple interactions from our proteomic screen through immunoprecipitation and western blotting.

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Bulk RNA-sequencing of unsorted and PAX7::GFP-sorted ESC-derived myogenic progenitors

Project description:Tissue engineering strategies that combine human pluripotent stem cell-derived myogenic progenitors (hPDMs) with advanced biomaterials provide promising tools for engineering 3D skeletal muscle grafts to model tissue development in vitro and promote muscle regeneration in vivo. We recently demonstrated (i) the potential for obtaining large numbers of hPDMs using a combination of two small molecules and (ii) the application of electrospun fibrin microfiber bundles for functional skeletal muscle restoration following volumetric muscle loss. In this study, we demonstrate that the biophysical cues provided by the microfiber bundles can induce unsorted hPDMs to form multinucleated myotubes that express desmin and myosin heavy chain. To reduce the batch-to-batch variability of these samples, we tested a genetic PAX7 reporter line (PAX7::GFP) to sort for more homogenous populations of hPDMs. RNA sequencing and gene set enrichment analyses confirmed that PAX7::GFP-sorted hPDMs exhibited higher expression of myogenic genes while unsorted hPDMs demonstrated increased expression of genes associated with embryonic, neural crest, and fibroadipogenic progenitor lineages. We tested engineered skeletal muscle grafts derived from either PAX7::GFP-sorted or unsorted hPDMs within in vivo skeletal muscle defects. The unsorted hPDM-derived grafts also exhibited greater fibrosis and more proinflammatory responses. In contrast, the PAX7::GFP-sorted groups had moderately high vascular infiltration, more implanted cell association with embryonic myosin heavy chain (eMHC) regions, and greater CD206:CD86 ratios compared to unsorted hPDMs and acellular fibers suggesting they induced more pro-regenerative microenvironments. These findings demonstrated some promise for the use of PAX7::GFP-sorted hPDMS on fibrin microfiber bundles and provided some insights for improving the cell-biomaterial system to stimulate more robust in vivo skeletal muscle regeneration.

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