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Myogenic Progenitor Cell Lineage Specification by CRISPR/Cas9-based Transcriptional Activators

ABSTRACT: Myogenic Progenitor Cell Lineage Specification by CRISPR/Cas9-based Transcriptional Activators

PROVIDER: PRJNA607567 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11116170_1.fastq.gz	Fastqsanger.gz
	SRR11116170_2.fastq.gz	Fastqsanger.gz
	SRR11116171_1.fastq.gz	Fastqsanger.gz
	SRR11116171_2.fastq.gz	Fastqsanger.gz
	SRR11116172_1.fastq.gz	Fastqsanger.gz

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Myogenic Progenitor Cell Lineage Specification by CRISPR/Cas9-based Transcriptional Activators

Project description:Engineered CRISPR/Cas9-based transcriptional activators can potently and specifically activate endogenous fate-determining genes to direct differentiation of pluripotent stem cells. Here, we demonstrate that endogenous activation of the PAX7 transcription factor results in stable epigenetic remodeling and directly reprograms human pluripotent stem cells into skeletal myoblast progenitor cells. Compared to the exogenous overexpression of PAX7 cDNA, we find that endogenous activation results in the generation of more proliferative myogenic progenitors that can maintain PAX7 expression over multiple passages in serum-free conditions while preserving the capacity for terminal myogenic differentiation. Transplantation of human myogenic progenitors derived from endogenous activation of PAX7 into immunodeficient mice resulted in a greater number of human dystrophin+ myofibers compared to exogenous PAX7 overexpression. RNA-seq analysis also revealed transcriptome-wide differences between myogenic progenitors generated via CRISPR-based endogenous activation of PAX7 and exogenous PAX7 cDNA overexpression. These studies demonstrate the utility of CRISPR/Cas9-based transcriptional activators for progenitor cell specification and their potential for regenerative medicine.

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Myogenic Progenitor Cell Lineage Specification by CRISPR/Cas9-Based Transcriptional Activators.

Project description:Engineered CRISPR/Cas9-based transcriptional activators can potently and specifically activate endogenous fate-determining genes to direct differentiation of pluripotent stem cells. Here, we demonstrate that endogenous activation of the PAX7 transcription factor results in stable epigenetic remodeling and differentiates human pluripotent stem cells into skeletal myogenic progenitor cells. Compared with exogenous overexpression of PAX7 cDNA, we find that endogenous activation results in the generation of more proliferative myogenic progenitors that can maintain PAX7 expression over multiple passages in serum-free conditions while preserving the capacity for terminal myogenic differentiation. Transplantation of human myogenic progenitors derived from endogenous activation of PAX7 into immunodeficient mice resulted in a greater number of human dystrophin+ myofibers compared with exogenous PAX7 overexpression. RNA-sequencing analysis also revealed transcriptome-wide differences between myogenic progenitors generated via CRISPR-based endogenous activation of PAX7 and exogenous PAX7 cDNA overexpression. These studies demonstrate the utility of CRISPR/Cas9-based transcriptional activators for controlling cell-fate decisions.

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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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