Project description:Synthetic transcription factors can be applied to many areas of biotechnology, medicine, and basic research.Â Currently, the most common method for engineering synthetic transcription factors has been based on programmable DNA-binding domains of zinc finger proteins, Transcription Activator-Like Effectors (TALEs), and most recently the CRISPR/Cas9 system. These transcription factor platforms consist of the DNA-binding domain fused to potent transcriptional activation domains, most commonly the tetramer of the minimal transactivation domain of the VP16 protein from herpes simplex virus, referred to as VP64. Although many applications are well-suited for the targeted activation of a single gene, genetic reprogramming requires the coordinated regulation of many nodes of natural gene networks as is typically performed by naturally occurring reprogramming factors. Thus we sought to combine principles from each of these approaches by attaching potent transcriptional activation domains to a natural reprogramming factor to increase the efficiency and/or rate of cell fate conversion. In this study, we evaluated the effects of fusing potent activation domains to the transcription factor MyoD, the master regulator of the skeletal myoblast lineage. In certain non-myogenic lineages, MyoD overexpression causes upregulation of the myogenic gene network and conversion to a myoblast phenotype including cell fusion into multinucleated myotubes. Compared to wild-type MyoD, the VP64-MyoD fusion protein induced greater overall reprogramming of global gene expression. This simple approach for increasing the potency of natural reprogramming factors circumvents the need for screening engineered proteins and leads to a more robust cellular reprogramming compared to treatment with the wild type transcription factor. Human dermal fibroblasts were transduced with a single tet inducible lentivirus that expresses either WT-MyoD or VP64-MyoD in response to treatment with doxycycline. Untreated human dermal fibroblast served as the negative control. Gene expression was measured using mRNA-seq, and differential expression was calculated using DESeq. All experiments were performed in biological duplicates.

Project description:Direct lineage reprogramming provides a unique system to study cell fate transitions and unearth molecular mechanisms that safeguard cellular identity. We previously reported on direct conversion of mouse fibroblasts into induced myogenic progenitor cells (iMPCs) by means of transient MyoD overexpression in concert with small molecules treatment. Here we employed integrative multi-omic assays to delineate the molecular landscape of fibroblast reprogramming into iMPCs in comparison to transdifferentiation into myogenic cells solely by MyoD overexpression. Utilizing bulk RNA-sequencing and mass spectrometry we characterized molecular regulators and pathways that endow a myogenic stem cell identity on fibroblasts only in the presence of small molecule treatment. We further demonstrate that iMPC reprogramming is a stepwise process, commencing with the appearance of myofibers and committed myogenic progenitors prior to the formation of satellite cell-like myogenic progenitors that express a suite of stem cell markers including Pax7, Dek, Myf5, Sox8, and Dmrt2. To directly compare iMPCs to satellite cell-derived primary myoblasts, we employed a fluorescent Pax7-GFP reporter to purify Pax7+ cells from the heterogenous iMPC cultures and assess their equivalency to FACS-purified Pax7-GFP+ myoblasts. We demonstrate that Pax7+ iMPCs share molecular attributes with myoblasts, however also express genes and signaling pathways that are indicative of activated satellite cells including Carm1, Dlk1, Lgr5, Fos, Dek, Calcr and Pitx3. We further establish that iMPC formation and maintenance is dependent on the Notch pathway, as small molecule inhibition of Notch abrogates iMPC formation and derails stable iMPC cultures via depletion of Pax7 expressing cells. Lastly, using single cell RNA-sequencing we determine the cell populations that comprise iMPCs in addition to reconstructing the differentiation trajectory present in these heterogenous cultures. We demonstrate that a highly proliferative activated satellite cell-like population differentiates into committed myoblasts and myocytes that further give rise to myofibers that express mature muscle markers, thus capturing a dynamic differentiation program in vitro. Collectively, this study charts a molecular blueprint for reprogramming fibroblasts into myogenic stem and progenitor cells and further establishes the fidelity of stable iMPC cultures in capturing skeletal muscle regeneration in vitro for disease modeling and basic research applications.

Dataset Information

Label-free quantification (LFQ) analysis on skeletal muscle stem cells and reprogrammed skeletal muscle stem and progenitor cells

Publications

Integrative molecular roadmap for direct conversion of fibroblasts into myocytes and myogenic progenitor cells.

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