Ubiquitination-coupled transcriptional control of muscle stem cell quiescence and activation

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Summary Postnatally, skeletal muscle retains a small population of muscle stem cells (MuSCs) which will exit cell cycle, acquire a quiescent state (G0) but are capable of cell cycle reentry. Unlike embryonic development where proliferating progenitors give rise to new muscle, adult MuSCs must rapidly transition from G0 to reenter cell cycle upon receiving extracellular cues to maintain tissue. How MuSCs swiftly and reversibly transition between quiescent and activated states is a fundamental question which remains poorly understood. We have recently discovered that an integrated mechanism via coupling of ubiquitin proteasome system (UPS) and transcriptional control by chromatin remodelling is essential for MuSCs quiescence. Inhibition of UPS in MuSCs leads to their spontaneous activation. Subsequent analyses showed that Nedd4l, an E3 ligase may play a central role in bridging the UPS with transcriptional control of gene expression in MuSCs by modulating the protein level of Wdr5 and Baf53a. The latter proteins are critical components of the Ash2l/MLL histone methyltransferase (HMT) and the SWI/SNF chromatin remodeling complexes respectively. We hypothesize that maintenance of MuSCs quiescence and their rapid activation during regeneration is achieved by a mechanism in which active recycling and degradation of key chromatin remodeling factors by UPS blocks MuSCs from entering the cell cycle. Therefore, UPS may be required to keep MuSCs in a "poised" state. In short term, we aim to molecularly define how Nedd4l links UPS with chromatin remodelling as defined by the three aims below. Our long-term goal is to unravel how MuSCs broadly integrate UPS with transcriptional mechanisms to maintain tissue homeostasis. I anticipate that the knowledge gained from this program can be used as a model to study stem cells and the homeostasis of other tissue types. AIM1: We will combine FACS with LC-MS/MS to determine the proteomic signature of Nedd4L in freshly sorted MuSCs from Nedd4L-cKO and WT mice. Aim1 is the extension of the analysis performed in Figure 5 and will be carried out on multiple biological and technical replicates. AIM2: We will determine the role of Nedd4L in quiescence, self-renewal and differentiation using an inducible Nedd4Lf/f; Pax7CreERT2 and constitutive conditional knockout, Nedd4Lf/f; Pax7Cre to study the effect of loss of Nedd4l temporally and during long-term development into late adulthood (8-month-old). We will perform differential gene expression and pathway analysis and validate high-priority candidates by RT-qPCR and immunofluorescence staining on myofiber-associated MuSCs from the Extensor Digitorum Longus (EDL) muscle. AIM3: We will determine the epigenetic alterations caused by genetic deletion of Nedd4L in freshly sorted MuSCs by applying Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) and ChIP-seq for H3K4me3 (active mark deposited by Ash2l/MLL/Wdr5) complex and by ChIP-seq of Baf53a.