Supplementary MaterialsSupplementary Information guide. differentiation factor Mef2c10,11. However, Notch signalling has broader functions in muscle cells, including maintenance of quiescence4,5. To explore these functions, we exploited a ChIP-seq screening12 and observed that intracellular Notch (NICD) and its downstream effector RBPJ, occupied and regulated enhancers proximal to collagen genes and (Extended data Fig. 2a-e). Moreover, transcriptional induction of and by NICD translated to elevated COLV protein levels, specifically the [(a1(V)a2(V)a3(V)] isoform (3-COLV), in foetal forelimb (Fig. 1c) and adult hindlimb (3-COLV production. As and transcripts are downregulated upon exit from quiescence (Extended data Fig. 1a and Extended data Fig. 2g), no 3-COLV was detected in freshly isolated or activated satellite cells. Instead, genetic overexpression of NICD resulted in abundant, newly synthetized 3-COLV (Fig. 1e, f). Open in a separate window Figure 1 NICD/RBPJ regulates transcription of and genes by binding to distal regulatory elements.(a) RBPJ/NICD ChIP-seq tracks from C2C12 cells indicating enhancers associated with the and loci. Orange rectangle, RBPJ/NICD enhancers; asterisk, enhancers used for luciferase assays (Extended data Fig. 1c). (b) foetuses show upregulation of COLV. Inset shows low 3-COLV expression (higher exposure time). Note, membrane GFP-marked fibres in control and mononucleated NICD/PAX7+ cells in muscles, (t0h, left) or after 24h in culture (right) and stained for GFP and 3-COLV. (f) Vertical and horizontal optical sections of myofibre presented in (e) from mice (24h culture) showing COLV surrounding NICD-GFP+ satellite cells. Scale bars: c, 50m; d-f, 10 m. Scale bar NU-7441 (KU-57788) insets: c, 100 m; d, 20 m. To assess the functional role of COLV, isolated satellite cells were incubated with COLI, COLV, or COLVI in the presence of EdU, and stained for PAX7, that marks muscle stem/progenitor cells, and the muscle commitment (MYOD) and differentiation (Myogenin). Strikingly, only the COLV-complemented medium delayed entry of quiescent cells into the cell cycle (32h, Fig. 2a) and consequently their proliferation and differentiation (72h, Fig. 2b; 10d Extended data Fig. 3a-c). As shown previously4,13, cells underwent precocious differentiation, and this was partially antagonized by COLV, consistent with the finding that genes are NICD/RBPJ targets (Fig. 2c, d and Extended data Fig. 3d-g). Taken together, these results show that COLV specifically sustains primary muscle cells in a more stem-like PAX7+ state, indicating that it could potentially play a role in the quiescent niche. Open in a separate window Figure 2 Collagen V delays proliferation and differentiation of satellite cells.(a) EdU pulse (2h) of isolated satellite cells cultured for 32h: COLI (35%), COLVI (34%), COLV (18%); (n=4 mice, 250 cells, 2 wells/condition). (b) Immunostaining of isolated satellite cells cultured for 72h. PAX7: 58%, 55% and 81%; Myogenin: 56%, 57% and 24% for COLI, COLVI and COLV, respectively (n=4 mice, 250 cells, 2 wells/condition). NU-7441 (KU-57788) (c) Experimental scheme for satellite cells plated overnight (o/n) before collagen treatment. Mouse monoclonal antibody to KDM5C. This gene is a member of the SMCY homolog family and encodes a protein with one ARIDdomain, one JmjC domain, one JmjN domain and two PHD-type zinc fingers. The DNA-bindingmotifs suggest this protein is involved in the regulation of transcription and chromatinremodeling. Mutations in this gene have been associated with X-linked mental retardation.Alternative splicing results in multiple transcript variants (d) Immunostainings of satellite cells incubated with collagens for 60h (n=3 mice, 200 cells, 2 wells/condition). Error bars, mean SD; two-sided paired t-test; #p-value: two-sided unpaired t-test. Scale bar: 50m. To determine if collagen V produced by satellite cells is a functional component of the niche, we generated compound (cKO) mice, in which COLV was NU-7441 (KU-57788) depleted and simultaneously lineage-traced in GFP+ satellite cells4,14 (Fig. 3a and Extended data Fig. 4a). As the 1-chain of COLV is present in all COLV isoforms, which are trimeric, deletion produces complete COLV-deficient cells14. Remarkably, given the general stability of collagens, targeted deletion of resulted in upregulation of the differentiation markers and only 18d after tamoxifen treatment (Fig. 3b). Mutant cells also showed ectopic expression of Myogenin (Fig. 3c), increased BrdU incorporation (Fig. 3d), and a significant decline in PAX7+ satellite cells (Fig. 3e). The cKO cells did not undergo apoptosis (data not shown), but fused to give rise to GFP-marked myofibres (Fig. 3f). Therefore, blocking satellite cell-produced COLV resulted in their spontaneous exit from quiescence and differentiation, a phenotype reminiscent of Notch loss-of-function4,5. Open in a separate window Figure 3 Satellite.