GSE159594
BMP4 and BMP6 stimulation of adult mouse muscle stem cells activates the Notch signaling pathway [RNA-seq I]
Muscle stem cell (MuSC) activation, proliferation, self-renewal, and differentiation are tightly regulated processes that enable muscle development and regeneration without exhaustion of the stem cell pool. BMP signaling keeps MuSCs in a proliferative state and prevents their differentiation. We investigated the effect of bone morphogenetic protein 4 and 6 (BMP4 and BMP6) on transcriptional regulation and intracellular signaling of adult murine MuSCs using RNA-sequencing, RNA expression arrays, pathway-specific qPCR arrays, and Cleavage Under Targets and Tagmentation (CUT&Tag), which identifies the binding sites of transcription factors and the presence of specific histone marks with high spatial resolution. Freshly isolated MuSCs were grown for 42 to 72 hours, kept serum deprived for 6 hours, and then stimulated with BMP4/6. BMP4/6 addition resulted in a rapid increase in Inhibitor of DNA binding 1 (Id1)-expression, a typical BMP target gene, which peaked after one hour. As expected, stimulation with BMP4/6 resulted in a significant increase in transcription of BMP target genes such as Smad7, Nog, Id1, Klf10, and Lif, but also a strong increase in transcription of Notch pathway genes such as Hes1, Lfng, and Hey1. We showed that the important BMP pathway effectors SMAD4 and pSMAD1/5/9 bind to the promoters of these Notch targets. Our results suggest that the rapid expression of genes encoding Notch signaling components is due to direct regulation by BMP signaling. Overall design: Muscle stem cells were isolated from muscle of female 6-8-week-old mice, cultured on Matrigel® for expansion over 2 days. In order to limit the effect of BMPs in the culture medium (e.g. from serum supplements), cells were cultured prior to the BMP4 or BMP6 stimulation for 6 h in serum-free medium containing BMP soluble receptor (recombinant mouse BMPR-IA/ALK3-Fc Chimera, R&D Systems, 437-MR) in a concentration of 200 ng/ml. Cells were then stimulated with a pulse of 1 h with 100 ng/ml BMP4 or BMP6 or only with vehicle used for reconstitution of BMP in the case of controls. Cells were harvested thereafter and total RNA was extracted.