RNA seq of SCs reinforced

RNA-seq of SCs reinforced transcriptional dynamics observed from whole tissues, whereby in the early period, immune-response and chemokine transcripts were upregulated, followed by proliferation and differentiation during the middle time period. Several TFs associated with quiescent biotin-LC-LC-tyramide cost (Pax3, Pax7, Foxo3, Spry1) and immune response (c-Jun, c-Fos, Atf3, Egr1) exhibited downregulation at the same time that genes associated with cell-cycle progression (Ccna2, Ccnb1, Dnmt1, Birc5), differentiation, and fusion (MyoG, Hes6, Tmem8c, Cdh15) were upregulated (>72 hr). During the same time period, we also observed a switch from high to low expression of lysine demethylase 6A (Kdm6a or Utx), which regulates removal of the repressive H3K27me3chromatin mark (Faralli et al., 2016), to low to high expression of a component of a histone methyltransferase complex (Ash2l). Ash2l is part of the Trithorax complex that specifically methylates the fourth lysine residue of histone H3 (H3K4) and is targeted by Pax7 to activate Myf5 target genes (Almada and Wagers, 2016). Combining these dynamics suggests that in the early time period, upregulated TFs and chromatin remodelers help to establish a more accessible chromatin state at myogenic loci, which is consistent with the increase in enrichments of H3K4me3, H3K4me1, and H3K27ac. Moving into the middle period, as stress-responsive and other immune-activated transcripts decrease in expression, another set of TFs and chromatin remodelers is then upregulated and titrated via miRNAs (see below) to enact appropriate satellite cell proliferation and differentiation. miRNA-seq on the FACS-sorted SCs demonstrated that the programs of activation, proliferation, and subsequent differentiation are tightly controlled. For the time points linked with activation and mobilization (3–48 hr), several miRNAs were downregulated, such as miR-22, which is activated by the transcription factors AP-1 and NF-κB. Since miR-22 inhibits expression of Hdac4 and targets c-myc, which also suppresses MyoD-initiated myogenic differentiation and promotes proliferation, a regulatory program can be inferred whereby stimuli from infiltrating immune cells induce activation of TFs that promote the progenitors to activate, mobilize, and begin to proliferate but are repressed from differentiating. Other post-transcriptional factors such as miR-181, miR-191, and miR-222 appear to reinforce this program in the early time periods and as inflammation and immune-stimulated signatures drop in expression, another program begins around 72 hr via increases of expression of regulators of cell-cycle genes (miR-16, miR-182, miR-486) and myogenic differentiation (let-7 family, miR-206). The efficacy of myogenic transcription factors to initiate and maintain muscle gene expression programs depends critically on the chromatin state (Blais et al., 2005; Blum et al., 2012) of their targets as well as their interactions with each other (Liu et al., 2014; Molkentin et al., 1995) and other TFs. Quantifying the genome-wide changes in histone modifications revealed that a robust induction of chromatin remodeling occurs at a genome-wide level after muscle trauma, and cis-regulatory elements such as promoters were largely invariant when compared with regions demarcated by enhancer marks (H3K4me1, H3K27ac). Immediately after injury, the enriched enhancer regions were associated with canonical mediators of early stress, immunity, and growth factor responses, which was consistent with the upregulation of cytokines and TFs such as AP-1 (Fos/Jun), SRF, NF-κβ, Egr, and Stats. The detection of these transcripts and enriched enhancer sites was expected since an appropriate inflammatory response is essential for functional recovery after muscle injury. A significant fraction of the enriched chromatin sites correlated with transcriptional dynamics and was most likely attributable to infiltrating monocytes that secrete and respond to cytokines and chemokines. These early immune-related changes were serially followed by changes in expression and chromatin remodeling of genes associated with anti-inflammatory macrophages (Arnold et al., 2007) and activation and proliferation of SCs. During this period, upregulation of Smads and Tgf-β signaling was observed along with increases in expression of TFs such as Tead4 (Benhaddou et al., 2012), Runx1 (Umansky et al., 2015), and MyoD (Mullen et al., 2011). Smads have been shown to interact with chromatin remodeling complexes such as histone acetyltransferases p300 and CBP to induce H3K27 acetylation (Pouponnot et al., 1998), which was consistent with the observation that the highest number of sites that acquired H3K27ac was in the middle time period. In addition, MyoD has been shown to associate with p300 and p300/CBP on E-box motifs of target genes (Puri et al., 1997), and interactions between Smads and Tgf-β signaling have been shown to alter myogenic differentiation kinetics, suggesting a cooperative interaction between these different TFs and chromatin remodeling complexes during this phase of the injury response. Integrating these results reveals a transient shift in the balance of pro- and anti-inflammatory cytokine programs (high expression of Il-6 and low expression of Tgf-β in the early period to low Il-6 expression and high expression of Tgf-β in the middle period), which has previously been shown to promote the recruitment and differentiation of different types of cells that potentiate muscle repair (neutrophils, M1 macrophages, TH17 cells for the early period, and M2 macrophages and Treg cells for the middle period), influences chromatin state and expression programs associated with proliferation and restraint of myogenic differentiation (Dionyssiou et al., 2013).