br Results br Discussion Muscle recovery after

Results
Discussion Muscle recovery after traumatic injuries such as an occupational crush or blast suffered during military combat canonically induces a predisposition for additional injuries and chronic pain owing to incomplete regeneration of the tissue. Our understanding of how different factors translate to influence the SB203580 architecture that regulates muscle regeneration has been limited. Herein, we used integrative genomic mapping technologies to profile coding and noncoding expression and the in vivo chromatin state of various cis-regulatory elements, and found successive waves of transcriptional and chromatin changes during the course of healing. To obtain broad, unbiased views of the diverse repair and regeneration pathways utilized by different cell types after trauma, we performed profiling of both whole, unfractionated muscle tissue and FACS-sorted SCs (Liu et al., 2015). Whole-tissue profiling enabled capture of signals emanating from multiple cell types and the capacity to profile multiple chromatin modifications from a single tissue without pooling tissues from multiple animals. The contralateral/uninjured tissue was used as the control for this study; however, recently (Rodgers et al., 2014) it was shown that systemic signals induced from acute trauma stimulate SCs within the contralateral muscle to undergo a transition to an alert state that primes their differentiation potential. Thus, while we normalized changes to the contralateral tissue, the normalization may not be reflective of an unperturbed muscle. Pathway analysis of the time-clustered whole-tissue RNA-seq data revealed waves of transcription associated with proinflammatory and immune responses in the early period, followed by transcriptional signatures associated with myogenic differentiation and ECM remodeling in the middle and late time periods. Disruption of the muscle integrity by acute trauma also produced noncoding RNA (ncRNA) dynamics very similar to those observed in other muscle myopathies (Neguembor et al., 2014; Cesana et al., 2011; Eisenberg et al., 2007), suggesting the modulation of common molecular pathways. miRanda-mirSVR (Betel et al., 2010) was used to study the interaction of dynamic miRNA-mRNA pairs and whether their expression patterns changed concomitantly with time. We identified 200 miRNA-mRNA mutually dynamic relationships and clustered the pairs into three categories of immune regulation, TGF-β signaling, and ECM and cytoskeletal remodeling followed by myogenic differentiation. These three clusters highlight a temporal regulatory program whereby infiltrating immune cells release signaling molecules that trigger SC activation, followed by a transition whereby the activated progenitors proliferate and are repressed from differentiating by TGF-β signaling and changes to the surrounding matrix. These repressive cues were extended via other regulatory mechanisms (see below) and began to subside at 72 hr when a new class of ncRNAs (miR-206, miR-205, miR-203, miR-1a, miR182, miR-31, H19, and linc-MD1) increased in expression. Similar to the mRNA and miRNA-seq results, three clusters of differentially expressed lncRNAs were detected. Interestingly, H19 (which was upregulated in the middle and late periods) and another lncRNA called lncMyoD (which was upregulated in the early and middle periods) bind mRNA IGF2 binding proteins, suggesting that induction and stabilization of this pathway may be regulated temporally via different combinations of lncRNAs. Since muscle repair and regeneration utilizes many feedforward and feedback loops, tightly regulated expression patterns across multiple levels may facilitate a precise way to prevent extrinsic signal propagation from adjacent tissues that are also regenerating or responding to the injury, reinforce or tune noisy expression patterns, and perhaps facilitate a metabolically efficient mechanism to quickly respond after injury.