Unique intergenic TSS
After identification of TSS profile in both muscles, we found that substantial part of identified peakes does not intersect neither with Ensembl 99 nor with RefSeq 106 or 6.0 transcriptome annotation or FANTOM and thus were specific for muscle tissue (including 2246 out of 4149 DEGs in the slow muscle) and have never been described before. (See ST7).
A large number (2092) of intergenic TSS also conformed to aforesaid clusters along with the gene expression (Figure 3). Almost half of those intergenic TSS (995) had a corresponding DEG as the closest downstream gene. Surprisingly, the inclusion of the differentially expressed intergenic TSS dramatically improved the transcriptome-based clusterization of analyzed samples. (See SF for clusterization of genic peaks), which suggests strong, and yet to be explored in detail, involvement of alternative and distant promoters in the processes associated with muscle atrophy. These intergenic TSS clustered along with robust genic signatures also had a downstream DEG (394 genes in total) (Figure 3). As much as 152 of those DEGs were classified by GSEA analysis as signatures of chromatin reorganization, cytoskeleton organization, and cellular component biogenesis (See ST6). The non-genic robust signature peaks upstream of those genes were also differentially expressed on the first day of recovery (See heatmap in ST6), which is consistent with the GSEA analysis on DEGs.
In total, more than a half (1263*100/2246 = 56.23%) of non-coding region-associated TSS in slow muscle and less than a half (210*100/474 = 44.30%) region-associated TSS in fast muscle, located upstream of a given DEG showed significant co-expression with that gene in the cycle of atrophy-recovery (See ST7).
Taking together, we observed strong evidence of co-expression of distant cis-regulatory elements and non-coding RNA and corresponding genes in the course of atrophy-recovery, that illustrated deep involvement of transicribed regulatory elements in the genetic control of muscle homeostasis (Figure 3A, Figure 3B).
