Genome-Wide Atlas of Promoter Expression Reveals Contribution of Transcribed Regulatory Elements to Genetic Control of Disuse-Mediated Atrophy of Skeletal Muscle

Abstract

The prevention of muscle atrophy carries with it clinical significance for the control of increased morbidity and mortality following physical inactivity. While major transcriptional events associated with muscle atrophy-recovery processes are the subject of active research on the gene level, the contribution of non-coding regulatory elements and alternative promoter usage is a major source for both the production of alternative protein products and new insights into the activity of transcription factors. We used the cap-analysis of gene expression (CAGE) to create a genome-wide atlas of promoter-level transcription in fast (m. EDL) and slow (m. soleus) muscles in rats that were subjected to hindlimb unloading and subsequent recovery. We found that the genetic regulation of the atrophy-recovery cycle in two types of muscle is mediated by different pathways, including a unique set of non-coding transcribed regulatory elements. We showed that the activation of "shadow" enhancers is tightly linked to specific stages of atrophy and recovery dynamics, with the largest number of specific regulatory elements being transcriptionally active in the muscles on the first day of recovery after a week of disuse. The developed comprehensive database of transcription of regulatory elements will further stimulate research on the gene regulation of muscle homeostasis in mammals.

Keywords: RNA transcription; atrophy; cis-regulatory elements; disuse; enhancers; promoters; rat; skeletal muscles; transcribed non-coding elements of genome; transcriptomics.

Figure 1

Design the U-experiment: morphometric and dystrophin fluorescence intensity analysis in muscle fibers during different time-points of disuse and recovery. (A) Time points of the muscle sampling during the disuse (Dx) and recovery (Rx) stage of the experiment. Estimation of relative muscle mass and relative cross-section area in m. Soleus (B) demonstrates significant decline after the seventh day of disuse to the first day of recovery, while in fast muscle, m. EDL (C) there are no significant changes. When assessing dystrophin fluorescence intensity in slow muscle m. Soleus (B), a significant decrease in dystrophin content during the third and seventh days of disuse, and the first day of the recovery period, is observed, followed by a slight increase until the seventh day of recovery. In m. EDL (C), a significant decline in dystrophin content after the third to the seventh day of disuse, and a rise on the first day of recovery, is also demonstrated. In total, 100% of the values for the control group were accepted. The significant differences are indicated by asterisks. See Supplementary Table ST1 for full data.

Figure 2

Clustering of differentially expressed peaks (both genic and intergenic), common to both phase-control and time-course comparisons (‘slow’ muscle—m. soleus). FDR threshold was 5 × 10⁻⁴. Clusters of the metabolism regulation through MAP kinase activity, actin biosynthesis, nucleosome assembly, and lipid metabolism are shown in black, green, blue, and red, respectively (see Supplementary Figure S1 for full figure). Sample names attributed to Dx—days of disuse, and Rx—recovery, and the numbers 1–3 at the end of the sample refer to the experimental animal.

Figure 3

Differentially expressed genes (DEGs) and differentially expressed enhancers in soleus and EDL muscles. (A) Number of DEGs. (B) Number of differentially expressed enhancers. (C) DEGs in the fast and slow muscles (as compared to control) on days 1, 3 and 7 of disuse (shown as D1, D3, D7, respectively) and on days 1, 3, and 7 of recovery (shown as R1, R3, R7, respectively). (D) Differentially expressed enhancers in the fast and slow muscles (as compared to control) on days 1, 3 and 7 of disuse (shown as D1, D3, D7, respectively) and on days 1, 3, and 7 of recovery (shown as R1, R3, R7, respectively).

Figure 4

Non-genic peaks annotated with GO biological process of DEGs, which are located upstream (see Supplementary Figure S2 for full figure). Samples names attribute to Dx—days of disuse, and Rx—recovery.

Figure 5

Alternative TSS in genes Myh4 (A) and Rtkn (B), shown in BioUML web browser. See Supplementary Figure S4 for the full figure.

Figure 6

Tissue-specific enhancers of soleus and EDL muscles. Strandless coordinate intersection with 1 nt cutoff. See Supplementary Table ST11 for the full data.