Fig. 1: Elucidation of enhancer transcription in muscle cells.

a GRO-seq detected eRNAs that were up-, down-regulated or unchanged in myotube (MT) vs myoblast (MB) cells, and the changes were correlated with H3K27ac remodeling. b Expression of neighboring genes associated with up- or down-regulated eRNAs in MT vs MB. c The eRNAs were categorized into ‘stable’ (captured by GRO-seq and total RNA-seq) and ‘unstable’ transcripts (captured only by GRO-seq); the divergent enhancer transcription was further categorized into three types of pairs: Bi-stable (both directions generate stable transcripts), Uni-stable (only one direction generates stable transcript) and Unstable (both directions generate unstable transcripts). d Distribution of the above types of divergent eRNAs. e Box plot showing the read density (RPM) of GRO-seq signals or Pol II binding on the above three types of eRNAs in MT. f Analyzing total RNA-seq or GRO-seq revealed that a higher percentage of SEs in MB or MT gave rise to eRNAs (eRNA+) compared to TEs. g seRNAs displayed higher level of GRO-seq signals compared to teRNAs. h TF hotspot regions showed markedly higher levels of GRO-seq signals compared to non-hotspot enhancer regions. i Genomic snapshots of representative eRNAs identified from MB- (left) or MT-expressed SE (right), showing H3K4me1, H3K4me2, H3K27ac, H3K4me3, Pol II and H3K36me3 ChIP-seq profiles, and GRO-seq in MB and MT cells. The red bar highlights the SE region. The “transcript” track indicates transcript units identified through GRO-seq. GRO-seq signals are displayed in “+” (red) and “−” (light green) strands separately. j qRT-PCR measurement of expression dynamics of several MT seRNAs during 120 h differentiation course of C2C12 myoblast. k seRNA expressions were measured in the muscles after cardiotoxin (CTX) injection induced regeneration. n = 3 per group. Data in j and k represent the average of three independent experiments ± s.d. Data in b, e, and h are presented in boxplots. Center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range. Statistical analyses in b, e, and h were done by Mann–Whitney non-parametric test; ***P < 0.001. Source data are provided as a Source Data file.