Intergenic bidirectional promoter and cooperative regulation of the IIx and IIb MHC genes in fast skeletal muscle

Abstract

This study investigated the dynamic regulation of IIx-IIb MHC genes in the fast white medial gastrocnemius (WMG) muscle in response to intermittent resistance exercise training (RE), a model associated with a rapid shift from IIb to IIx expression (11). We investigated the effect of 4 days of RE on the transcriptional activity across the skeletal MHC gene locus in the WMG in female Sprague-Dawley rats. Our results show that RE resulted in significant shifts from IIb to IIx observed at both the pre-mRNA and mRNA levels. An antisense RNA (xII NAT) was detected in the intergenic (IG) region between IIx and IIb, extending across the entire IIx gene and into its promoter. The expression of the xII NAT was positively correlated with IIb pre-mRNA (R = +0.8), and negatively correlated with IIx pre-mRNA (R = -0.8). Transcription mapping of the IIx-IIb IG region revealed the generation of sense IIb and xII NATs from a single promoter region. This bidirectional promoter is highly conserved among species and contains several regulatory elements that may be implicated in its regulation. These results suggest that the IIx and the IIb genes are physically and functionally linked via the bidirectional promoter. In order for the IIx MHC gene to be regulated, a feedback mechanism from the IG xII NAT is needed. In conclusion, the IG bidirectional promoter generating antisense RNA appears to be essential for the coordinated regulation of the skeletal muscle MHC genes during dynamic phenotype shifts.

Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle

“…From a teleological perspective, coregulation of bII NAT and Neo MHC would ensure that transcription of Neo MHC is coordinated with inhibition of IIb MHC transcription. In addition, the coregulation observed between bII NAT and Neo MHC transcription supports our previously reported model of MHC gene transcriptional coordination, which entails a functional linkage between two neighboring MHC genes mediated by cotranscription of a NAT with one MHC gene that negatively regulates the other MHC gene (19,42,47). However, the relatively large distance between the TSS of the bII NAT and Neo MHC stands out compared with the previously reported MHC NATs, which are much more closely spaced to their coregulated MHC gene.…”

“…The aII NAT appears to repress IIa MHC gene transcription (42). Sense-antisense pairs were also observed to regulate IIx and IIb MHC in both fast and slow skeletal muscle and in cardiac muscle in coordination of ␣-and ␤-MHC expression (18,20,42,47). These reports are accompanied by other recent reports in nonmuscle cells describing mechanisms of cis-acting long noncoding antisense RNA-mediated transcriptional repression (23,28,37,58,61,68).…”

“…Our previous analyses of the MHC NATs, which appear to regulate IIx, IIa, and type I (␤) MHC were found to have TSSs within 1,000 bp of the IIb, IIx, and ␣-MHC TSSs, respectively (19,42,47). The sense and antisense transcripts are suggested to be regulated by bidirectional promoters, such that the MHC gene promoter and antisense promoter are coregulated.…”

“…These procedures were also utilized in the analyses of antisense RNA expression across the skeletal MHC gene locus between the IIb and Neo MHC. The manufacturer's protocol was followed with some modifications as described previously (42,47). This protocol has been optimized to avoid amplification of nonspecific transcripts (21,42).…”

“…The genomic order, relative spacing, and the coding sequence are highly conserved across a broad evolutionary span. We have previously shown that functional significance for this gene order and intergenic spacing is apparent by the transcriptional coordination of MHC genes with long noncoding RNA transcripts that are transcribed in the antisense orientation to the IIa, IIx, and IIb MHC genes during alterations to muscle loading state (42,47). For example, muscle inactivity and unloading induce a coordinated IIa to IIx MHC shift in the soleus muscle whereby IIa decreases while IIx increases concurrently.…”