Making sense (and antisense) of myosin heavy chain gene expression. Comments on “Intergenic bidirectional promoter and cooperative regulation of the IIx and IIb MHC genes in fast skeletal muscle” by Rinaldi et al.

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Abstract
For over 40 years, our primary understanding of how eukaryotic gene transcription is regulated has centered on the promoter concept. In this model, transcriptional activators bind to consensus sequences in the proximal upstream regulatory region of a gene, altering chromatin structure to allow recruitment of the RNA polymerase complex to a promoter sequence, which then creates a complementary transcript of the negative strand ([Fig. 1 A ][1]). However, many genes in eukaryotes exist in gene clusters formed via gene duplication, and a key question centers on how coordinated regulation of expression of genes within these clusters occurs. Specifically, gene isoform switching within a given cell or tissue in response to developmental, physiological, or pathological stimuli often requires sequential temporal and/or spatial expression of adjacent genes; thus, expression of two or more genes must be coordinated, which requires an additional level of integration. Locus control regions (LCRs), or groups of regulatory elements upstream of the gene cluster that regulate the sequential activation of the genes in the cluster, represent one mechanism by which this occurs ([Fig. 1 B ][1]). LCRs have been identified for several key gene clusters, including the globin and interleukin genes ([5][2]). Rinaldi et al. ([14][3]) provide further evidence for another mechanism regulating isoform switching in a set of clustered genes, the striated muscle myosin heavy chain (MHC) genes. Eight MHC genes are expressed in cardiac and skeletal muscle and are clustered in two groups: the predominantly cardiac-expressed β- and α-MHC genes are found in this order on chromosome 14 in the mouse and humans and chromosome 15 in the rat, while six skeletal-specific isoforms are found clustered on chromosome 10 in the rat, chromosome 11 in the mouse, and chromosome 17 in humans ([18][4]). Differential expression of these isoforms in both heart and skeletal muscle occurs in response to a number of developmental, neural, hormonal, and mechanical stimuli, but the mechanisms coordinating this transcriptional switching have remained obscure. In previous work ([7][5]), this group demonstrated that switches between the predominantly cardiac-expressed β- and α-MHC genes occur via an intergenic bidirectional promoter ([Fig. 1 C ][1]) that simultaneously produces a sense α-transcript, as well as an antisense β-transcript that decreases β-MHC expression. Previous investigators had identified the presence of antisense MHC transcripts in the heart ([4][6], [10][7]–[12][8]), but it was not clear what, if any, function these had. The paper by Haddad et al. ([7][5]) demonstrated that increased activity of this bidirectional promoter increases expression of α- while decreasing expression of β-MHC by increasing the sense and antisense transcripts for these loci, respectively. These authors have subsequently shown that this mechanism contributes to the α- and β-MHC switching occurring in the heart in response to a wide variety of physiological and pathological conditions, including development ([9][9]), hypo- and hyperthyroidism ([6][10], [7][5], [9][9]), pressure overload ([8][11]), and diabetes ([6][10], [7][5]). The present work extends this work by examining whether a bidirectional intergenic sense/antisense promoter exists between the IIx and IIb skeletal muscle MHC genes. Three “fast” MHC isoforms, IIa, IIx and IIb, are expressed in adult skeletal muscle in the rodent. Switching between these three MHC isoforms can occur in response to changes in activity levels, with increased activity favoring a switch toward type IIa and reduced activity favoring a switch toward type IIb; these shifts always occur sequentially through the three isoforms in the order IIa-IIx-IIb, which is the order of these genes in the cluster. In a previous paper, this group showed that an intergenic bidirectional promoter exists between the IIa and IIx MHC genes that inactivates IIa and activates IIx MHC expression during conditions of muscle atrophy in response to decreased usage ([13][12]). The present paper expands upon this previous work in two ways: it examines whether a second bidirectional intergenic promoter exists between the IIx and IIb MHC genes, and if so, whether it can account for the shift from IIb to IIx occurring in response to models of increased muscle activity. Using a model of muscle stimulation simulating a 4-day regimen of resistance exercise training in rats, Rinaldi et al. ([14][3]) show that levels of IIx pre-mRNA and mature mRNA are increased while levels of IIb pre-mRNA and mature mRNA are decreased. Moreover, using strand-specific primers, they demonstrate that an antisense RNA spanning all of the IIx MHC gene and extending into the 3′ end of the IIa MHC gene is present in control rats but is suppressed following resistance training. Sequence analysis identified an evolutionarily conserved region between the IIx and IIb MHC genes that contains several putative consensus binding sequences that may function as the sense/antisense promoter for producing these two transcripts ([14][3]). Thus this work supports the hypothesis that intergenic bidirectional promoter activity is a strong contributor to regulation of the skeletal MHC gene cluster as a whole in response to both increased and decreased muscle activity and may be another mechanism for regulating gene switching among clusters in general. Other examples of antisense RNA production or “opposite strand transcription” have been reported in other systems ([17][13]), including the hox genes ([16][14]), and thus, this may represent a fairly common mechanism for regulating expression of genes and/or switching between adjacent isoform genes. However, several questions have yet to be addressed. First, the mechanisms by which the antisense RNA interferes with gene expression have yet to be...

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