Paul W. Bodell

Role of Antisense RNA in Coordinating Cardiac Myosin Heavy Chain Gene Switching

A novel mechanism of regulation of cardiac α and β myosin heavy chain gene by naturally occurring antisense transcription was elucidated via pre-mRNA analysis. Herein, we report the expression of an antisense β myosin heavy chain RNA in the normal rodent myocardium. The pattern of expression of the antisense βMHC RNA (β RNA) under altered thyroid state and in diabetes directly correlates with that of the α pre-mRNA/mRNA, whereas it negatively correlates with the β mRNA expression. Rapid amplification of the 5′ end shows that this antisense transcript originates 2 kb downstream of the β gene, and it is transcribed across the entire β gene from the opposite strand. Our results demonstrate that the β-α myosin heavy chain intergenic DNA possesses a bidirectional transcriptional activity, one direction transcribing the α gene, and the opposite direction transcribing the antisense β RNA. This process turns on the α expression, and it simultaneously turns off that of the β and thus coordinates α and β expression in an opposite fashion. Comparative analyses of the intergenic DNA sequence across five mammalian species revealed a conserved region that is proposed to be a common regulatory region for the α and antisense β promoter. This finding unravels the mechanism of cardiac α-β gene switching and implicates the role of cardiac myosin gene organization with their function.

Rapid muscle atrophy response to unloading: pretranslational processes involving MHC and actin.

Skeletal muscles, especially weight-bearing muscles, are very sensitive to changes in loading state. The aim of this paper was to characterize the dynamic changes in the unloaded soleus muscle in vivo following a short bout of hindlimb suspension (HS), testing the hypothesis that transcriptional events respond early to the atrophic stimulus. In fact, we observed that after only 1 day of HS, primary transcript levels of skeletal alpha-actin and type I myosin heavy chain (MHC) genes were significantly reduced by more than 50% compared with ground control levels. The degree of the decline for the mRNA expression of actin and type I MHC lagged behind that of the pre-mRNA levels after 1 day of HS, but by 2 and 7 days of HS, large decreases were observed. Although the faster MHC isoforms, IIx and IIb, began to be expressed in soleus after 1 day of HS, a relatively significant shift in mRNA expression from the slow MHC isoform type I toward these fast MHC isoforms did not emerge until 7 days of HS. One day of HS was sufficient to show significant decreases in mRNA levels of putative signaling factors serum response factor (SRF), suppressor of cytokine signaling-3 (SOCS3), and striated muscle activator of Rho signaling (STARS), although transcription factors yin-yang-1 (YY1) and transcriptional enhancing factor-1 (TEF-1) were not significantly affected by HS. The protein levels of actin and type I MHC were significantly decreased after 2 days of HS, and SRF protein was significantly decreased after 7 days HS. Our results show that after only 1 day of unloading, pre-mRNA and mRNA expression of muscle proteins and muscle-specific signaling factors are significantly reduced, suggesting that the downregulation of the synthesis side of the protein balance equation that occurs in atrophying muscle is initiated rapidly.

Differential epigenetic modifications of histones at the myosin heavy chain genes in fast and slow skeletal muscle fibers and in response to muscle unloading

Recent advances in chromatin biology have enhanced our understanding of gene regulation. It is now widely appreciated that gene regulation is dependent upon post-translational modifications to the histones which package genes in the nucleus of cells. Active genes are known to be associated with acetylation of histones (H3ac) and trimethylation of lysine 4 in histone H3 (H3K4me3). Using chromatin immunoprecipitation (ChIP), we examined histone modifications at the myosin heavy chain (MHC) genes expressed in fast vs. slow fiber-type skeletal muscle, and in a model of muscle unloading, which results in a shift to fast MHC gene expression in slow muscles. Both H3ac and H3K4me3 varied directly with the transcriptional activity of the MHC genes in fast fiber-type plantaris and slow fiber-type soleus. During MHC transitions with muscle unloading, histone H3 at the type I MHC becomes de-acetylated in correspondence with down-regulation of that gene, while upregulation of the fast type IIx and IIb MHCs occurs in conjunction with enhanced H3ac in those MHCs. Enrichment of H3K4me3 is also increased at the type IIx and IIb MHCs when these genes are induced with muscle unloading. Downregulation of IIa MHC, however, was not associated with corresponding loss of H3ac or H3K4me3. These observations demonstrate the feasibility of using the ChIP assay to understand the native chromatin environment in adult skeletal muscle, and also suggest that the transcriptional state of types I, IIx and IIb MHC genes are sensitive to histone modifications both in different muscle fiber-types and in response to altered loading states.

In vivo regulation of β-MHC gene in rodent heart : role of T3 and evidence for an upstream enhancer

Cardiac β-myosin heavy chain (β-MHC) gene expression is mainly regulated through transcriptional processes. Although these results are based primarily on in vitro cell culture models, relatively little information is available concerning the interaction of key regulatory factors thought to modulate MHC expression in the intact rodent heart. Using a direct gene transfer approach, we studied the in vivo transcriptional activity of different-length β-MHC promoter fragments in normal control and in altered thyroid states. The test β-MHC promoter was fused to a firefly luciferase reporter gene, whereas the control α-MHC promoter was fused to the Renilla luciferase reporter gene and was used to account for variations in transfection efficiency. Absolute reporter gene activities showed that β- and α-MHC genes were individually and reciprocally regulated by thyroid hormone. The β-to-α ratios of reporter gene expression demonstrated an almost threefold larger β-MHC gene expression in the longest than in the shorter promoter fragments in normal control animals, implying the existence of an upstream enhancer. A mutation in the putative thyroid response element of the -408-bp β-MHC promoter construct caused transcriptional activity to drop to null. When studied in the -3,500-bp β-MHC promoter, construct activity was reduced (∼100-fold) while thyroid hormone responsiveness was retained. These findings suggest that, even though the bulk of the thyroid hormone responsiveness of the gene is contained within the first 215 bp of the β-MHC promoter sequence, the exact mechanism of triiodothyronine (T3) action remains to be elucidated.

Intergenic Bidirectional Promoter and Cooperative Regulation of the IIx and IIb MHC Genes in Fast Skeletal Muscle

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.

Calcineurin plays a modulatory role in loading-induced regulation of type I myosin heavy chain gene expression in slow skeletal muscle.

The role of calcineurin (Cn) in skeletal muscle fiber-type expression has been a subject of great interest because of reports indicating that it controls the slow muscle phenotype. To delineate the role of Cn in phenotype remodeling, particularly its role in driving expression of the type I myosin heavy chain (MHC) gene, we used a novel strategy whereby a profound transition from fast to slow fiber type is induced and examined in the absence and presence of cyclosporin A (CsA), a Cn inhibitor. To induce the fast-to-slow transition, we first subjected rats to 7 days of hindlimb suspension (HS) + thyroid hormone [triiodothyronine (T(3))] to suppress nearly all expression of type I MHC mRNA in the soleus muscle. HS + T(3) was then withdrawn, and rats resumed normal ambulation and thyroid state, during which vehicle or CsA (30 mg x kg(-1) x day(-1)) was administered for 7 or 14 days. The findings demonstrate that, despite significant inhibition of Cn, pre-mRNA, mRNA, and protein abundance of type I MHC increased markedly during reloading relative to HS + T(3) (P < 0.05). Type I MHC expression was, however, attenuated by CsA compared with vehicle treatment. In addition, type IIa and IIx MHC pre-mRNA, mRNA, and relative protein levels were increased in Cn-treated compared with vehicle-treated rats. These findings indicate that Cn has a modulatory role in MHC transcription, rather than a role as a primary regulator of slow MHC gene expression.

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

Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development.

The CAAT-binding transcription factor 1/nuclear factor 1 binding site is important in β-myosin heavy chain antisense promoter regulation in rats

The rat heart expresses two myosin heavy chain (MHC) isoforms, β and α; these genes are arranged in tandem on the same chromosome. We have reported that an antisense (AS) β RNA starts in the intergenic (IG) region between β and α genes and extends to overlap the β gene. We propose that in adult rats, both the α sense and IG βAS RNA expression are activated by an IG bidirectional promoter and that the transcription of βAS RNA interferes with the sense β, resulting in low levels of β mRNA and high levels of α, a phenotype seen in a typical rat heart. A previous report examined the activity of the βAS promoter and showed that a 559 bp fragment of the βAS promoter (–2285 to –1726; relative to αMHC gene start site) injected into rat ventricle was activated in control heart, and decreased significantly in response to hypothyroidism (propylthiouracil induced) and diabetes (streptozotocin induced) and increased in hyperthyroid rats (T3 induced), similar in pattern to the endogenous βAS RNA. In the present paper, we demonstrate with electrophoretic mobility shift analyses that ventricular nuclear proteins are interacting with a nuclear factor 1/CAAT‐binding transcription factor 1 (NF1/CTF1) binding site, and a supershift assay indicates that the protein binding at this site is antigenetically related to the CTF1/NF1 factor. Moreover, a mutation of the CTF1/NF1 site within the 559 bp promoter region nearly abolished promoter activity in vivo in control, STZ‐ and PTU‐treated rats. Based on these findings, we conclude that the NF1 site is critical to βAS promoter regulation.