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.
- gene transcription
- natural antisense RNA
- comparative genomics
skeletal muscle tissue is able to adapt to different types of stimuli, such as neuronal, mechanical, and hormonal interventions (8). This plasticity is made possible, in part, by the existence of several isoforms of myosin heavy chain (MHC), the molecular motor of muscle contraction. At least eight MHC isoforms are expressed in mammalian striated muscle (skeletal and cardiac), including two developmental isoforms (embryonic and neonatal), one slow MHC (type I or β-cardiac), one cardiac isoform (α-MHC), three fast-type II isoforms (IIa, IIx, and IIb), and one specialized isoform (the extraocular MHC). These MHC isoforms are the products of distinct genes that are clustered in multigenic complexes on two chromosomes. The skeletal MHC gene cluster resides on chromosome 10 in the rat (10q24) (17p13 in human, 11 35.0 cM in mouse), spanning ∼420 kb. These MHC genes are linked in a head-to-tail fashion in the following order: embryonic, IIa, IIx, IIb, neonatal, and extraocular MHC (28, 29, 41, 49, 51). The cardiac MHC gene cluster resides on chromosome 15 in the rat (15p13) (14q12 in human and 14 20.0 cM in mouse), spanning ∼50 kb and consists of the β- (slow-type I MHC) ∼4.5 kb upstream of the α-MHC gene (16, 34, 39).
Different combinations of MHC isoforms may occur in the same fiber, and the MHC isoform profile is a major determinant of muscle fiber functional properties, such as the speed of contraction, power output, and fatigue resistance. In adult rodents and small-body-size mammal skeletal muscle, all four types of protein isoforms, MHC-I, IIa, IIx, and IIb, are expressed (40). In humans, even though the MHC IIb gene has been identified (41), the general belief, based on current detection methods, is that the IIb MHC gene is not expressed in human muscle fibers (31, 42, 50). However, the IIb isoform was abundantly detected in a subset of human masseter muscle fibers and was rarely detected in limb muscle and in few external oblique fibers, but these detections occurred only at the mRNA level (24).
The expression of MHC isoforms is both developmentally regulated and highly responsive to mechanical, neuronal, or hormonal stimuli (6, 7, 10, 40). Based on the type of intervention and the starting phenotype, muscles can shift toward a slower or a faster contractile profile (6). MHC isoform regulation occurs in a muscle type-specific fashion in response to various stimuli. For example, the IIx MHC expression is upregulated in a slow muscle fiber type in response to unloading or inactivity (18, 23, 25, 45); whereas, the same IIx MHC expression is also upregulated in a fast muscle in response to overloading and increased activity, such as resistance training (11, 22). The upregulation of the IIx gene in response to opposing stimuli in two different muscle types is intriguing and is thought to occur at the transcriptional/pretranslational level (37, 38). The exact mechanism behind this regulation is not clear, but it may be the result of both muscle-type-specific factors acting on the genetic apparatus, as well as cross talk between the IIx gene and its adjacent genes (IIa and IIb).
The MHC gene family organization, tandem linkage, individual gene order, and spacing have been conserved through millions of years of evolution; thus, these physical characteristics are believed to play a key role in their coordinated regulation and dynamic switching that occurs between the MHC isoforms during development and in response to altered physiological conditions (13, 17, 20, 26, 50). In fact, in recent years, our studies examining MHC gene switching uncovered that the intergenic (IG) DNA physically linking the two MHC genes is transcriptionally active and thus, may provide a functional link between these tandem MHC genes, thus leading to their coordinated regulation. For example, the IG space between cardiac β- and α-MHC genes is transcriptionally active on both strands, thus exhibiting some inherent bidirectional transcriptional activity (20). In a normal control rodent heart, as well as in response to thyroid hormone 3,5,3′-triiodothyronine (T3), the IG activity is turned on, leading to an increase in both sense and antisense IG transcription. The sense IG RNA is correlated to an increased α-MHC gene transcription, whereas the antisense transcription is carried out through the entire β-MHC gene into its promoter, whereby it is believed to interfere with its transcription via a mechanism likely involving epigenetic processes (20, 21, 38). The IG transcriptional activity is thought to play a critical role in the coordinated antithetical regulation of these two cardiac MHC genes.
In a more recent report, we show that the IG region between the IIa and IIx genes also exhibits bidirectional transcriptional activity in slow muscles, such as the soleus and vastus intermedius (38). This IG transcription is thought to be a player in the coordinated regulation of IIa-IIx MHC in response to unloading and inactivity (38).
The goal of this present study was to further examine the MHC isoform shifts in the fast white medial gastrocnemius (WMG) muscle in response to increased activity, such as high resistance exercise training (RE). Under normal control conditions, the adult rodent WMG MHC isoform profile is composed of ∼80–90% IIb MHC and 10–20% IIx MHC, and this composition is found at both the protein and mRNA level of expression. In rodents, RE can induce rapid shifts in MHC isoform from IIb to IIx, during which the IIb is downregulated, whereas the IIx is upregulated (11, 22). These IIb→IIx shifts occur rapidly and can be detected at the mRNA level after only 2 days of RE (11). The IIx (Myh1) and IIb (Myh 4) MHC genes are located in tandem on the skeletal MHC gene locus and are separated by ∼14 kb of IG DNA in the rat [National Center for Biotechnology Information (NCBI) rat genomic database, contig accession no. NW_047334]. Consequently, one goal of this study was to determine whether the IG DNA between the IIx and IIb genes is transcriptionally active on both strands, and whether this IG locus serves as a focal point in their coordinated regulation in WMG in response to RE. Thus, we tested the working hypothesis that the same mechanism of coordinated regulation, described for the cardiac MHC genes and for the skeletal muscle MHC gene IIa and IIx, is also involved in the switching of the MHC genes IIx and IIb in the WMG. Type IIx and IIb pre-mRNA and mature mRNA were analyzed as markers of transcriptional and pretranslational events, respectively. In addition, sense and antisense RNA were analyzed along the IIx-IIb MHC gene locus, including the IG DNA. We investigated the effect of 4 days of RE on these MHC RNA markers. The second hypothesis tested was that the coregulated modulation of the IIb sense and IIx antisense transcripts is linked to the presence of a common bidirectional promoter situated in the IIx-IIb IG region. Transcription was mapped through the entire IIx-IIb IG region and rapid amplification of cDNA 5′ ends analyses (5′RACE) were conducted to identify transcription start sites (TSSs). The obtained findings corroborate the generation of the sense and antisense transcripts from a single promoter region. Multispecies comparative sequence analyses (in silico) was conducted to identify potential transcription factor binding sites that could be involved in the regulation of the bidirectional promoter.
Animal model and experimental design.
This study was conducted in conformity with the “Guiding Principles in the Care and Use of Animals” of the American Physiological Society, and the protocol was approved by the University of California Irvine, Animal Use Committee. This study used young adult female Sprague-Dawley rats weighing 256 ± 5 g (n = 12). Rats were on a 12:12-h light-dark cycle and were allowed access to food and water ad libitum. The rats experienced unilateral leg RE via imposed electrical stimulation (see below) targeting the left gastrocnemius muscle. The experiment lasted for 6 days, RE was administered on days 1, 2, 4, and 5. Day 3 was a rest day. For each RE bout, the rats were anesthetized with ketamine-xylazine-acepromazine (30·4·1 mg kg−1). On day 6, i.e., 24 h after the last RE session, the experiment was terminated, and the animals were euthanized for tissue collection via an intraperitoneal injection of Pentosol euthanasia solution (Med-Pharmex) at a dose of 0.4 ml/kg (∼160 mg/kg pentobarbitol). The medial gastrocnemius muscle (MG) from both legs was dissected out, and was divided into WMG, red MG, and a mix of MG portions. Muscle pieces were immediately frozen between blocks of dry ice and were stored at −80°C for subsequent RNA extraction and analyses. The contralateral limbs served as an internal control, and data values are therefore designated as control as opposed to RE for the trained leg data.
We have used a combined muscle action training protocol (isometric, concentric, eccentric) similar to the one described in Adams et al. (2). Each bout of stimulation lasted 3 s, the 1st s was of the isometric-type contraction followed by a 1-s concentric contraction, followed by a 1-s eccentric mode contraction. A period of 27 s of rest was allowed between each contraction. Rats performed five contractions per set. Five minutes of recovery was allowed between sets. On day 1, the session was limited to five sets, on the subsequent three training days, the animals received six sets of contractions during each training session.
Total RNA was extracted from frozen WMG muscle using the Tri Reagent protocol (Molecular Research Center). The extracted RNA was DNase-treated using 1 unit of RQ1 RNase-free DNase (Promega) per microgram of total RNA and was incubated at 37°C for 30 min followed by a second RNA extraction using Tri Reagent LS (Molecular Research Center).
The MHC mRNA isoform distribution was evaluated by reverse transcription (RT) with random primers/oligo(dT) mix, followed by PCR with primers targeting the embryonic, neonatal, I, IIa, IIx, and IIb MHC mRNAs, as described previously (3, 15). In these PCR reactions, each MHC mRNA signal was corrected to an externally added control DNA fragment that was coamplified with the MHC cDNAs using the same PCR primer pair. This provides a means to correct for any differences in the PCR reaction efficiency and/or pipetting of each PCR reaction. A correction factor was used for each control fragment band on the ethidium bromide-stained gel to account for the staining intensity of the variably-sized fragments (224 to 324 bp), as reported previously (15).
While the above RT-PCR can provide information on the MHC mRNA distribution pattern, it does not give information on how each isoform is regulated. Strand-specific RT-PCR was used to analyze the expression of specific MHC pre-mRNAs and mRNAs, as well as antisense RNAs that are of opposite orientation to the MHC genes.
Strand-specific RT-PCR used the one-step RT-PCR kit from Qiagen. These assays were utilized in the determination of the relative level of expression of pre-mRNA, antisense RNA, and mRNA in a known amount of total RNA, in comparing control vs. RE WMG. Also, these analyses were carried out in the mapping analyses of RNA expression across the skeletal MHC gene locus. The provided instruction was followed except in the RT step, only the reverse primer was added to target the sense RNA, whereas only the forward primer was added to target the antisense RNA. At the end of the RT, the reaction was heated to 90°C for 15 min to denature the RT enzyme; then the missing primer was added before initiating the PCR. In these assays, a negative control reaction was carried out to ensure the strand specificity of the product. In the RT step, no primer was added. After denaturing the RT, both the forward and reverse PCR primers were added. Any resulting product from this negative control reaction may be indicative of the formation of a cDNA in a primer-independent fashion or. alternatively. may be indicative of the presence of genomic DNA in the RNA. Our tests show the absence of any products in the negative control reactions, confirming that any amplified product corresponded to the target sense or antisense RNA in question (see Ref. 19 for more details).
For all of the PCR analyses, the primers were designed using DNA-star Primer Select software in which the target sequence was repeat masked using the Repeat Masker Web server at http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker.
These assays were conducted to identify the TSS of both the antisense IIx RNA and the sense IIb RNA. We adapted the method described by Invitrogen supplied with the kit (5′RACE). The cDNA was synthesized using Superscript II at 50°C and an RT primer reverse complementary to a region of the target RNA near the 5′ end. The cDNA was RNase H treated and purified using the Qiaquick PCR Purification Kit (Qiagen). The pure cDNA was tailed with dCTP using terminal transferase (New England Biolab). The tailed cDNA was amplified using a gene-specific reverse primer that was nested to the RT primer and an adapter primer (5′RACE primer, Invitrogen) (see supplementary Table S1 for the RT and gene-specific primer sequence. The online version of this article contains supplemental data.). The PCR products were cloned into pGEMT Easy (Promega), and the insert of individual clones was sequenced to determine the 5′ end corresponding to the end of the cDNA that is adjacent to the poly dC tail. Sequencing reactions used the ABI Big Dye sequencing kit version 3.1. Sequencing data acquisition was obtained from the UCI Sequencing Facility using an Applied Biosystem 3700 sequencer.
Genomic DNA was extracted from rat tissue using the DNAeasy Tissue Kit (Qiagen). DNA was eluted with water and its concentration was determined by UV absorption at 260 nm (using a factor of 50 μg/ml per OD unit). The obtained genomic DNA was used as template to test all the PCR primer sets targeting pre-mRNA and antisense RNA to ensure that these primers worked with similar efficiency to each other.
Computational analyses of DNA sequence.
Rat genomic sequence (genomic contig number: NW_047334, location: 10q24) was aligned with human (contig number: NT_010718, location: 17p13), mouse (contig number: NT_096135, location: 11 35cM) and monkey (GenBank accession no. AC212180) using Mulan comparative genomics site at http://mulan.dcode.org/ (36). The purpose for this comparative analysis was to identify evolutionarily conserved sequences at 85% in these promoters and to identify conserved transcription factor binding sites (TFBSs) in these conserved regions.
Data are reported as means ± SE. Differences between two groups (control vs. RE) were analyzed using a paired t-test. Relationships between two variables were assessed using linear regression and correlation analyses (GraphPad Software). One-way ANOVA followed by Newman-Keuls post hoc test was used for the analyses of promoter activity across muscle types. Statistical significance was set at P < 0.05.
After 4 days of RE, the ratio of MG muscle wt/body wt was increased by 5% in the trained leg relative to the contralateral control leg, although this increase was not statistically significant. Control MG muscle was 630 ± 16 mg; RE muscle weight was 662 ± 17 mg.
MHC mRNA analysis.
The MHC mRNA isoforms distribution in the WMG and their shift following 4 days of RE were assessed through a competitive PCR approach, and they are shown in Fig. 1. The mRNA of each MHC gene was expressed as the percentage of the total MHC mRNA pool. Confirming previous findings (11, 22), only fast MHC isoforms, IIx and IIb, were expressed in both groups of samples. Since other MHC mRNA isoforms were not detected either in control or in the RE group, they were not reported in the graph. The MHC mRNA distribution in control WMG muscles was represented by 76% of IIb. The acute RE paradigm caused a statistically significant shift in the MHC composition, with a decrease of MHC IIb mRNA to 50% and a consequent increase of MHC IIx mRNA from 24 to 47%. These results were consistent with a previous report on MHC isoform shifts following mechanical loading of fast skeletal muscles (11). Furthermore, these results demonstrate that the RE in these present experiments was effective in inducing MHC isoform shifts, which warrant further analyses of RNA across the gene locus.
MHC RNA expression in response to resistance loading.
The IIx and IIb MHC pre-mRNA and mRNA were used as markers of transcriptional and of pretranslational events, respectively. In theses analyses, both the mRNA and the pre-mRNA are expressed as arbitrary units per nanogram of total RNA, whereas in Fig. 1, the MHC mRNA was expressed as relative percentage of the total MHC mRNA pool. Results in Fig. 2 show that for the IIx MHC gene products, both the pre-mRNA and the mature mRNA signals (Fig. 2, A and B) were significantly increased in response to RE (P < 0.0001). The IIx MHC pre-mRNA was approximately fivefold higher in RE muscles than in the control group and the IIx MHC mRNA recorded was 6.5-fold greater (Fig. 2B). For the IIb MHC gene products, the expression of pre-mRNA and the mRNA was significantly decreased in RE muscles (P < 0.001). The RE WMG expressed only 25% and 40% of the IIb pre-mRNA and mRNA, respectively, compared with control muscle (Fig. 2, D and E).
In addition to the sense RNA species for the IIx and IIb MHC genes, the antisense RNA was analyzed in several locations across the IIx MHC gene and in the IIx-IIb IG region. The IIx MHC antisense RNA (xII NAT), as measured in the IG region (at ∼2 kb from the IIx TGA stop codon), was decreased significantly in the RE muscles (RE is 9% of the control group; P < 0.001) (Fig. 2C). Interestingly, the antisense transcription persisted along the entire IIx gene and into its promoter region 5′ upstream of the IIx gene (see below). No signal of antisense RNA was found in the IIb MHC gene, either in control or in RE muscles. The antisense transcript corresponding to the IIb MHC gene was targeted at several locations across the IIb gene, including its 3′ flanking region.
The relationship between xII NAT vs. IIx MHC pre-mRNA and the relationship between xII NAT vs. IIb MHC pre-mRNA were analyzed by linear regression. As presented in Fig. 3, there was a significant inverse correlation between the xII NAT and the IIx MHC pre-mRNA (R = −0.8; P < 0.001). In contrast, there was a significant positive correlation between the xII NAT and that of the IIb pre-mRNA (R = +0.8; P < 0.0001) (Fig. 3). These relationships show that the behavior of the IIx-IIb gene locus transcriptional activity is remarkably similar to those described for the cardiac β- and α-MHC genes (17), as well as for those for the IIa-IIx MHC gene in the soleus muscle (38). The coordinated regulation of the IIx-IIb gene concurs with the previously described models for two linked genes.
Transcription mapping across the IIa-IIx-IIb genes in WMG muscle.
The above-reported results demonstrate that an antisense IIx RNA is expressed in fast WMG muscle fibers, and its expression is downregulated in response to RE. It was of interest to map the extent of sense and antisense transcription along the adult skeletal MHC gene locus (IIa-IIx-IIb) in the fast WMG. Based on the rat genomic DNA sequence in NCBI (contig accession no. NW_047334, Rattus norvegicus chromosome 10 genomic contig, reference assembly; RGSC v3.4), these genes cover a region of ∼90 kb of genomic DNA of which the IIa gene is ∼27 kb, whereas the IIx and the IIb genes are ∼23 kb each in size. The IIa and IIx MHC genes are separated by ∼2.7 kb, whereas the IIx-IIb MHC genes are separated by ∼14 kb of IG space. Within the 14 kb IG space, there is a gap of ∼661 bp of unknown sequence centered at approximately −3 kb relative to the IIb MHC gene TSS. Several DNA repetitive elements are found on the IIx-IIb IG sequence as revealed by using a repeat masker tool (Repeat Masker web server; url: http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker). The masking results showed that these genomic repeats make up ∼31% of the total IIx-IIb IG region (Fig. 4).
A strand-specific RT-PCR-based approach was utilized to target and amplify the sense and antisense RNA in the WMG total RNA at sites corresponding to various regions of the IIa-IIx-IIb MHC gene locus. Strand-specific RT-PCR results demonstrated that the expression of antisense RNA in WMG muscles can be detected across the entire IIx gene, its promoter region consisting of the IIa-IIx IG region (2,677 bp), and in the region corresponding to the 3′ end of the IIa gene (see Fig. 4). Also, antisense RNA was detected corresponding to a large segment of the IG region between the IIx and IIb MHC genes to within approximately −200 bp relative the IIb MHC gene TSS (+1). No sense RNA was detected in the control WMG across either the IIa gene or the IIa-IIx IG region. In a few trained WMG samples, some trace levels of IIa sense RNA was detected. A significant amount of sense RNA was detected across the entire IIx MHC gene, the entire IIb MHC gene, and to ∼3 kb past the IIx or IIb gene stop codon (Fig. 4). Furthermore, these mapping analyses detected no antisense RNA that corresponds either to the IIb gene or to its 3′ flanking sequence. To our surprise, a low level of sense RNA was detected across ∼400 bp of the IIb MHC proximal promoter. This sense RNA merged with the IIb gene at +1 and it became undistinguishable from the IIb pre-mRNA. This low level of sense RNA corresponding to the IIb proximal promoter could not be attributed to a nonspecific signal or contamination since no bands were observed in the negative control reactions.
A high resolution mapping of sense and antisense transcriptional activity in the proximal IIb promoter (−1 kb to +1 kb vs. IIb TSS) is shown in Fig. 4. These mapping analyses suggest that both sense and antisense IG transcriptions are occurring in the proximal IIb promoter. Both of these sense and antisense IG RNA start within 400 bp from the IIb MHC gene TSS. 5′RACE analyses using primer extension and tailing reactions followed by sequencing, revealed the presence of multiple start sites of the antisense RNA that are clustered between −403 to −271 bp relative to the IIb MHC gene TSS. Similarly, the 5′RACE analyses of the sense RNA targeting sense IIb RNA revealed the presence of multiple start sites with the major site corresponding at +1, but several minor sites clustered between −400 and −100 relative to the major TSS at +1. The close proximity of the antisense TSSs to those of the IIb MHC TSSs confirmed the idea that the two opposite-in-direction transcripts were initiated in the same area of the IIx-IIb IG region from a unique bidirectional promoter or from two adjacent head-to-head opposing promoters. These findings strongly suggest that the IIx-IIb gene regulation is, in a way, similar to the cardiac β- and α-MHC genes, whereby both sense and antisense transcription were detected in the IG region (20). The linear regression analyses showing that the xII NAT was strongly correlated with IIb pre-mRNA in the WMG when subjected to RE (R = +0.8 P < 0.0001) (see Fig. 3B) highlights the intriguing notion of common regulation between the two head-to-head situated promoters.
Comparative analyses of the IIb MHC gene promoter sequence across species.
Based on the above results, the 400 bp proximal promoter of the endogenous IIb MHC gene is transcriptionally active in two directions and could be implicated in the regulation of not only the downstream IIb gene but also the upstream IIx gene as suggested by Figs. 3 and 4. Therefore, sequence analyses of the IIb promoter was undertaken in silico, using a comparative genomics approach with a goal to gain further insight into its function, such as identifying potential cis elements/TFBSs that may be important for transcriptional regulation. With a larger availability of genomic sequences it is becoming clear that a high number of coding and structural sequences are conserved through many species. In addition, regions of noncoding DNA with a particularly high similarity among species are recognized as good candidates for functional significance, which is the basis of the evolving field of comparative genomics. Thus, we studied and compared the orthologous DNA sequences spanning the IIb MHC promoter from −1,400 to +2,800 bp relative to the IIb MHC TSS across four different species: rat, mouse, human, and rhesus monkey. The rat sequence was used as the reference for the comparison.
Sequences were subjected to comparative analyses using the computational resources publicly available at http://mulan.dcode.org (36). Mulan is dynamically interconnected with the multi-TF utility (http://multitf.dcode.org), which identifies TFBSs that are shared among all the input species involved in the alignment. Thus, it is primarily designed to identify potential functional domains in a sequence predicated on sequence conservation. Analyses of the rat sequence against those sequences from three other mammalian species (mouse, rhesus monkey, and human; all extracted from the GenBank via Blast searches) identified two ECRs that show >85% similarity across the four species (Fig. 5). These two evolutionarily conserved regions (ECRs) among all the four species are located in the area where the supposed bidirectional promoter is situated (Fig. 5). The first ECR was located between −294 bp and +47 bp relative to the sense IIb MHC TSS and included the first exon of the IIb MHC gene extending from +1 to +23. The second ECR was located at the 5′ end of the IIb MHC gene, between +211 bp and 312 bp, and included the entire second exon extending from +224 to +275. Several muscle-specific transcription factors (Myogenin, myoD, MEF-2, GATA) binding sites were found conserved across four species on ECR1, whereas both ECR1 and ECR2 displayed a variety of conserved binding sites for general and ubiquitous transcription factors, such as C/EBP, SRF, CAAT, Sp1, Oct1, TBP, and these were located on both the sense DNA strand (+) and the complementary strand (−) (see Fig. 5B). While these observations are based on computational analyses of the sequence, the biological function of these associated binding sites and their role in skeletal MHC gene regulation remain to be determined in future studies. It is important to note that some of the identified TFBSs on ECR1 by Mulan, matches well with what was determined before in studies focusing mainly on the mouse IIb promoter (4, 5, 43, 44), as well as the human IIb proximal promoter (26).
Regulation of the switch between IIx and IIb MHC isoforms in response to acute resistance training.
Skeletal muscle is the most abundant organ system in the body, and, although considerable biochemical information has been already acquired concerning its structure and function, the complete knowledge of the molecular events underlying muscle plasticity has not yet been achieved. Therefore, it is of great interest to identify and understand the molecular events that underlie the shift among myosin isoforms that are responsible for the change in the fiber profile among targeted muscles.
In light of previous studies (17, 20, 38) and of our observations on the RNA expression by the use of strand-specific RT-PCR approach, the goal of this study was to test the hypothesis that the switch from IIb to IIx fiber type in the WMG in response to RE is coordinated and regulated at the transcriptional level. Also we tested the hypothesis that this regulation involves a bidirectional promoter region, generating both sense and antisense RNA, thereby linking the regulation of the two genes. The RE paradigm used enabled us to observe a statistically significant modification in the MHC mRNA isoform composition in the WMG (Fig. 1). These results are consistent with the findings that Caiozzo and Haddad reported (10), i.e., that RE induces an increase in IIx MHC expression with a concomitant decrease in IIb MHC in the whole WMG, as well as in individual muscle fibers. The analysis of the primary transcription products of these two genes revealed the same pattern; i.e., the IIx pre-mRNA expression was upregulated; whereas IIb pre-mRNA expression was downregulated in the RE muscle (Fig. 2). In addition, we also detected an antisense transcript overlapping with the IIx gene that was downregulated in RE muscles. The antisense transcript was detected corresponding to a long stretch on the IIx-IIb MHC gene locus. The RT-PCR results and RNA mapping analyses suggest that the antisense RNA is a long transcript that originated from the IIb proximal promoter, spanned the IIx-IIb IG region, overlapped the entire IIx MHC gene, and continued through the IIx promoter into the IIa-IIx IG region (Fig. 4). In addition, analyses revealed a significant inverse relationship between the antisense transcript and the IIx pre-mRNA (Fig. 3). These findings are consistent with the hypothesis that the antisense RNA is involved in the antithetical regulation of the IIx-IIb MHC genes, as seen for the cardiac α- and β-MHC genes and for the IIa-IIx genes (17, 20, 38). A closer examination of the inverse relationship between xII NAT vs. IIx pre-mRNA shown in Fig. 3A reveals that when analyzing either the control group alone or the RE group alone, the correlation between the two variables is not significant. The lack of correlation in the normal control group may be due to the fact that when the xII NAT is expressed above a certain threshold, the IIx sense expression is blunted and maintained at a low level, regardless of small fluctuation in xII NAT expression. On the opposite end, in the RE group, when xII NAT expression is reduced below a certain threshold, IIx sense gene expression is activated. However, in that state, any small fluctuation in the xII NAT is not correlated with those of IIx sense expression. In contrast, the negative correlation between xII NAT and IIx pre-mRNA becomes evident during dramatic shifts in IIx expression, such as in response to RE.
In the IIb-IIx switching model involving the WMG, as depicted in Fig. 6, the xII NAT appears to act like an inhibitory cis factor in the transcription process of the IIx gene. Noncoding antisense transcript expression “in cis” seems able to obstruct the sense transcription, thereby silencing the overlapping gene; how this happens precisely is still unresolved, but it likely involves epigenetic processes, such as DNA methylation and chromatin remodeling. Recently, several RNA-dependent epigenetic processes have been described and are involved in transcription regulation. For example, in yeast, antisense transcripts mediate the PHO84 gene silencing via an epigenetic process involving the recruitment of histone deacetylases at the gene promoter, thus promoting a repressed state (12). In another instance, a chromatin remodeling factor, Isw2, has been implicated in repressing antisense transcription that may occur at the 3′ end of the genes in the yeast genome (52). Isw2 provides a regulatory mechanism to control gene expression by occluding the TSS or regulatory sequences through nucleosome repositioning along the DNA (52). Furthermore, antisense noncoding RNA has been implicated with chromatin silencing in the X-inactivation, in imprinting, and in the hypermethylation and silencing of CpG islands in promoters of target genes (for a review, see Ref. 47).
It is intriguing to speculate that similar processes of noncoding-RNA-dependent gene silencing are involved in MHC gene-switching in muscle fibers. However, future research is needed to identify these processes.
Study of the locus of the IIX-IIB IG region.
Our data show that the sense and the antisense transcripts share the same proximal promoter region (Fig. 4). Through RNA mapping using a one-step-RT PCR approach and 5′RACE analyses, we could circumscribe the area of transcriptional origin and limit it to a region of approximately −403–271 bp relative to the major IIb TSS located at +1. The TSS analysis carried out with a 5′RACE approach on the rat sequence indicated a cluster of multiple start sites for the antisense RNA in the area situated between −270 and −413 bp upstream of the IIb MHC TSS. This finding is not surprising; it is in agreement with the cardiac antisense β-RNA in which at least two TSSs were identified (17), and the skeletal muscle antisense IIa RNA (aII NAT), whereby more than one TSS was found (38). In addition, mapping analyses and 5′RACE targeting the sense IIb RNA revealed that IIb transcription is carried out with the major start site at +1. However, several minor sites were also found in the IIb promoter, which result in sense RNA that merges with the IIb pre-mRNA and is subjected to similar splicing as suggested by the PCR results using primer 10 (Fig. 4). The observation of the presence of multiple TSS for IIb is not surprising and confirms the finding by Dennehey et al. (14) for the mouse gene showing that the IIb MHC mRNA is expressed in several isoforms that are different in their 5′ untranslated region. Some of these RNA start as far as −1 kb from the IIb TSS (+1) (14). The presence of a bidirectional promoter that coregulates both the sense and antisense RNA transcription is very interesting and is common in human genes. Recently, it has been reported that >10% of human genes are divergent, i.e., controlled by bidirectional promoters (1, 30, 46). Such promoters are defined as IG, < 1-kb long, and flanked by a TSS on both of the DNA strands (32). What makes the bidirectional proximal IIb gene promoter even more interesting in the present study is that its activity is in an inverse relation with the activity of the upstream IIx sense gene (Figs. 3A and 6). That is, when the bidirectional promoter is active, both the IIb sense and the antisense IIx RNA are transcribed, whereas the IIx MHC gene is shut down. In contrast, when the bidirectional promoter undergoes inhibition, both the IIb gene and the antisense transcript are downregulated, but the IIx gene located upstream is activated. There is a simultaneous ying-yang relationship, facilitated by cross talk through more than 37 kb of genomic DNA. Therefore, the IG region that makes up the proximal bidirectional promoter may act not only as a physical linkage between the two MHC genes (IIx and IIb), but it seems to be essential also for their coordinated regulation. Therefore, the IIx and IIb MHC genes are physically and functionally linked through the IG bidirectional promoter region as suggested by the model presented in Fig. 6. In particular, it appears that in order for the IIx MHC gene to be regulated, a feedback from the IG antisense RNA is needed, especially in states whereby shifts in IIx-IIb expression occurs in response to a change is physiological stimuli.
Comparative sequence analysis.
Genome sequence comparisons are typically used to identify noncoding genomic DNA regions that have been evolutionarily conserved across species, presumably to maintain some critical biological function. Such conserved regions often correspond to TFBS and regulatory regions (33, 35, 48). Thus, in this study, a comparative genomic approach was applied to provide insights on IG IIx-IIb promoter sequence conservation among species. MULAN, a computational tool available at http://mulan.dcode.org, which is able to generate multiple sequence alignments, was used to identify conserved regulatory elements by comparing genomic sequences between related species (36). Four orthologous sequences of 4 kb in length, spanning from ∼−1.4 to +2.8 relative to the IIb TSS (+1), were compared. The rat sequence was analyzed against mouse, rhesus monkey, and human DNA sequences. The sequence conservation through evolution presumes a regulatory function of that region. The comparative analysis showed, in fact, that between −1.4 kb and +2.8 kb from the IIb MHC TSS, two regions were highly conserved (85% of similarity) (Fig. 5). The first one (ECR1) was shown to be across the IG region and into the beginning of the IIb MHC gene, located from −294 bp to +47 bp (Fig. 5). The second one (ECR2) was found within the 5′ end of the IIb MHC gene from +211 to +312 bp relative to IIb TSS (+1). Several muscle-specific TFBSs were found on ECR1 upper and lower strands, whereas both ECR1 and ECR2 exhibited several general TFBSs. The ECR1 analysis identified several potential cis regulatory elements such as CAAT box, C/EBP, SRF, and the muscle-specific TFBS for myogenin and MEF-2 (Fig. 5). These conserved regulatory elements were found on the sense strand as well as on the reverse strand. Since ECR1 and ECR2 are highly conserved, their properties may be important promoter regions controlling both the antisense as well as the sense transcription.
The location of the TSS, the presence of conserved cis regulatory elements driving transcription on both the DNA strands and also the position of the very high conserved region across four species provides evidence that the sequence considered (ECR1) is highly probable to function as the common promoter region for the sense IIb and the xII NAT.
It is worth mentioning that previous studies on the mouse IIb promoter have identified some important cis regulatory regions that coincide with some of the ones conserved in ECR1 in the proximal promoter (see Fig. 5). For example Takeda et al. (44) have identified three AT-rich regulatory regions and a proximal E box, and later it was determined that these AT-rich sites interact with MEF-2 and Oct1 (27). Furthermore, the functional role of MEF2, MRF, and SRF transacting factors in the regulation of the IIb proximal promoter in muscle fibers was highlighted in studies by Allen et al. (4, 5). Konig et al. (26) have followed a comparative genomic approach to identify conserved domains in the proximal promoter of mouse and human IIb. However, these previous studies were mostly concerned with the regulation of the IIb promoter activity in the sense direction, whereas in this present study, we unraveled bidirectional activity, and thus we are more concerned in finding that some of these factors can operate for both the sense and antisense transcription.
Relevance to IIx-IIb coexpression in single fibers.
The IIx and IIb MHC genes are closely linked on the skeletal MHC gene locus, and in the WMG are regulated in opposite direction as the muscle undergoes phenotype shift in response to training. Both isoforms are expressed in fast skeletal muscle fibers (9), and therefore may respond to the same transacting factors. In fact, the similarity of TFBS in the proximal promoter among the fast MHC genes, in particular the MEF2-AT-rich sites was noted in several reports (5, 26, 44). Being part of a gene cluster, these genes may be regulated via a common enhancer (26); however, the antisense xII NAT may be important when opposite regulation of the two genes is required. At present, it is not clear whether within the same locus both IIx and IIb are being transcribed at different levels, such that in control WMG 70–80% of the total MHC pool is IIb, whereas the rest is IIx. On the other hand, because the muscle fibers are multinucleated, one might consider that the mosaic nature of MHC isoform expression within a single fiber is because individual myonuclei may be expressing different components of the sense and antisense regulatory circuit differentially at any given moment. On the other hand, because the muscle fibers are multinucleated, one might consider that the mosaic nature of MHC isoform expression within a single fiber is due to individual myonuclei expressing different components of the sense and antisense regulatory circuit differentially at any given moment. Another point to consider is that the dual expression of these two isoforms in many of the individual IIx/IIb fibers is not an all or none process; that is, the antisense model does not operate in a fashion that IIb is fully repressed, while the IIx is optimally expressed. Clearly the regulation is more complex as our data indicate.
Relevance to the differential IIb MHC gene expression between rodent and human.
The results of this study show that xII NAT and the pre-mRNA of the IIb gene are generated by a unique bidirectional promoter located in the IG region flanking the 5′ end of the IIb gene. The IIx-IIb promoter region is highly conserved across mammalian species, even in species phylogenetically distant from each other, such as rat and human. In view of the fact that the IIb MHC gene is rarely expressed in human skeletal muscle (24), it is intriguing to find out why the human IIb gene regulation is different from that of the highly expressed rodent IIb, considering that major regulatory components of the IIb promoter are conserved between the two species. Preliminary studies of the human IIb promoter in a transient reporter assay following direct gene transfer in skeletal muscle show that the human IIb promoter is active in rodent fast muscle and exhibited muscle-type specificity, as the mouse IIb was shown to do previously (5, 43). Thus, it would be useful and intriguing to ascertain the factor/s that really promotes the transcription of the IIb MHC gene in rodent vs. human muscle fibers.
Collectively, this study provides significant insight on the dynamic regulation of the fast IIx and IIb skeletal muscle MHC genes. The expression of the two tandem genes, IIx and IIb, in the WMG is well coordinated to allow rapid shifts between the two isoforms when needed in response to resistance loading. This regulation occurs at the transcriptional level and is tightly coordinated by a cis-acting natural antisense RNA (xII NAT). 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 IIb genes are physically and functionally linked via the bidirectional promoter generating antisense RNA, which appears to be essential for the coordinated regulation of the skeletal muscle MHC genes during dynamic phenotype shifts. In order for the IIx MHC gene to be regulated, a feedback mechanism from the IG xII NAT is needed.
This study was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-30346 (to K. M. Baldwin).
The authors thank Li Ying Zhang, Phuc Tran, Alvin Yu, Bryce Buchowicz, Daniel Jimenez, Sandy Liu, Nkiruka Ojukwu, Jasleen Saini, and Tiffany Yu for excellent technical assistance.
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- Copyright © 2008 the American Physiological Society