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Am J Physiol Heart Circ Physiol 292: H3065-H3071, 2007. First published February 16, 2007; doi:10.1152/ajpheart.01224.2006
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Activity of the beta-myosin heavy chain antisense promoter responds to diabetes and hypothyroidism

Julia Giger, Anqi X. Qin, Paul W. Bodell, Kenneth M. Baldwin, and Fadia Haddad

Department of Physiology and Biophysics, University of California, Irvine, California

Submitted 6 November 2006 ; accepted in final form 15 February 2007


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Two genes encoding cardiac myosin heavy chain (MHC) isoforms, beta and {alpha}, are arranged in tandem 4.5 kb apart. We examined pre-mRNA and mature mRNA levels of beta and {alpha} genes in control, diabetic (streptozotocin), hypothyroid (propylthiouracil), and hyperthyroid rat hearts and analyzed the naturally occurring antisense (AS) beta RNA species that starts in the middle of the 4.5-kb intergenic region and extends upstream to the beta-gene promoter. The beta and {alpha} genes are expressed antithetically in control, diabetic, hypothyroid, and hyperthyroid hearts. Expression of AS beta-RNA was positively correlated with {alpha}-mRNA and negatively correlated with sense beta mRNA. These results support the novel idea of common promoter-regulatory elements situated in the intergenic region that likely control transcription of both sense {alpha} and AS beta genes and that AS beta transcription negatively regulates beta-MHC gene expression. To test whether an intergenic promoter drives transcription of AS beta RNA, a 1340-bp sequence of the intergenic region was inserted into a luciferase plasmid in the 3'-to-5' AS direction and was injected into rat ventricle. This promoter was activated in control heart and decreased greatly in response to propylthiouracil and streptozotocin and increased in hyperthyroid rats, similar in pattern to the endogenous AS beta RNA. When a putative retinoic acid receptor (RAR) site (a known thyroid hormone receptor cofactor) in this promoter was mutated, the reporter activity was almost abolished in control, propylthiouracil, and streptozotocin hearts. We conclude that there is an intergenic promoter that is active in the AS direction and that the putative RAR element is a vital regulatory site.

mRNA; hyperthyroidism; in vivo gene injection; retinoic acid receptor; peroxisome proliferator-activated receptor

MYOSIN HEAVY CHAIN (MHC) is an integral contractile component of the muscle sarcomere, and the enzymatic activity of the MHC ATPase is fundamental in determining the rate of cross-bridge cycling, and thus the speed of muscle contraction. Normal adult rat hearts typically express a predominance of the fast {alpha}-MHC isoform and consequently have a higher rate of contraction. The stimuli of constrictive overload, diabetes, and hypothyroidism cause a shift in expression of MHC isoforms such that these hearts have a significantly higher proportion of the beta-isoform and thus have lower rate of contraction but a greater economy of force production (19, 24, 25, 31, 33, 34). Evidence suggests that the MHC isoforms are regulated pretranslationally, yet the molecular mechanisms that are involved are unclear. The two cardiac MHC genes, beta and {alpha}, are arranged in tandem in a head-to-tail orientation and are only 4 kb apart, and this genomic organization has been conserved for millions of years of evolution (23). Using strand- and gene-specific RT-PCR techniques, we recently reported (17) that in rodent myocardium, in addition to the normal transcription of the {alpha}- and beta-MHC genes into the pre-mRNA and corresponding mRNAs, a transcription of the opposite strand is also detected. The transcription of this other strand generates an antisense (AS) RNA that starts in the intergenic region between the beta and {alpha} genes and extends to overlap with the beta-MHC gene (17) and thus is called AS beta-RNA. Streptozotocin (STZ)-induced diabetes and propylthiouracil (PTU)-induced hypothyroidism caused an increase in sense beta pre-mRNA and a marked decrease in both the sense {alpha} and the AS beta RNAs (17). The inverse relationship between beta mRNA and AS beta RNA suggests that the expression of the AS beta RNA interferes with the transcription of the sense beta pre-mRNA and thus hinders the accumulation of mature beta mRNA. Also, the AS beta RNA is positively correlated with the sense {alpha} pre-mRNA, suggesting that these RNA species are controlled by common intergenic promoter elements, which are capable of affecting transcription in both directions.

On the basis of previous observations (17) in the normal control rodent heart, the beta-isoform expression appears to be inhibited by the amount of the AS beta RNA that is transcribed. This inhibition is removed in response to PTU and diabetes because the AS beta expression is downregulated (17). We hypothesize that a region between the beta and {alpha} genes contains common bidirectional regulatory elements that control the long-recognized coordinated and antithetical regulation of {alpha} and beta gene expression in control and disease states. The current study aims to examine the in vivo regulation of an AS beta promoter via a direct gene transfer of promoter-reporter plasmids into the left ventricle of rat hearts. To our knowledge, the AS promoter activity of the MHC intergenic region has not previously been examined. In this study, the reporter activity of the AS beta promoter was initially characterized in control rat hearts to determine baseline activity and was subsequently examined in response to diabetes (STZ treatment) and thyroid hormone status (PTU and T3 treatment). We show that the AS beta promoter is active in hearts in vivo and that its activity reflects a pattern of response similar to that of the endogenous AS RNA. This supports, in part, the novel hypothesis that an intergenic sequence may serve to control the coordinated antithetical expression pattern of the beta- and {alpha}-MHC genes in response to different stimuli that has been long recognized in the literature.


   METHODS
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 METHODS
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Animal models. Young adult female Sprague-Dawley rats (~150–180 g body wt) were used for experimentally induced diabetes and hypo- and hyperthyroid states spanning a period of 7 days. Diabetes was induced by a single dose of STZ administered via the jugular vein in anesthetized rats immediately before the plasmid gene-injection procedure. STZ (75 mg/kg) was diluted in vehicle solution consisting of 50 mM citrate buffer, pH 4.5. Rats administered only vehicle served as the control group. Hypothyroidism was induced by a daily PTU intraperitoneal injection at a dose of 12 mg/kg body wt (37), and hyperthyroidism was induced by a daily T3 (150 µg/kg body wt) intraperitoneal injection (37). Anesthesia was induced via treatment with ketamine-acepromazine-xylazine (50:1:4 mg/kg), and all animals were euthanized on the seventh experimental day, 6–8 h after the last PTU or T3 injection, by an overdose of sodium pentobarbital (100 mg/kg ip). This study followed the NIH Animal Care Guidelines and was approved by the University of California Irvine Animal Care and Use Committee.

MHC RNA analyses. Total RNA was extracted from frozen ventricles by using the Tri Reagent protocol (Molecular Research Center). The extracted RNA was DNase-treated by using 1 unit RQ1 RNase-free DNase (Promega)/µg 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 RNA concentration was determined by OD260 analysis, and its quality was tested by gel electrophoresis and ethidium bromide staining of 0.5 µg total RNA on the gel. A strand-specific RT-PCR approach was used to analyze the expression of cardiac MHC RNA, which included the following RNAs: beta-MHC mRNA, {alpha}-MHC mRNA, sense beta pre-mRNA, sense {alpha} pre-mRNA, and the AS beta RNA. RT-PCR reactions were performed with the OneStep RT-PCR kit (Qiagen), where the RT and PCR are performed in one reaction tube, with some modifications to the manufacturer's protocol. Detailed methods for RT-PCR reactions and analysis were as described previously (18). See Table 1 for information about the primers.


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  		Table 1. PCR primer information

 
In vivo DNA injection in the myocardium. The DNA injection into the myocardium was performed via a subdiaphragmatic approach exactly as described previously (36, 37). At the time of DNA injection, the rats were anesthetized and the abdomen was opened with a lateral incision below the diaphragm with the use of sterile techniques. The diaphragm was slightly pushed against the heart, and as upward pressure was placed on the sternum, the apex of the ventricle presented against the diaphragm wall. Then 40 µl of sterile phosphate-buffered saline containing an equimolar mixture of two supercoiled DNA plasmids was injected through the diaphragm into the left ventricular wall by using a 29-gauge needle attached to a 0.5-ml insulin syringe. A collar attached around the needle prevented the needle end from advancing into the ventricular chamber. After the injection, the abdomen was closed with sterile surgical sutures and the rats were allowed to recover.

The animals were euthanized on the seventh day following direct DNA transfer, and the apex was removed for analysis. This time period was based on pilot studies demonstrating that optimal luciferase activity is achieved within this seven-day time period. Tissue was processed as described previously for reporter-gene assays (36, 37).

DNA plasmid constructs. Several different sequence fragments of the –4.5 kb intergenic region (depicted in GoFig. 2) were isolated and inserted into renilla luciferase plasmid (Promega) in the 3'-to-5' direction to drive renilla luciferase expression. The promoter of the cardiac myosin light chain (MLC2) gene (a gift from Dr. K. Esser, University of Kentucky) was cloned into a firefly luciferase expression plasmid (pGL3; Promega) and was coinjected with the test plasmid. The firefly activity was used to normalize renilla luciferase (test promoter) activity to correct for variation in the test plasmid uptake/transfection.


Figure 1
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  		Fig. 1. Antisense (AS) beta-myosin heavy chain (MHC) and sense {alpha}-MHC transcripts are coregulated. A: representative gels of pre-mRNA, AS beta RNA, and mature mRNA transcripts of control (NC) vehicle-treated, diabetic [streptozotocin (STZ)-treated], hyperthyroid (T3-treated), and hypothyroid [propylthiouracil (PTU)-treated] rats. Sense (S) and AS primary transcripts and mature mRNA of beta- and {alpha}-MHC genes are depicted (see Table 1 for primer information). B: sense beta, AS beta, and sense {alpha} pre-mRNA levels under different conditions: control (Veh; n = 16), STZ (n = 10), PTU (n = 6), T3 (n = 6). AU, arbitrary units. *Significantly different from vehicle sample in group (P < 0.05). C: relationship between AS beta and sense {alpha} RNA for all groups combined. Data points represent individual samples, and lines were generated by linear regression analysis. D: relationship between beta pre-mRNA and AS beta RNA. Data points represent individual samples, and lines were generated by linear regression analysis.

 

Figure 2
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  		Fig. 2. AS beta promoter activity and deletion analysis in control rat hearts. Top: intergenic (IG) region between the tandem cardiac MHC beta and {alpha} genes, which possesses transcriptional activity in the sense and AS directions. AS promoter fragments as derived from the intergenic sequence were tested in control rat hearts. The arrow labeled +1 denotes the start site of the {alpha}-MHC gene; the left-pointing arrows denote the start sites of the AS beta transcript. Bottom: deletion analysis of AS beta promoter in control rat heart. AS-driven renilla luciferase activity divided by reference myosin light chain (MLC2) promoter-driven firefly luciferase. *Significantly different from 1340-bp AS promoter activity (P < 0.05).

 
For promoter deletions and site-directed mutagenesis, standard recombinant DNA techniques and methods were used as previously described (12–14, 36). Putative transcription-factor consensus binding sites were identified by using Internet sources (TESS, MatInspector, http://www.cbil.upenn.edu/cgi-bin/tess/tess). When necessary, DNA was sequenced by using services provided by the University of California Irvine DNA Core facility to verify the mutation within the insert. Mutations are shown in Table 2.


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  		Table 2. Mutation of putative PPAR and RAR binding sites created in the 1340-bp promoter

 
Statistical analysis. Data are reported as means ± SE. For bar graphs, comparisons within groups used a one-way analysis of variance with Newman-Keuls post hoc test (GraphPad Software). Statistical significance was set at P < 0.05; n ≥ 8 samples/group.


   RESULTS
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Strand-specific RNA analysis of MHC in diabetic, hypothyroid, hyperthyroid, and normal adult rats. We have previously examined the RNA species of MHC in rat ventricles and discovered a naturally occurring AS beta RNA transcript that starts in the middle of the intergenic region (between beta- and {alpha}-MHC genes) and extends upstream to the beta-MHC gene-promoter region, overlapping and surpassing the entire beta-MHC gene (17, 18). Transcription of the lower strand, which proceeds upstream toward the beta-MHC gene, produces AS RNA, a process that appears to interfere with beta gene transcription. In the current study we examined sense beta, AS beta, and sense {alpha} pre-RNA species and mature beta and {alpha} mRNA in vehicle-treated normal control, type 1 diabetic (7-day STZ-treated), hypothyroid (PTU-treated), and hyperthyroid (T3-treated) rats (Fig. 1). Treatment with STZ and PTU caused similar effects on the level of transcripts, as both perturbations caused an increase in sense beta pre-mRNA and beta mRNA and a decrease in AS beta, sense {alpha}, and {alpha} mRNA (Fig. 1) (17). T3 treatment caused a shift in expression to a greater extent than the vehicle control group, and both states resulted in low levels of sense beta and beta mRNA, which was in contrast to the high levels of AS beta, sense {alpha}, and {alpha} mRNA (Fig. 1). The pattern of transcription of the AS beta and sense {alpha} RNA, as they varied with the different experimental manipulations, was significantly correlated (P < 0.001; Fig. 1C). This strong positive correlation (r = 0.97; R2 = 0.94;) supports the hypothesis that common promoter regulatory elements are situated in the intergenic region between beta and {alpha} genes and likely control transcription of both the sense {alpha} and the AS beta genes. The hypothesis proposes that in a normal rat, this regulatory region is active and induces transcription of {alpha} pre-mRNA and AS beta RNA. In a hypothyroid or diabetic state, as the AS beta transcription is inhibited, the beta-MHC gene transcription is activated, which results in the accumulation of sense beta pre-mRNA and mature beta mRNA. This inverse relationship is best illustrated by the strong negative correlation between the sense beta pre-mRNA and the AS beta RNA (r = –0.95; R2 = 0.90; P < 0.001, Fig. 1D). On the basis of RNA expression, we propose that in the diabetic and hypothyroid rat, the common intergenic regulatory region is inactive or inhibited, thereby blocking transcription of both the {alpha}- and the AS beta genes. Since the intergenic region appears to be pivotal in the regulation of {alpha} and beta gene expression, we sought to determine if an AS promoter-reporter system can be studied in vivo under manipulations involving altered thyroid and diabetic states and the mutation of specific regulatory elements.

Characterization of AS beta promoter in vivo. AS beta promoter was examined in vivo to address the question of whether the amount of AS beta RNA is controlled by regulatory sequences in the intergenic region. The boundaries of the AS promoter are not clear, but two start sites have been identified at –2197 and at –2160 by using 5'-rapid amplification of cDNA ends analyses (17). Several different sequence fragments of the –4.5-kb intergenic region were isolated and inserted into renilla luciferase plasmid in the 3'-to-5' direction (Fig. 2). The reporter activity of a shorter –2.3-kb promoter was higher than that of the full-length –4.5-kb promoter. Further deletion of this fragment (–1.7 kb) caused a significant reduction in activity, likely due, in part, to the removal of the start sites. A 1340-bp promoter corresponding to the –2285/–945 region, which includes the start sites, was active in control hearts at a level comparable with the 2.3-kb promoter. A further deletion of this 1340-bp promoter, which was 559 bp (–2285/–1726) in length, was not significantly different from the 1340-bp promoter despite the fact that more than half of the base pairs were deleted. These latter results indicate that the 559-bp promoter contains essential elements for the activation of the AS promoter in control hearts.

Activity of the AS beta-MHC promoter in normal control vs. diabetic, hypothyroid, and hyperthyroid rat hearts. If the hypothesis that the intergenic promoter regulates the AS RNA transcription is correct, STZ and PTU treatment should cause a decrease and T3 treatment should induce an increase in AS promoter activity relative to the control state. The AS beta promoter-reporter construct was injected into ventricles of control (vehicle-treated), STZ-, PTU-, and T3-treated rats. The activity of the 1340-bp (–2285/–945) AS beta-MHC construct was significantly decreased in response to both PTU and STZ treatment, whereas it increased ninefold with T3 treatment (see Fig. 3). This result supports the hypothesis that AS RNA expression in response to diabetes and thyroid status is likely regulated through an intergenic promoter. PTU, STZ, and T3 treatments caused similar effects on promoter activity of the 559-bp deletion promoter as well, indicating that diabetes and thyroid-responsive regulatory elements are within the 559-bp (–2285/–1726) region.


Figure 3
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  		Fig. 3. AS beta promoter responds to manipulations. Activity of wild-type 1340-bp and 559-bp AS beta promoters and 1340-bp promoters containing peroxisome proliferator-activated receptor (PPAR) mutation and a retinoic acid receptor (RAR) mutation in response to diabetes (7 days STZ treatment), hypothyroidism (7 days PTU treatment), or hyperthyroidism (7 days T3 treatment). *Significantly different from vehicle sample in group (P < 0.05). Inset: reference promoter, MLC2 promoter-driven firefly luciferase, is useful as an effective transfection control because its activity does not change with manipulations by STZ, PTU, and T3. RLU, relative light units. Bottom: location of putative response elements within the 1340-bp AS promoter. X, mutated sites at PPAR and RAR sites; T3R, thyroid hormone receptor; RXR, retinoid X receptor; –1726, end of the 559-bp promoter (–2285 to –1726); MEF2, myocyte enhancer factor 2; NFAT, nuclear factor of activated T cells.

 
The 1340-bp promoter sequence was analyzed by using Internet sources (TESS, MatInspector) to identify putative transcription factor consensus binding sites, and numerous putative cis-regulatory elements were identified, including E-boxes, nuclear factor of activated T cells (NFAT), peroxisome proliferator-activated receptor (PPAR), and retinoic acid receptor (RAR)/retinoid X receptor (RXR)/thyroid hormone receptor (TR) binding sites. A mutation of a putative PPAR site at –1823/–1802 (Table 2), was created in the 1340-bp promoter and was tested in vehicle-, STZ-, PTU-, and T3-treated rat hearts (Fig. 3). PPAR has been associated with diabetes-induced changes in gene expression. If changes in promoter activity in response to diabetes and thyroid hormone are mediated through this PPAR site, then one would expect the PPAR mutant to abolish the promoter responsiveness to these manipulations. However, the activity levels of the mutated PPAR promoter resembled those of the wild-type promoter. Taking into account that alterations in thyroid status affected the AS promoter activity, we then tested a mutation of two putative RAR/TR binding sites. RAR/TR are nuclear receptors that are known to form a heterodimer with each other (22). Although we designated our mutation "RAR," the site also may be a TR binding site, because TRs, RARs, and RXRs recognize the same sequence (22) (as indicated by TESS). Mutation of the two RAR binding sites (–1937/–1923) within the 1340-bp promoter almost completely abolished reporter activity in all groups, and the STZ and PTU groups were no different from the vehicle group. The T3 group did respond to T3 despite the mutation, but there was only a threefold increase. Recall that this is markedly lower than the ninefold response to T3 in the wild type (Fig. 3). The RAR sites are evidently critical in the regulation of AS beta transcription.


   DISCUSSION
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We postulate that the cardiac MHC intergenic bidirectional transcription serves a relevant molecular function in modulating the transcription of cis-linked beta- and {alpha}-MHC genes and plays a critical role in their reciprocally regulated expression. The RNA results suggest that there is negative regulation of beta-MHC gene expression by AS beta RNA. This notion is supported by the fact that there is an inverse relationship between beta pre-RNA and AS beta RNA levels, because these two vary in response to STZ and thyroid state manipulations. AS RNA could regulate the amount of beta pre-mRNA expressed by two possible mechanisms (18). One is transcriptional interference, whereby transcription of a gene encoded on one strand of DNA inhibits the concomitant transcription of the overlapping gene on the opposite strand, possibly due to collision of the large transcriptional complexes (2). The second mechanism is via an AS RNA-mediated methylation of CpG islands within the beta-MHC gene promoter. This has primarily been associated with gene silencing at imprinted loci, although examples exist for nonimprinted loci (35). DNA methylation can alter the histone structure and the chromatin to a conformation that is not favorable for transcription (4). Several CpG islands have been identified in the beta-MHC gene promoter and in the first intronic region via in-silico analyses (data not shown). These CpG islands may be the targets for the AS RNA-mediated methylation, because the AS beta RNA has been shown to be transcribed through the beta promoter region, up to 9 kb upstream of the transcription start site. Although the natural AS RNA may inhibit the corresponding sense gene expression via several mechanisms (21), the above two mechanisms are more likely at play based on the RNA expression pattern and other analyses (18). In the models used in this study (hypothyroid and diabetic hearts, where less AS RNA is transcribed), the inhibition of the beta-MHC gene transcription is reduced via one of the two proposed mechanisms. Future studies should be designed to differentiate between these regulatory mechanisms involving the AS RNA, transcriptional interference, and CpG methylation. This novel regulatory mechanism provides the basis behind the tight antithetical regulation of the two genes and plays a pivotal role in providing plasticity and adaptive advantage to the myocardium MHC phenotype.

The present study concentrated on the regulation of the AS transcriptional activity of the proposed bidirectional intergenic regulatory region. Our aim was to characterize an AS promoter in vivo and to determine whether it would respond to deletion analysis and hormonal stimuli appropriately. The data presented herein indicate that the injected 1340-bp AS promoter was active in normal hearts. The 1340-bp AS promoter responded to experimental manipulations (STZ, PTU, T3) similarly to the endogenous AS RNA species. The fact that the AS promoter is active and responsive to altered physiological states allows us to directly examine its promoter sequence to determine which elements are involved in regulating its transcriptional activity in response to conditions such as diabetes and thyroid hormone changes. Whether the same potential elements are also relevant in regulating transcription in the sense direction, as the hypothesis proposes, should be determined in future experiments.

STZ treatment was used in this study to mimic a type 1 diabetic stimulus. Diabetes causes an increase in beta-MHC and a decrease in {alpha}-MHC expression in both rats and humans, which has been shown to have a negative impact on cardiac contractile function (unpublished data and Refs. 29 and 31). In the diabetic state, there is a decrease in glucose oxidation and an increase in fatty acid oxidation in the myocardium, and this shift in cardiac energy substrate use is believed to lead to structural and metabolic alterations in cardiac myocytes and ultimately cardiac contractile dysfunction in mice (3), sheep (28), and humans (32). Increased uptake and use of fatty acids is associated with an increase in gene expression of enzymes and factors involved in fatty acid oxidation. PPARs have been shown to play an important role in the transcriptional control of these fatty acid oxidation genes, and PPAR-{alpha} may mediate the cardiomyopathies resulting from diabetes (10, 11). In the present study, the PPAR binding site was mutated in the AS promoter, but this mutation was found to have no obvious effect on AS promoter activity compared with wild type. Therefore, this putative PPAR binding sequence is not likely to be involved in the regulation of the AS promoter, but this does not definitively exclude PPAR as a possible transcription factor/cofactor (see below).

Thyroid status had a profound effect on the 1340-bp promoter, causing reporter activity to decrease and increase dramatically in response to hypo- and hyperthyroid states, respectively (Fig. 3). Thyroid hormone has been shown to have a large impact on cardiac MHC gene expression (this study and Refs. 6, 9, 15, and 16) and is a significant positive regulator of {alpha}-MHC promoter (6, 26, 27, 30). Although the beta-MHC gene is downregulated in response to T3 and the beta-MHC promoter activity was inhibited by T3, there was no clear-cut sequence that contained a thyroid-response element that can be implicated in the T3 response (8, 27, 37). A regulatory role of T3 on the beta-MHC promoter is not clear and may only affect the beta-MHC posttranscriptionally (5).

The molecular action of thyroid hormones is mediated by nuclear receptors TR-{alpha} and TR-beta. TR forms homodimers and more commonly heterodimers with either RXR or RAR. The dimers bind to two adjacent half-sites, often repeats, within the gene sequence. TRs, RARs, and RXRs recognize the same sequences. Two half-sites create a functional hormone-response element. The ligand for the TR is T3, the ligand for RAR is all-trans retinoic acid, and the ligand for RXR is 9-cis retinoic acid. Ligand dependence, heterodimer partner, half-site repeat base pair spacing, and coactivator/corepressor interactions all influence the potential transcriptional activity of these receptors. For example, when no ligand is present, the TR/RAR heterodimer is bound by a corepressor; however, the presence of T3 or all-trans retinoic acid causes the partial release of the corepressor. Furthermore, the presence of both T3 and all-trans retinoic acid results in full release of corepressor and the binding of a coactivator. Therefore, these heterodimers have large regulatory capacity of gene transcription (see Refs. 7 and 22).

The TR/RAR binding site mutation in the 1340-bp AS promoter caused such a large decrease in the reporter activity in all groups that we believe this regulatory element undoubtedly plays a vital role in the general transcriptional activity of the AS promoter in control rats. It is possible that this TR/RAR site may confer the promoter's responsiveness to thyroid hormone and diabetes. The diabetes response may be transduced through this RAR site, perhaps via the diabetes-induced PPAR. PPAR commonly forms a heterodimer with RXR. In fact, PPAR has been shown to substitute for RAR in an RAR-RXR heterodimer, mediating transactivation in response to both PPAR and RXR ligands, whereas RAR-RXR heterodimers normally mediate transcriptional repression (7).

The fact that mutating the TR/RAR site has such a dramatic effect on promoter activity should not be surprising given that these sites are contained in a distal domain that is highly conserved among five mammalian species (rat, mouse, hamster, rabbit, and human; all extracted from the GenBank via BLAST searches). This distal domain is divided into three functional blocks, B1, B2, and B3, on the basis of a high rate of sequence similarity, and TR/RAR binding sites are conserved in all three blocks, giving this region a high probability of sensitivity to thyroid hormone. Furthermore, it is of interest to know that this TR/RAR site is located within the DNase-hypersensitive site that is sensitive to T3 during developmental stages of the hamster as reported by Huang et al. (20). Future experiments are required to definitively identify what factor(s) are binding the relevant RAR element within the promoter.

We have shown that the endogenous mRNA species of beta- and {alpha}-MHC genes are expressed in an antithetical manner in control, diabetic, hypothyroid, and hyperthyroid hearts (Fig. 1). There is a positive correlation in the expression of AS beta RNA and sense {alpha}-pre-mRNA (Fig. 1C), indicating a likely regulatory link between these two genes. Our hypothesis that an intergenic promoter exists for AS RNA was supported by the data, which shows that the 1340-bp AS promoter is active in vivo and responds to hormonal manipulations in a similar fashion to the endogenous AS RNA species. The AS promoter sequence is within a highly conserved domain of the region between the beta- and {alpha}-MHC genes and is likely involved in the coordinated, antithetical regulation of the beta- and {alpha}-MHC genes in heart.


   GRANTS
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This study was funded by National Heart, Lung, and Blood Institute Grant HL-73473-01 to K. Baldwin, a University of California, Irvine, Center for Cardiovascular Hormone Research Award, and American Diabetes Association Grant no. 7-06-JF-22 to J. Giger.


   ACKNOWLEDGMENTS
 
We thank LiYing Zhang, Toni Garma, Cori Kobayashi, Phuc Tran, Alvin Yu, Juliane Lynn, Rudy Senstad, and Natasha Moningka for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Giger, Dept. of Physiology and Biophysics, Univ. of California, Irvine, D-346, Med. Sci. I, Irvine, CA 92697 (e-mail: jmeehan{at}uci.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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