An M-CAT binding factor and an RSRF-related A-rich binding factor positively regulate expression of the alpha-cardiac myosin heavy-chain gene in vivo.

Cardiac muscle-restricted expression of the alpha-myosin heavy-chain (alpha-MHC) gene is regulated by multiple elements in the proximal enhancer/promoter. Within this region, an M-CAT site and an A-rich site were identified as potential regulatory elements. Site-specific mutations in each site, individually, reduced activity from the wild-type promoter by approximately 85% in the adult rat heart, demonstrating that these sites were positive regulatory elements. alpha-MHC, beta-MHC, and chicken cardiac troponin T (cTnT) M-CAT sites interacted with an M-CAT-binding factor (MCBF) from rat heart nuclear extracts that was immunologically related to transcriptional enhancer factor 1, a factor that binds within the simian virus 40 enhancer. The factor that bound the A-rich region (ARF) was antigenically related to the RSRF family of proteins, ARF was distinct from myocyte-specific enhancer factor 2 (MEF-2) on the basis of DNA-binding specificity and developmental expression. Like MEF-2, ARF DNA-binding activity was present in the heart and brain; however, no ARF activity was detected in extracts from skeletal muscle or C2C12 myotubes. MCBF and ARF DNA-binding activities were developmentally regulated with peak levels in the 1- to 2-day neonatal heart. The activity of both factors increased nearly fivefold in adult rat hearts subjected to a pressure overload. By comparison, the levels of alpha-MHC binding factor 2 did not change during hypertrophy. Binding sites for MCBF and ARF are present in several genes that are upregulated during cardiac hypertrophy. Our results suggest that these factors participate in the alterations in gene expression that occur during cardiac development and hypertrophy.

Transcription factor GATA-4 regulates cardiac muscle-specific expression of the alpha-myosin heavy-chain gene.

The alpha-myosin heavy-chain (alpha-MHC) gene is the major structural protein in the adult rodent myocardium. Its expression is restricted to the heart by a complex interplay of trans-acting factors and their cis-acting sites. However, to date, the factors that have been shown to regulate expression of this gene have also been found in skeletal muscle cells. Recently, transcription factor GATA-4, which has a tissue distribution limited to the heart and endodermally derived tissues, was identified. We recently found two putative GATA-binding sites within the proximal enhancer of the alpha-MHC gene, suggesting that GATA-4 might regulate its expression. In this study, we establish that GATA-4 interacts with the alpha-MHC GATA sites to stimulate cardiac muscle-specific expression. Mutation of the GATA-4-binding sites either individually or together decreased activity by 50 and 88% in the adult myocardium, respectively. GATA-4-dependent enhancement of activity from a heterologous promoter was mediated through the alpha-MHC GATA sites. Coinjection of an alpha-MHC promoter construct with a GATA-4 expression vector permitted ectopic expression in skeletal muscle but not in fibroblasts. Thus, the lack of alpha-MHC expression in skeletal muscle correlates with a lack of GATA-4. GATA-4 DNA binding activity was significantly up-regulated in triiodothyronine- or retinoic acid-treated cardiomyocytes. Putative GATA-4-binding sites are also found in the regulatory regions of other cardiac muscle-expressed structural genes. This indicates a mechanism whereby triiodothyronine and retinoic acid can exert coordinate control of the cardiac phenotype through a trans-acting regulatory factor.

The cardiac tissue-restricted homeobox protein Csx/Nkx2.5 physically associates with the zinc finger protein GATA4 and cooperatively activates atrial natriuretic factor gene expression.

Specification and differentiation of the cardiac muscle lineage appear to require a combinatorial network of many factors. The cardiac muscle-restricted homeobox protein Csx/Nkx2.5 (Csx) is expressed in the precardiac mesoderm as well as the embryonic and adult heart. Targeted disruption of Csx causes embryonic lethality due to abnormal heart morphogenesis. The zinc finger transcription factor GATA4 is also expressed in the heart and has been shown to be essential for heart tube formation. GATA4 is known to activate many cardiac tissue-restricted genes. In this study, we tested whether Csx and GATA4 physically associate and cooperatively activate transcription of a target gene. Coimmunoprecipitation experiments demonstrate that Csx and GATA4 associate intracellularly. Interestingly, in vitro protein-protein interaction studies indicate that helix III of the homeodomain of Csx is required to interact with GATA4 and that the carboxy-terminal zinc finger of GATA4 is necessary to associate with Csx. Both regions are known to directly contact the cognate DNA sequences. The promoter-enhancer region of the atrial natriuretic factor (ANF) contains several putative Csx binding sites and consensus GATA4 binding sites. Transient-transfection assays indicate that Csx can activate ANF reporter gene expression to the same extent that GATA4 does in a DNA binding site-dependent manner. Coexpression of Csx and GATA4 synergistically activates ANF reporter gene expression. Mutational analyses suggest that this synergy requires both factors to fully retain their transcriptional activities, including the cofactor binding activity. These results demonstrate the first example of homeoprotein and zinc finger protein interaction in vertebrates to cooperatively regulate target gene expression. Such synergistic interaction among tissue-restricted transcription factors may be an important mechanism to reinforce tissue-specific developmental pathways.

The transcription factor GATA4 is activated by extracellular signal-regulated kinase 1- and 2-mediated phosphorylation of serine 105 in cardiomyocytes.

The zinc finger-containing transcription factor GATA4 has been implicated as a critical regulator of multiple cardiac-expressed genes as well as a regulator of inducible gene expression in response to hypertrophic stimulation. Here we demonstrate that GATA4 is itself regulated by the mitogen-activated protein kinase signaling cascade through direct phosphorylation. Site-directed mutagenesis and phospho-specific GATA4 antiserum revealed serine 105 as the primary site involved in agonist-induced phosphorylation of GATA4. Infection of cultured cardiomyocytes with an activated MEK1-expressing adenovirus induced robust phosphorylation of serine 105 in GATA4, while a dominant-negative MEK1-expressing adenovirus blocked agonist-induced phosphorylation of serine 105, implicating extracellular signal-regulated kinase (ERK) as a GATA4 kinase. Indeed, bacterially purified ERK2 protein directly phosphorylated purified GATA4 at serine 105 in vitro. Phosphorylation of serine 105 enhanced the transcriptional potency of GATA4, which was sensitive to U0126 (MEK1 inhibitor) but not SB202190 (p38 inhibitor). Phosphorylation of serine 105 also modestly enhanced the DNA binding activity of bacterially purified GATA4. Finally, induction of cardiomyocyte hypertrophy with an activated MEK1-expressing adenovirus was blocked with a dominant-negative GATA4-engrailed-expressing adenovirus. These results suggest a molecular pathway whereby MEK1-ERK1/2 signaling regulates cardiomyocyte hypertrophic growth through the transcription factor GATA4 by direct phosphorylation of serine 105, which enhances DNA binding and transcriptional activation.

Analysis of mRNA synthesis following induction of the Escherichia coli SOS system.

Escherichia coli responds to impairment of DNA synthesis by inducing a system of DNA repair known as the SOS response. Specific genes are derepressed through proteolytic cleavage of their repressor, the lexA gene product. Cleavage in vivo requires functional RecA protein in a role not yet understood. We used mRNA hybridization techniques to follow the rapid changes that occur with induction in cells with mutations in the recA operator or in the repressor cleavage site. These mutations allowed us to uncouple the induction of RecA protein synthesis from its role in inducing the other SOS functions. Following induction with ultraviolet light, we observed increased rates of mRNA synthesis from five SOS genes within five minutes, maximum expression ten to 20 minutes later and then a later decline to near the initial rates. The presence of a recA operator mutation did not significantly influence these kinetics, whereas induction was fully blocked by an additional mutation in the repressor cleavage site. These experiments are consistent with activation of RecA protein preceding repressor cleavage and derepression of SOS genes. The results also suggest that the timing and extent of induction of individual SOS genes may be different.

Myocyte-specific enhancer-binding factor (MEF-2) regulates alpha-cardiac myosin heavy chain gene expression in vitro and in vivo.

A myocyte-specific enhancer-binding factor (MEF-2) DNA binding site was identified in the rat alpha-myosin heavy chain (MHC) gene adjacent to the E-box binding site for alpha-MHC binding factor-2 (BF-2). Mutation of the MEF-2 site, within the context of the full-length promoter, reduced activity by 85 and 80% in neonatal cardiomyocytes and the adult heart, respectively. Mutation of the BF-2 site reduced activity approximately 70% in both models. A MEF-2/BF-2 double mutant gave significantly less activity than the BF-2 mutant but not the MEF-2 mutant, suggesting the possibility that BF-2 and MEF-2 interact. Mutations in MEF-2, which decreased functional activity, also abolished MEF-2 DNA binding activity. MEF-2 DNA binding activity was present in the developing heart, reached a peak in the late fetal and early neonatal stages, and then declined to low levels in the adult heart. The adult levels were sufficient to support alpha-MHC gene expression. MEF-2 activity was increased 2-3-fold in the adult heart subjected to a pressure or volume overload. Two working models are proposed as possible explanations of the antithetic relationship between MEF-2 levels and alpha-MHC gene expression.

p300 Functions as a coactivator of transcription factor GATA-4.

Transcription factor GATA-4 plays critical roles in controlling heart development and cardiac hypertrophy. To understand how GATA-4 functions under diverse conditions, we sought to identify its coactivators. We tested p300 as a coactivator in GATA-4-dependent transient transcription assays in NIH3T3 cells and found that p300 synergistically activated GATA-4-dependent transcription on both synthetic and natural promoters. Direct physical interactions between the N- and C-zinc finger domains of GATA-4 and the cysteine/histidine-rich region 3 (C/H3) of p300 were identified in immunoprecipitation and glutathione S-transferase pull-down experiments. Deletion of the C/H3 region of p300 abolished its coactivator activity indicating that the physical interaction was required for functional synergy. Through the use of a series of GATA-4 zinc finger mutants, the amino acids WRR in the C finger were identified as critical to the interaction. The adenoviral E1A protein or a peptide encoding the C/H3 region of p300 could inhibit GATA-4-dependent transcription, presumably by competing for p300 binding. Furthermore, deletion of the region of p300 encoding the histone acetyltransferase activity abolished its effect on GATA-4-dependent transcriptional activity. These results establish that p300 acts as a GATA-4 coactivator and that the p300 histone acetyltransferase activity is necessary for the functional interaction.

Influence of chromosome integrity on Escherichia coli cell division.

The antitumor agent cis-platinum(II)diamminodichloride (PDD) caused wild-type and recA+ deoxyribonucleic acid (DNA) repair-deficient mutant cells of Escherichia coli K-12 to grow as long, multinucleated filaments. At 5 micrograms/ml, the times required for reduction of viability to 37% for wild-type, polA, recB,C, uvrA, and recA organisms were > 200, 200, 120, 25, and 5 min, respectively. Only recA cells exhibited @reckless" degradation of DNA at this concentration of PDD. As shown by sedimentation in alkaline sucrose gradients, generation of single-strand breaks in DNA of the remaining organisms was a major consequence of growth in PDD. Upon incubation in fresh medium after removal of the compound and storage for 4 h at 4 degrees C, a respective lag of 3, 4, 6, and 9 h occurred before filaments of wild-type, polA, recB,C, and uvrA cells commenced cell division. Maintenance at 4 degrees C, which evidently delayed postshift initiation of chromosome replication, was only essential for fragmentation of uvrA filaments. In all cases, these periods of division delay corresponded to those required for restoration of normal chromosomal molecular weight as determined in alkaline sucrose gradients.

Physiology of the SOS response: kinetics of lexA and recA transcriptional activity following induction.

The products of the lexA and recA genes play central roles in the regulation of the Escherichia coli SOS response. We have measured the rate of mRNA synthesis from each gene at intervals following various inducing treatments in order to obtain a more precise timing of the induction process. Further, we provide quantitative evidence for kinetics of decay from fully induced levels of mRNA synthesis to basal levels as the cells shut down the SOS response which are in agreement with previously published data on the expression of specific SOS functions. The induction kinetics of lexA and recA gene expression are parallel except for nalidixic acid (NAL) treatment, with the actual levels of lexA mRNA synthesis being about 10-fold lower than that of recA. Reestablishment of repression from RecA commenced over 30 min earlier than from lexA. These results are fully consistent with the model that the functions result from the increased gene expression.

Analysis of thyroid hormone effects on myosin heavy chain gene expression in cardiac and soleus muscles using a novel dot-blot mRNA assay.

Effects of thyroid hormone on myosin heavy chain (MHC) gene expression were compared in ventricle and soleus muscle of hypothyroid rats, since in this condition, both muscle types express predominately beta-MHC mRNA. Changes in MHC mRNAs were analyzed using synthetic oligonucleotide probes complementary to the 3' untranslated region of the MHC mRNAs. The results indicated that daily treatment with 3,5,3'-triiodo-L-thyronine (T3) at a dose of 2 micrograms/100 g body weight increased alpha-MHC mRNA content in heart muscle by 600% and decreased beta-MHC mRNA by 70% within 48 h. In soleus muscle, beta-MHC mRNA levels were not affected by 9 wks of treatment, however, Fast IIa-MHC mRNA was increased by 150% at 7 wks and 300% after 9 wks of T3 administration. Thus, regulation of MHC gene expression by thyroid hormone is both gene and tissue specific.

Alpha-myosin heavy chain gene regulation: delineation and characterization of the cardiac muscle-specific enhancer and muscle-specific promoter.

The alpha-myosin heavy chain (alpha-MHC) gene encodes a cardiac muscle-specific protein involved in active force generation. The mechanism responsible for restricting expression of this gene to the heart should provide clues for the identification of transcriptional regulatory events involved in the induction and maintenance of the cardiac cell lineage. In this report we dissect the alpha-MHC regulatory region to identify the components necessary for directing high levels of cardiac muscle-restricted expression. Deletion, site-specific mutant and heterologous promoter constructs were assayed for expression after injection into adult rat heart and skeletal muscle or transfection into non-muscle cells. These studies indicated that sequences from -344 to -156 directed high levels of cardiac-muscle specific expression from a heterologous promoter that was independent of position and orientation. This region includes a previously uncharacterized CArG box, alpha-MHC sequences from -86 to +16 promoted activity from two heterologous enhancers in a muscle-specific fashion. Mutational analysis of an E-box and a CArG box within the promoter revealed that they act as negative and positive regulatory elements, respectively. Based on competitive binding and supershift electrophoretic mobility shift assays, serum response factor was shown interact with the CArG boxes found in the promoter and enhancer. Similar experiments demonstrated that the E-box bound to a factor immunologically related to upstream stimulatory factor. Together, these results identify two distinct regions with different regulatory function that are critical for the tissue restricted expression of the alpha-MHC gene.

Nucleotide sequence of the lexA gene of Escherichia coli K-12.

A number of E. coli genes exhibit increased expression when the cellular DNA is damaged. In undamaged cells, lexA repressor limits the extent of their transcription, whereas, in damaged cells, the repressor is cleaved by a cellular protease, the product of the recA gene. We have sequenced 943 base pairs of cloned E. coli DNA containing the lexA gene. A regulatory region has been identified, followed by a translational open reading frame which encodes a polypeptide of 202 amino acids with a molecular weight of 22,300. The protein contains a single alanyl-glycyl peptide near its middle. This peptide is also found in certain phage repressors which are cleaved by the recA protease and has been shown to be the site of cleavage in these repressors. We have determined the nucleotide sequence of a portion of the lexA3 gene, whose product is 100-fold less susceptible to recA protease than the wild type repressor. We report a single base change (G to A) which alters the unique alanine-glycine sequence to alanine-aspartic acid.

Effects of thyroid hormone on alpha-actin and myosin heavy chain gene expression in cardiac and skeletal muscles of the rat: measurement of mRNA content using synthetic oligonucleotide probes.

Effects of thyroid hormone on alpha-actin and myosin heavy chain gene expression were compared in ventricle, soleus, and extensor digitorum longus muscles of hypothyroid rats. Changes in mRNA content were analyzed using synthetic oligonucleotide probes complementary to the unique 3' untranslated regions of four striated myosin heavy chain mRNAs and cardiac and skeletal muscle alpha-actin mRNAs. The results indicate that daily treatment with 3,5,3'-triiodo-L-thyronine (2 micrograms/100 g body weight) increased alpha-myosin heavy chain mRNA content in heart muscle by 500% and decreased beta-myosin heavy chain mRNA by 65% within 48 hours. beta-mRNA in extensor digitorum longus was decreased by 60% at 48 hours while in soleus, beta-mRNA levels were not affected by 9 weeks of treatment. Fast IIa mRNA was present in small amounts in hypothyroid soleus and increased by 150% and 200% after 7 and 9 weeks of thyroid hormone administration, respectively. Fast IIb mRNA also was found in hypothyroid soleus and a small increase (60%) was observed after 1 day of treatment. In extensor digitorum longus, Fast IIb mRNA increased by 200% and Fast IIa mRNA decreased by 50% after 1 week of treatment. When larger daily doses of thyroid hormone (15 micrograms/100 g body weight) were administered, similar changes in mRNA levels were observed, except that beta-mRNA content of soleus muscle was decreased slightly (25%). Expression of the cardiac form of alpha-actin was induced transiently in ventricle, but the skeletal form of alpha-actin mRNA in soleus and extensor digitorum longus did not change significantly after thyroid hormone treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Complete amino acid sequence of human thyroxine-binding globulin deduced from cloned DNA: close homology to the serine antiproteases.

Antibodies directed against thyroxine-binding globulin (TBG) have been used to screen a human liver lambda gt11 expression library. A 1.46-kilobase clone was identified which encodes nearly the complete amino acid sequence, beginning at amino acid 17 of the mature protein. To complete the protein sequence, the cDNA clone was used to identify a genomic clone coding for TBG in a human X chromosome library. The overlapping recombinant clones contained an open reading frame coding for 415 amino acids followed by a polyadenylylation signal (AATAAA) located 275 nucleotides from a TAG termination codon. Beginning at residue 21, the deduced amino acid sequence agrees closely with the known NH2-terminal sequence of the mature peptide. The preceding 20 amino acid residues are hydrophobic in character and presumably represent a leader sequence. Four glycosylation sites were identified, corresponding to the number determined for the purified protein. DNA blot hybridization revealed a single-copy gene, which by chromosomal analysis was found to be located on the long arm of the X chromosome. Unexpectedly, the nucleotide sequence of TBG is closely homologous to those encoding the plasma serine antiproteases alpha 1-antichymotrypsin and alpha 1-antitrypsin. However, there is little overall homology between TBG and transthyretin (prealbumin), the other major thyroxine-binding protein of human plasma.

An anti-sense c-erbA clone inhibits thyroid hormone-induced expression from the alpha-myosin heavy chain promoter.

The effects of thyroid hormone on expression of cardiac myosin heavy chain genes generally are thought to be mediated by nuclear 3,5,3'-triiodo-L-thyronine (T3) receptors that have been identified as the products of the protooncogene, c-erbA. This hypothesis has been tested by transfection of cardiomyocytes in primary culture with a plasmid, pRSVhEACAT-, expressing anti-sense c-erbA mRNA. Because only a low percentage of cells (20%) could be transfected in primary culture an alpha-myosin heavy chain-chloramphenicol acetyltransferase fusion construct was used as a reporter gene. The results indicate that the anti-sense plasmid almost completely blocks T3-induced activity of the reporter gene (less than 1% control) while transfection of a similar amount of the sense construct, pRSVhEACAT+, has no effect. When the c-erbA plasmids were cotransfected with constructs containing T3-independent promoters, no effects on expression were observed. The combined use of an anti-sense construct and a report gene provides a means of studying the role of c-erbA products in intracellular signal transduction even in differentiated, nondividing cells like those of the heart.

Interaction of a protein factor within a thyroid hormone-sensitive region of rat alpha-myosin heavy chain gene.

Using a gel mobility-shift assay, a nuclear protein factor was identified in cardiac myocyte which binds specifically to a DNA fragment from the 5' region of the alpha-myosin heavy chain gene shown previously to contain a thyroid hormone-sensitive element. Methylation interference experiments located the binding site within a 24-base pair sequence from positions -599 to -576. A double-stranded synthetic oligonucleotide containing this 24-base pair sequence bound to the factor and effectively competed with the natural binding site for factor binding. The factor was present in rat and human fibroblasts, and rat GH1 cells as well as L6E9 myoblasts and myotubes. The specificity with which this factor binds to DNA suggests that it could be involved in regulation of the alpha-myosin heavy chain gene.

cis-Acting sequences that mediate induction of beta-myosin heavy chain gene expression during left ventricular hypertrophy due to aortic constriction.

BACKGROUND: Marked alterations in the expression of specific genes occur during the development of cardiac hypertrophy in vivo. Little is known, however, about the cis-acting elements that mediate these changes in response to clinically relevant hypertrophic stimuli, such as hemodynamic overload, in intact adult animals. METHODS AND RESULTS: The left ventricular expression of a directly injected reporter gene driven by 3542 bp of rat beta-myosin heavy chain (beta-MHC) promoter was increased 3.0-fold by aortic constriction (P<.005), an increment similar to the 3.2-fold increase in the level of the endogenous beta-MHC mRNA in the same left ventricles. Subsequent analysis identified a 107-bp beta-MHC promoter sequence (-303/-197) sufficient to convert a heterologous neutral promoter to one that is activated by aortic constriction. These sequences contain two M-CAT elements, which have previously been demonstrated to mediate inducible expression during alpha1-adrenergic-stimulated hypertrophy in cultured neonatal cardiac myocytes, and a GATA element. Although simultaneous mutation of both M-CAT elements markedly decreased the basal transcriptional activity of an injected 333-bp beta-MHC promoter, it had no effect on aortic constriction-stimulated transcription (3.5-fold increase, P<.005 for both wild type and mutant). In contrast, mutation of the GATA motif markedly attenuated aortic constriction-stimulated transcription (1.6-fold, P=NS) without affecting the basal transcriptional activity. This GATA site can interact with in vitro translated GATA-4 and compete with an established GATA site for GATA-4 binding activity in nuclear extracts from aortic constricted hearts. CONCLUSIONS: Basal and aortic constriction-stimulated transcription of the beta-MHC gene is mediated, at least in part, through different mechanisms. A GATA element within beta-MHC sequences -303/-197 plays a role in the transcriptional activation of this gene by aortic constriction.

Expression of the alpha-myosin heavy chain gene in the heart is regulated in part by an E-box-dependent mechanism.

Proximal regulatory element B (PRE-B), located from positions -318 to -284 in the alpha-myosin heavy chain (MHC) promoter, stimulated expression from an otherwise weak alpha-MHC promoter fragment in primary rat neonatal cardiomyocytes but not in the C2C12 myogenic cell line. PRE-B interacted with alpha-MHC binding factor 2 (BF-2), a protein found in nuclear extracts from several neonatal rat tissues and cell types including cardiomyocytes. BF-2 DNA binding activity was greatly reduced in adult versus neonatal tissues. Methylation interference footprints indicated that BF-2 bound to an element that included an E-box consensus sequence. Site-directed mutations in the BF-2-binding site, that abolish BF-2 binding, reduced expression from the full-length alpha-MHC promoter by 70%. A BF-2-like protein interacts within the HF-1a element of the myosin light chain-2 (MLC-2) promoter suggesting that one of the proteins that regulates the alpha-MHC and MLC-2 genes is identical or closely related. Analysis of binding by competition gel shift experiments indicated that both BF-2 and HF-1a are E-box-binding proteins. The alpha-MHC and MLC-2 genes encode contractile proteins which are precursors of myosin. Regulation by the same transcription factor might indicate that the expression of alpha-MHC and MLC-2 is coordinately controlled.

Thyroid hormone regulates expression of a transfected alpha-myosin heavy-chain fusion gene in fetal heart cells.

In ventricular muscle, 3,5,3'-triiodo-L-thyronine (T3) stimulates the expression of the alpha-myosin heavy-chain (alpha-MHC) gene. To test for gene elements required for induction, a fragment of the alpha-MHC gene containing 2.9 kilobases of 5' flanking sequences and 420 base pairs of DNA 3' to the transcription initiation site was linked to the coding sequences of the bacterial chloramphenicol acetyltransferase (CAT) gene. The alpha-MHC fusion gene was introduced into primary cultures of fetal rat heart myocytes. Induction of the transfected gene was monitored by assaying CAT activity while endogenous alpha-MHC mRNA expression was measured by using a synthetic oligonucleotide probe complementary to sequences in the 3' untranslated region of the mRNA. Without T3, CAT activity was only slightly greater than background. When T3 at a final concentration of 10 nM was added to the cultures, CAT activity was increased 8-fold by 48 hr. The response time and doses of T3 required for induction of CAT activity and alpha-MHC mRNA in transfected cells were similar, suggesting that the synthetic and endogenous genes may have a common mechanism of control. When simian virus 40 enhancer and early promoter sequences were included in the construct, CAT activity was constitutively expressed, but it could be increased 7-fold by the addition of T3. Several deletions were introduced into the 5' flanking sequences of the alpha-MHC fragment and the effects on induction of CAT activity were examined. Progressive deletions of 5' sequences from positions -947 to -374 reduced but did not eliminate induction of CAT activity, suggesting that more than one region may be required for optimal induction by thyroid hormone. The results indicate that DNA sequences required for efficient induction by T3 are present in the 5' flanking sequences of the alpha-MHC gene.

Hormonal regulation of myosin heavy chain and alpha-actin gene expression in cultured fetal rat heart myocytes.

Thyroid hormone regulates the expression of ventricular myosin isoenzymes by causing an accumulation of alpha-myosin heavy chain (MHC) mRNA and inhibiting expression of beta-MHC mRNA. However, the mechanism of thyroid hormone action has been difficult to examine in vivo because of its diverse actions. Accordingly, hormonal control of expression of six MHC isoform mRNAs and cardiac and skeletal alpha-actin mRNAs was studied in primary cultures of fetal rat heart myocytes grown in defined medium. The results indicate that in the absence of thyroid hormone, cultured heart cells express predominantly beta-MHC and cardiac alpha-actin mRNAs. Addition of 3,5,3'-triiodo-L-thyronine (T3) caused a rapid induction of alpha-MHC mRNA and decreased beta-MHC mRNA levels without affecting the skeletal muscle MHC mRNAs. There was an almost parallel change in the myosin isoenzymes. Cardiac alpha-actin mRNA levels were transiently increased by T3 treatment, but skeletal alpha-actin was unaffected. Elimination of insulin and epithelial growth factor from the medium did not alter the effects of T3 on cardiac MHC mRNA expression. Addition of various adrenergic agents to the medium had no appreciable effect on cardiac MHC mRNA expression despite the presence of functionally coupled alpha- and beta-adrenergic receptors. Addition of steroid hormones, muscarinic agents, and glucagon to the medium also had no effect. Thus, under defined conditions, T3 is able to regulate MHC gene expression at a pretranslational level without the need for other exogenous factors.