©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Alternatively Processed Isoforms of Cellular Nucleic Acid-binding Protein Interact with a Suppressor Region of the Human -Myosin Heavy Chain Gene (*)

(Received for publication, June 7, 1994; and in revised form, January 12, 1995)

Irwin L. Flink Eugene Morkin (§)

From the University Heart Center, and the Departments of Internal Medicine, Physiology and Pharmacology, University of Arizona, Tucson, Arizona 85724

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Analysis of a series of human beta-myosin heavy chain (MHC) constructs with progressive deletions in the 5`-flanking region has localized a strong positive element at positions -298/-277 with a repressor region located immediately upstream at -332/-300 (Flink, I. L., Edwards, J. G., Bahl, J. J., Liew, C.-C., Sole, M., and Morkin, E.(1992) J. Biol. Chem. 267, 9917-9924). A 49-base pair restriction fragment containing the suppressor element was used to screen a cardiac expression library. The 0.65-kilobase pair cDNA identified by this procedure was similar in sequence, except for the absence of a 21-base pair region encoding seven amino acids, to cellular nucleic acid-binding protein (CNBP), a 19-kDa zinc finger DNA-binding protein isolated earlier from liver, which may be involved in negative regulation of cholesterol biosynthesis (Rajavashisth, T. B., Taylor, A. K., Andalibi, A., Svenson, K. L., and Lusis, A. J.(1989) Science 245, 640-643). An additional clone identical to the one originally found in liver, referred to as CNBPalpha, was isolated from the cardiac library by hybridization screening. Gel mobility shift analysis indicated that CNBPalpha and CNBPbeta isoforms preferentially interact with single-stranded DNA corresponding to the proximal and distal regions of the suppressor. When cotransfected with a beta-MHC reporter construct, CNBPalpha repressed activity in a dosage-dependent manner, whereas repression was not observed with the shorter construct (CNBPbeta). Cotransfection of a combination of CNBPalpha and CNBPbeta repressed reporter activity to an extent similar to cotransfection with CNBPalpha alone, suggesting that CNBPbeta is not translationally active under these conditions. The results of RNase protection assays and genomic sequencing indicated that the alpha and beta isoforms are formed by alternative use of 5` donor sites within a single exon. These results suggest that CNBP isoforms may modulate the activity of the beta-MHC gene by interaction with a repressor region.


INTRODUCTION

The beta-myosin heavy chain (MHC) (^1)gene is the major myosin isoenzyme expressed in human slow skeletal muscle and ventricular myocardium (Liew et al., 1990; Bouvagnet et al., 1984; Gorza et al., 1984). The beta-MHC gene is expressed in a developmental and muscle-specific manner (Periasamy et al., 1984; Emerson et al., 1987; Mahdavi et al., 1986; Yu et al., 1989; Lyons et al., 1990), and in rats and rabbits has been shown to be transcriptionally down-regulated by thyroid hormone (Lompre et al., 1984; Everett et al., 1984) (see Morkin(1993) for review). In the human beta-MHC gene, a strong positive cis-regulatory element has been localized in the 5`-flanking sequences that is required for high level expression in cultured heart cells (Flink et al., 1992). A repressor with partial positional dependence is located immediately upstream (Edwards et al., 1992).

Presently, little is known about the protein(s) that trans-activate the repressor element of the beta-MHC gene or that inhibit transcription of other contractile protein genes in the heart. In skeletal muscle, Id, a helix-loop-helix protein that lacks the basic domain necessary for DNA binding, can associate specifically with MyoD-1 and related proteins, resulting in attenuation of their ability to bind DNA and trans-activate muscle-specific genes (Benezra et al., 1990; Evans et al., 1991). Despite considerable effort, no comparable system of positive and negative regulatory proteins has been found in cardiac muscle (Thompson et al., 1991).

In earlier studies of the human beta-MHC promoter, a region was identified by DNase I footprinting between positions -278/-296 that coincided with sequences between positions -274/-300 that were required for high level expression in primary fetal rat heart cell cultures (Flink et al., 1992). An additional region was protected in DNase I footprints using nuclear extracts from rat heart (-301/-313) and liver (-303/-315), which corresponded to a suppressor domain. Analysis of a larger series of deletion constructs and site-specific mutations suggested that there were proximal (-301/-314) and distal (-315/-332) negative elements within this region (Edwards et al., 1992). Additionally, the suppressor has been shown to down-regulate both the thymidine kinase and SV40 promoters when positioned upstream from these basal promoters. The beta-MHC suppressor region was used in the present study to identify a negative transcriptional factor. The clone initially characterized was similar in sequence to the message corresponding to cellular nucleic acid-binding protein (CNBP) (Rajavashisth et al., 1989), except for the absence of 21 nucleotides encoding seven amino acids near the 5` end of the cDNA. Subsequently, a longer cDNA was obtained from a heart library with a sequence identical to the version originally found in liver (CNBPalpha).

The CNBP gene encodes a 19-kDa protein containing seven tandem zinc finger repeats of 14 amino acids. Each finger region contains the same arrangement of Cys-X(2)-Cys-X(4)-His-X(4)-Cys residues and has extensive sequence similarity to the finger domains of retroviral nucleic acid-binding proteins (Covey, 1986). The results of RNase protection assays and genomic sequence analyses indicate that the CNBPalpha and CNBPbeta isoforms result from variations in mRNA splicing in which alternative 5` donor sites are utilized within a single exon. A single copy of the human CNBP gene has been localized to chromosome 3q13.3-q24 (Lusis et al., 1990).

In transient assays using primary heart cell cultures, CNBPalpha inhibited expression of a beta-MHC reporter plasmid containing the negative domain in a dosage-dependent manner, whereas the shorter isoform (CNBPbeta) did not repress transcription. These results suggest that isoforms of CNBP in cardiac muscle may differentially regulate the transcriptional activity of the beta-MHC gene by competing for a suppressor element. The alpha and beta isoforms of CNBP also are present in liver and other non-muscle tissues where they may play a role in modulating the transcriptional activity of non-contractile protein genes.


EXPERIMENTAL PROCEDURES

Materials

T(4) polynucleotide kinase, T(4) ligase, and restriction enzymes were obtained from New England Biolabs (Beverly, MA). Sequenase was purchased from U. S. Biochemical Corp. Taq polymerase was obtained from Promega (Madison, WI), and the protease inhibitor AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride) from ICN Biochemicals Inc. (Aurora, OH).The glutathione S-transferase gene fusion system was obtained from Pharmacia Biotech Inc., and the ribonuclease protection kit (RPA II) was supplied by Ambion (Austin, TX). A human fetal heart cDNA library was obtained from Clontech (Palo Alto, CA) and genomic Fix and Bluestar-1 libraries were purchased from Stratagene (La Jolla, CA) and Novagen (Madison, WI), respectively.

Cloning and Sequencing of CNBP

A 49-bp restriction fragment (-294/-342) containing the suppressor region of the human beta-MHC promoter was generated by digestion of pbeta-MHC-468 (Flink et al., 1992) with BstXI/PvuII. The fragment was concatenated, labeled by nick-translation, and used as a probe to screen about 1 times 10^6 recombinants from a commercial human fetal heart cDNA library as described by Singh et al.(1988). One positive clone was identified and used to screen the same library by the DNA hybridization method. An additional full-length clone was obtained by this procedure. Both strands of each cDNA clone were sequenced by the dideoxy chain termination method (Sanger et al., 1977).

The gene encoding CNBP was isolated by screening a human Fix genomic library using a 472-bp EcoRI restriction fragment located at the 5` end of the CNBPalpha cDNA. A single clone containing the 5` end of the CNBP gene was isolated. To obtain the 3` end of the CNBP gene, a BlueSTAR-1 library was screened with a 1028-bp EcoRI restriction fragment located at the 3`-end of the cDNA. One positive recombinant was obtained. The DNA insert was converted to a plasmid subclone by site-specific recombination following in vivo autoexcision utilizing loxP sites and bacterial host P1 Cre recombinase. Both strands of the purified phage DNA were directly sequenced using the Fmol DNA sequencing system (Promega) and the Applied Biosystems model 373A DNA sequencing system. Sequence alignments and analyes were carried out using the software package PC/Gene (IntelliGenetics, Mountain View, CA).

Cell Preparation and Culture

Heart cells were prepared from 18-19-day gestational age fetal rat hearts. About 1.0 times 10^8 cells/plate were seeded and transfected with 10-15 µg of reporter plasmid by electroporation using methods described earlier (Edwards et al., 1992). Cells were harvested after 72 h and assayed for CAT activity by the method of Gorman et al.(1982). The amount of chloramphenicol acetylated per plate was about 2.8-5.6 pmol/h. The growth hormone expression plasmid XGH5 was used to measure transfection efficiency. Growth hormone was assayed by the I-double antibody method (Nichols Institute, San Juan Capistrano, CA).

Plasmid Construction

The human beta-MHC/CAT fusion construct, pHbeta-MHC-332, containing the suppressor region, was used as a reporter plasmid in transfection assays. The construction of pbeta-MHC-332, pbetaX322, and pbetaX305 has been described in detail earlier (Edwards et al., 1992, 1994). The coding regions of the alpha and beta CNBP isoforms were ligated into the expression vector, pGS5 (Promega).

RNase Protection Assays

Total RNA was extracted from human papillary muscles excised during surgery by either the method of Chomczynski and Sacchi(1987) or with the reagents and instructions provided by Ambion. The 322-bp EcoRI-SmaI fragment (positions 151-472) of CNBPalpha was subcloned directionally into the Bluescript plasmid and linearized by digestion with BamHI. An antisense probe was synthesized from the T3 promoter using T3 RNA polymerase, annealed to RNA, incubated overnight at 45 °C, and digested with RNase. The protected fragments were precipitated with ethanol and separated by electrophoresis on a 6% polyacrylamide-urea gel. The dried gel was exposed for 16 h to Kodak XR-5 film at -70 °C.

Expression of CNBP in E. coli

The glutathione S-transferase gene fusion system was used for the expression of CNBPalpha and CNBPbeta in E. coli. Expressed proteins were purified and detected on 10% polyacrylamide-SDS gels. The coding regions of CNBPalpha (531 bp) and CNBPbeta (510 bp) were amplified by the polymerase chain reaction, using primers containing BamHI restriction sites at their 5`-ends, purified, and cloned in-frame into the prokaryotic expression vector, pGex-1T (Pharmacia Biotech Inc.). Protein was induced at mid-log phase (optical density 0.5, 550 µm) with 1.0 mM isopropyl-1-thio-beta-D-galactopyranoside in the bacterial strain JM109 for 30 min. Cells were harvested by centrifugation, washed in ice-cold phosphate-buffered saline buffer, and resuspended in 1/40 volume of phosphate-buffered saline at 5 °C in the presence of protease inhibitors containing 1 mM AEBSF, 1 µg/ml leupeptin, pepstatin, and aprotinin, and 1 mM dithiothreitol). Extracts were prepared by sonication, and recombinant protein was purified from bacterial lysates by affinity chromatography at 5 °C using glutathione-Sepharose 4B according to instructions provided by the manufacturer and stored in aliquots at -70 °C.

Mobility Shift Analyses

Electrophoretic mobility shift assays (EMSAs) were carried out using rat liver and heart nuclear extracts as described earlier (Flink et al., 1992). About 0.5-1.0 µg of affinity purified CNBP protein was used in the EMSA.


RESULTS

Cloning of Alternatively Spliced Transcripts of Human CNBP

To identify translationally active proteins that bind to the negative region of the human beta-MHC promoter, a human fetal heart cDNA expression library was screened with a radiolabeled 49-bp restriction fragment (-342/-294) containing the proximal and distal negative elements of the repressor. A single positive clone was isolated and both strands were sequenced. As shown in Fig. 1, the clone contained a 0.65-kilobase pair insert that was almost identical in nucleic acid sequence to cellular nucleic acid-binding protein (CNBP), a 19-kDa zinc finger protein (Fig. 1, top) identified earlier in liver (CNBPalpha) (Rajavashisth et al., 1989). Compared with CNBPalpha, however, the NH(2)-terminal sequence of the version found in heart (CNBPbeta) was slightly shorter because of the absence of a 21-bp region near the 5` end of the cDNA between nucleotide positions 205-227 (Fig. 1, bottom). This results in the loss of seven amino acids in the linker region between the first and second zinc finger domains (amino acid positions 36-42).


Figure 1: Comparison of the cDNAs encoding CNBP isolated from liver (top) and heart (bottom) tissues. The position of the polyadenylation signal (AATAAA), translation initiation triplet (ATG), and termination codon (TAA) of ``liver'' CNBP (CNBPalpha) is indicated. The coding and untranslated regions are indicated by the open and closedboxes, respectively. Nucleotides 206-226 are absent in CNBPbeta resulting in the loss of seven amino acids (residues 36-42) in the linker region between the first and second zinc fingers. The boldlines at the top show the protection of the alpha and beta products observed in the RNase protection assay shown in Fig. 3. The shorter, 55-bp protected fragment, corresponding to CNBPbeta, could not be visualized on the gel.




Figure 3: RNase protection assay of human heart and liver RNA. Total RNA was prepared from human heart papillary muscle and liver, and about 4-5 µg were hybridized to antisense probe encoding CNBPalpha. The purified, full-length probe of 389 bp is shown in lane1. Lanes2 and 3 demonstrate the two protected bands of 322 bp (CNBPalpha) and 246 bp (CNBPbeta) with human papillary muscle and liver RNA, respectively. Approximately equal amounts of CNBPalpha and CNBPbeta mRNA are present in liver and heart tissues. No protected bands were observed in the absence of RNA or with yeast RNA (results not shown).



Using a 451-bp EcoRI restriction fragment of the CNBPbeta isolate as a probe, a second clone was obtained from the cardiac cDNA library by DNA hybridization. This clone contained an insert that was identical to CNBPalpha (Fig. 1, top). Northern blot analysis of RNA from heart, using the same CNBPbeta EcoRI restriction fragment as a probe, revealed a single band of 1.5 kilobase pairs, which was identical to the size of the CNBP mRNA found in liver (results not shown).

CNBPalpha and CNBPbeta mRNAs Arise by Alternative Processing

To determine the intron-exon boundaries of the CNBP gene, two genomic clones were isolated using EcoRI restriction fragments of 472 and 1028 bp, respectively, which spanned the 5` and 3` portions of the cDNA. The intron-exon structure of the human CNBP gene is shown schematically in Fig. 2(upper panel). Five exons have been identified, four of which account for the entire coding sequence. (^2)There is a consensus internal 5` donor splice site at the 3` end of exon 2 (Fig. 2, lower panel). The removal of the 3` portion of exon 2 and the joining of the remainder to the 5` end of exon 3 would account for the exclusion of 21 nucleotides encoding seven amino acids of the CNBPalpha isoform. CNBPbeta seems to arise by use of this internal 5` donor site. A sequence representing a shortened exon 2 is not found upstream from exon 2 within the CNBP gene (results not shown).


Figure 2: Schematic diagram showing the genomic structure, alternative splicing, and sequences at exon-intron boundaries of the human CNBP gene. Boxes indicate exons, and the thickline indicates intervening sequences. The beta isoform is generated by the use of an internal 5` donor site in exon 2, which results in the fusion, in-frame, of the shortened 5` portion exon 2 to exon 3. The sequence of the intron/exon junctions between exons 2 and 3 is indicated at the bottom.



The presence of two alternatively spliced CNBP mRNA species in human heart was verified by RNase protection analyses. For this purpose, an antisense riboprobe was constructed spanning the coding sequence of CNBPalpha from position 151 in exon 2 to position 472 in exon 4 (Fig. 1). This probe contains an additional 67 bp of Bluescript vector sequence, so that the full-length probe was 389 bp in length. Fig. 3demonstrates the presence of two major protected fragments in heart and liver of 322 and 246 bp, respectively, which corresponds to the predicted sizes of the alpha and beta CNBP splice variants. The shorter, 55-bp protected fragment from CNBPbeta could not be visualized on the gel. No protected bands were observed in the absence of RNA or in the presence of unrelated yeast RNA (results not shown). Interestingly, the signal intensities of the 322- and the 246-nucleotide fragments were about the same, indicating that similar amounts of mRNAs encoding the alpha and beta isoforms of CNBP are present in heart and liver.

Interaction of Bacterially Expressed CNBP Isoforms with the beta-MHC Suppressor

The cDNA encoding CNBPbeta was initially identified by Southwestern DNA-protein screening using the beta-MHC repressor sequence as a radiolabeled probe. To further evaluate the interaction of protein with the distal suppressor region, unfractionated nuclear proteins from liver and ventricular myocardium were used in EMSAs with single- and double-stranded DNAs as labeled probes and both single- and double-stranded DNAs as competitors. Single-stranded DNAs were used in these experiments because it has been demonstrated previously that CNBP interacts preferentially with the sense strand of the hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase promoter sterol regulatory element (SRE) (Rajavashisth et al., 1989). Fig. 4(lanes1 and 7) demonstrates binding of protein from liver and heart nuclear extracts in the absence of competitor DNA. This interaction was effectively competed with a 50-100-fold molar excess of unlabeled sense and antisense DNAs, corresponding to the distal repressor element (Fig. 4, lanes2, 3, and 8). Competition was also observed with the single-stranded HMG-CoA SRE (lane4), which contains a related G-rich motif. By contrast, there was no loss of binding activity when similar amounts of double-stranded distal suppressor element (lane5) or unrelated adenovirus DNA were used as competitors (lanes6 and 9). These results demonstrate that the interaction of liver and heart nuclear protein(s) is specific for single-stranded DNA corresponding to the distal suppressor region. Additionally, two bands were present (indicated by the arrow) in the heart (lane7) nuclear extract and in the liver extract upon shorter exposure, suggesting that more than one protein may interact with the suppressor region; alternatively, CNBP may dimerize with itself or an accessory protein.


Figure 4: Electrophoretic mobility-shift analysis of P-labeled distal suppressor sense strand (-331/-311) with heart and liver nuclear extracts. Nuclear extracts from euthyroid (Eu) normal rabbit heart and liver were incubated with the distal suppressor sense strand, in the absence of competitor (lanes1 and 7), a 50-100 fold molar excess of distal suppressor sense strand (lane2), distal suppressor antisense strand (lanes3 and 8), HMG-CoA sense strand (lane4), distal suppressor double-strand (lane5), and nonspecific adenovirus LTR double-strand (lanes6 and 9) ``cold'' competitors. Competition was observed with the distal suppressor sense and antisense strands and the HMG-CoA sense strands. Arrow indicates the position of the specifically bound major band. Light exposure of the autoradiogram demonstrates two bands in the position of the arrow in liver and heart tissues.



It was of interest to study in more detail the interaction of expressed CNBP protein with the distal and proximal suppressor regions by EMSA. Purified CNBPalpha and CNBPbeta proteins were found to bind preferentially to single-stranded oligonucleotides corresponding to the sense strand of the distal element (Fig. 5, left panel, lanes1 and 11), and sense (Fig. 6, lanes1 and 9) and antisense strands (Fig. 6, lanes5 and 13) of the proximal element. Binding to each of these cis-elements was competed by the HMG-CoA reductase SRE (Fig. 5, left panel, lanes3 and 13), but not by the adenovirus promoter element (Fig. 5, left panel, lanes4 and 14 and Fig. 6, lanes4, 8, 12, and 15). CNBPbeta may bind to the repressor with slightly higher affinity than CNBPalpha, since an equal amount of CNBPbeta protein produced more intense bands by EMSA (Fig. 5, left panel and Fig. 6). Furthermore, mutations in the SRE-related region of the distal suppressor abolished binding (Fig. 5, right panel). Thus these results indicate that the CNBPalpha and CNBPbeta isoforms interact preferentially with single-stranded DNA corresponding to the proximal and distal negative elements of the beta-MHC repressor, and they support earlier conclusions, based upon functional assays, that these elements act synergistically to repress transcriptional activity (Edwards et al., 1992).


Figure 5: Electrophoretic mobility-shift analysis of P-labeled distal suppressor sense, antisense, and double-stranded DNA (-331/-311) with bacterially expressed CNBPalpha and CNBPbeta proteins. EMSAs were carried out as described under ``Experimental Procedures'' and in Fig. 4. Left, CNBPalpha and CNBPbeta protein interacted only with single-stranded sense DNA (lanes1 and 11). Competition was observed with the distal suppressor sense single-stranded DNA (lanes 2 and 12) and the HMG-CoA sense strand (lanes3 and 13). No binding was observed with the antisense (lane5) or distal suppressor double-stranded DNA (lane8). No competition was observed with the adenovirus LTR (lanes4 and 14). Arrow indicates the position of the specifically bound major band. Right, EMSA of mutated distal suppressor. The wild-type sequence (5`-GGTGGTCGTGG-3`) of the distal suppressor, which is homologous to the SRE found in the HMG-CoA reductase gene, was mutated to 5`-TTATTTATATT-3` (underline represents mutated nucleotides). No binding was observed with expressed CNBPalpha or CNBPbeta protein.




Figure 6: Electrophoretic mobility-shift analysis of P-labeled proximal suppressor sense- and antisense strands (-314/-301) with bacterially expressed CNBPalpha and CNBPbeta proteins. EMSA assays were carried out as described under ``Experimental Procedures'' and in Fig. 4. Binding was observed with sense (lanes1 and 9) and antisense (lanes5 and 13) DNA. Competition was observed with the proximal suppressor sense (lanes2, 6, 10, and 14) strand. No competition was observed with double-stranded proximal suppressor (lanes3, 7, and 11) or the adenovirus LTR (lanes4, 8, 12, and 15). Arrow indicates the position of the specifically bound major band.



Transcriptional Regulation of the Human beta-MHC Gene Is Controlled by Distinct Functional Properties of CNBPalpha and CNBPbeta

To assess the function of CNBP on transcriptional regulation of the beta-MHC gene promoter, cotransfections were carried out with a reporter plasmid containing the proximal and distal negative elements (pbeta-MHC-332) and vectors expressing either CNBPalpha or CNBPbeta at dosages ranging from 0.1 to 10 µg/plate. In the presence of CNBPalpha, dosage-dependent repression was observed with a decrease in activity of about 70% at the highest dosage (Fig. 7, top). By contrast, the activity of the reporter pbeta-MHC-332 was unaffected upon cotransfection with CNBPbeta. Cotransfection of pSG5, the vector used to express CNBP, also did not affect activity.


Figure 7: Top, comparison of the CAT activity of pbeta-MHC-332 cotransfected with plasmids expressing alpha and beta isoforms of CNBP in primary cardiomyocyte cultures. Dosage-dependent repression of pbeta-MHC-332 CAT activity occurred in the presence of increasing amounts of a vector (pSG5) expressing CNBPalpha (). Cotransfection with CNBPbeta in the same vector resulted in no change in CAT activity (bullet) compared to transfection of pSG5 vector alone (box). Results are expressed as percent activity (means ± S.E.) of pbeta-MHC-332 in the absence of CNBP for triplicate determinations in three to six experiments. Bottom, comparison of the CAT activity of wild-type pbeta-MHC-332 with constructs containing mutations within the distal (pbetaX322) and proximal (pbetaX305) suppressor regions, respectively. Cotransfection of CNBPbeta in combination with CNBPalpha and pbeta-MHC-332. Fetal heart cells were transfected with 10 µg of reporter plasmids (pbeta-MHC-332, pbetaX322, pbetaX305) and 5 µg of CNBP constructs as indicated. LaneA, pbeta-MHC-332; laneB, pbetaX322; laneC, pbetaX322 and CNBPalpha; laneD, pbetaX305; laneE, pbetaX305 and CNBPalpha; laneF, pbeta-MHC-332 and CNBPalpha; laneG, pbeta-MHC-332 and CNBPalpha and CNBPbeta. The activity of pbetaX322 was increased 310% compared with the activity of pbeta-MHC-332, whereas no change was observed with pbetaX305. Cotransfection of CNBPalpha with pbetaX322 and pbetaX305 decreased their activity by approx41% and approx27% repression, respectively. Cotransfection of equal amounts of CNBPalpha with CNBPbeta and pbeta-MHC-332 resulted in approx26% decrease in activity. This value is approximately the same as the activity observed when CNBPalpha alone was transfected. Results are expressed as percent activity (means ± S.E.) of pbeta-MHC-332 in the absence of CNBP for triplicate determinations in two to three experiments.



To study the activity of the distal and proximal repressor elements independently, site-specific mutations were designed within these two regions. In agreement with earlier results (Edwards et al., 1992), the activity of pbetaX322 (Fig. 7, bottompanel, laneB), which contains a mutation in the distal element, was approx3-fold greater than pbeta-MHC-332 (Fig. 7, bottom panel, laneA), indicating disruption of protein-DNA interactions within the distal element. By contrast, the construct pbetaX305, which contains a mutation in the proximal element, did not show a significant increase in activity (Fig. 7, bottom panel, laneD) compared to pbeta-MHC-332. These results suggest that the distal element may be a stronger repressor than the proximal element. When CNBPalpha was cotransfected with pbetaX322, approx41% repression was observed (Fig. 7, bottom panel, lane C), possibly because of interaction with the wild-type proximal suppressor element. Cotransfection of pbetaX305 with CNBPalpha caused a slight reduction in activity (approx27%) (Fig. 7, bottom panel, laneE) compared to the activity of pbetaX305 alone. Cotransfection of CNBPbeta in combination with CNBPalpha, resulted in a level of reporter activity simlar to cotransfection with CNBPalpha alone (lanesF and G). Taken together, these results suggest that both elements may be required for full silencing activity.


DISCUSSION

In this report, CNBP has been identified as a trans-acting factor that negatively regulates the beta-MHC gene. Two isoforms of CNBP, termed alpha and beta, are shown to be produced by alternative processing and to interact preferentially with single-stranded DNA corresponding to a repressor element (-332/-301) found within the proximal 5`-flanking region of the human beta-MHC gene. RNase protection experiments indicate that approximately equal amounts of the alpha and beta isoforms are present in human ventricular myocardium and liver. Interestingly, the two isoforms appear to have different effects on beta-MHC gene transcription. When increasing amounts of CNBPalpha were cotransfected into cultured cardiomyocytes with a beta-MHC/CAT reporter plasmid, activity of the beta-MHC gene was decreased in a dosage-dependent manner, whereas cotransfection of CNBPbeta did not cause repression. Additionally, mutations centered within the distal element (pbetaX322) increased reporter activity (Fig. 7, bottom panel, laneB), whereas nucleotide changes located within the proximal region (pbetaX305) did not appear to affect beta-MHC gene transcription (Fig. 7, bottom panel, laneD). These results suggest that both elements may be required for full silencing activity. Cotransfection of a combination of CNBPalpha and CNBPbeta repressed reporter activity to a level similar to CNBPalpha alone, indicating that CNBPbeta is not translationally active under these conditions.

Alternative processing has been shown to generate multiple mRNAs from single pre-mRNA transcripts and is an important mechanism in the regulation of gene expression (Green, 1992; McKeown, 1992). The CNBP isoforms provide an interesting example of a transcriptional regulator with alternatively spliced products that have different trans-activating functions. The splicing process for formation of CNBP isoforms involves selective use of alternative internal 5` donor sites within exon 2. It has been reported earlier that splice site selection can be influenced by both exon sequences and splice site proximity (Eperon et al., 1986; Mardon et al., 1987). In the CNBP gene, the donor site located at the intron/exon junction of exon 2 and the internal donor site within exon 2 contain an identical consensus recognition sequence (Fig. 2); hence, it is not surprising that these two sites are recognized with similar frequency by splicing trans-factors. Another example of an alternative donor site located within an exon is found within the mouse heat shock protein 47 (HSP47) gene (Takechi et al., 1994). In this case, the internal site is selected preferentially at elevated temperatures to produce a spliced mRNA product in which the adjacent downstream exon is skipped.

The three-dimensional solution structure (Lee et al., 1989) and crystal structure (Pavletich et al., 1991) of the zinc finger nucleic acid binding motif, Cys-XCysX-His-X(3)His, which is related to the motif found in CNBP, has been determined. This motif forms a compact globular domain containing a central zinc ion tetrahedrally coordinated by Cys/His residues that lie within an antiparallel beta ribbon and an alpha helix. The zinc fingers bind in the major groove of B-DNA with residues from the NH(2)-terminal portion of the alpha helix making primary contacts to the guanine-rich strand of the DNA. Additionally, a computer simulation of the three-dimensional structure of the finger domains of CNBP, and their interaction with the sterol regulatory element, has been described (Kothekar, 1990). According to this model, each of the seven fingers, including a portion of the linker region between fingers one and two, contact in succession, a single nucleotide of the 8-bp SRE. We have confirmed the interaction of CNBPalpha with the SRE, and also have shown that the alpha and beta CNBP isoforms bind preferentially with different affinities to single-stranded sequences of the proximal and distal negative elements within the repressor region of the beta-MHC promoter ( Fig. 5and Fig. 6). CNBPbeta lacks seven amino acids at the 3` end of the linker region involved in DNA contact, which may explain its inability to repress beta-MHC gene expression. Thus repression of transcription by CNBP may involve a functional domain located between the first and second zinc fingers that includes amino acid residues 36-42.

The beta-MHC suppressor, located at positions -300/-332, is comprised of adjacent proximal and distal negative cis-acting elements, each of which spans about 10-15 nucleotides. Both negative elements seem to be required for full suppressor activity (Edwards et al., 1992). CNBP protein-DNA interactions are somewhat unusual in that both CNBPalpha and CNBPbeta isoforms interact preferentially with single-stranded DNA and that the sequences of the proximal (5`-GTCAGTTCCCTCTC-3`) and distal (5`-GTGGTCGTG-3`) negative elements are not closely related. This may indicate that local DNA conformation or unusual secondary structural features in this region play a role in CNBP binding. The distal negative element is somewhat homologous to the consensus sequence (5`-GTG(C/G)GGTG-3`) of the sterol regulatory cis-element (Osborne et al., 1992). The latter element is found in promoter regions of several genes involved in cholesterol uptake and synthesis, including the low density lipoprotein receptor, HMG-CoA synthase, and HMG-CoA reductase genes (Dawson et al., 1988). Although the precise role of CNBP in regulating the level of cholesterol is unknown, reduction of plasma cholesterol is known to be associated with increased levels of CNBP mRNA (Rajavashisth et al., 1989). The interaction of CNBP with SRE is thought to result in down-regulation of the HMG-CoA reductase gene, thereby maintaining cholesterol homeostasis. It remains to be established whether changes in amounts of CNBP isoforms occur in association with variations in expression of beta-MHC mRNA.

The ability of a transcriptional factor to interact with two unrelated sequences is not unique to CNBP. Binding to two unrelated sequences has also been observed with transcriptional enhancer factor I (TEF-1) (Thompson et al., 1991; Flink et al., 1992), a putative regulator of the beta-MHC and cardiac troponin-T genes (Mar et al., 1990; Farrance et al., 1992). TEF-1 binds to the unrelated GT-IIC and SphI-II enhansons of the SV40 early promoter (Xiao et al., 1991). The sequence-specific single-stranded DNA-binding protein, muscle factor 3 (Santoro et al., 1991), also interacts with multiple elements, including the CArG motif, E box, and MCAT domains found in the promoters of skeletal muscle actin, muscle creatine kinase, and cardiac troponin-T genes, respectively. It is perhaps noteworthy that each of these elements is known to interact with other transcriptional regulators in addition to muscle factor 3.

In addition to acting as a repressor, CNBP may play other roles in regulating gene expression. For example, certain single-stranded DNA-binding proteins have been shown to be involved in stabilizing DNA at replication forks (Chase et al., 1986). CNBP may play a comparable role in maintaining the most favorable DNA conformation for modulating the transcriptional activity of RNA polymerase. CNBP interaction clearly requires DNA sequence specificity, however, because mutations within the beta-MHC suppressor sequences completely abolish protein-DNA interactions and eliminate suppression of reporter constructs (Edwards et al., 1992). A protein found in Schizosaccharomyces pombe, Byr3, (Xu et al., 1992) has seven zinc finger domains containing the consensus CX(2)CX(4)HX(4)C motif. This protein is able to suppress sporulation defects of ras-1 null diploids and also is required for efficient conjugation. Interestingly, CNBP is able to substitute for Byr3 in these functions. Another related protein, Xenopus posterior, is involved in anterior-posterior axis formation at mid-gastula stage in the developing embryo (Sato and Sargent, 1991). Further studies will be required to fully elucidate the role of CNBP in the regulation of gene expression and cell function within the heart and other tissues.


FOOTNOTES

*
This investigation was supported by Grant PO1 HL20984 from the National Institutes of Health, a grant from the Gustavus and Louise Pfeiffer Research Foundation, Grants G-2-03-90 and G-2-02-92 from the American Heart Association, Arizona Affiliate, and Grant 9411 from the Arizona Disease Control Research Commission. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: 1501 N. Campbell, Tucson, AZ 85724. Tel.: 602-626-4144; Fax: 602-626-2666.

(^1)
The abbreviations used are: MHC, myosin heavy chain; bp, base pair(s); CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; CNBP, cellular nucleic acid-binding protein; SRE, sterol regulatory element; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; HMG, hydroxymethylglutaryl.

(^2)
I. L. Flink and E. Morkin, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank M. Kimura and X. Zhou for excellent technical assistance and Dr. J. Bahl and Y. Wu for cultured heart cells.


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