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Volume 270, Number 12, Issue of March 24, 1995 pp. 6959-6965
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

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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)

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

ABSTRACT

Analysis of a series of human -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 CNBP, was isolated from the cardiac library by hybridization screening. Gel mobility shift analysis indicated that CNBP and CNBP isoforms preferentially interact with single-stranded DNA corresponding to the proximal and distal regions of the suppressor. When cotransfected with a -MHC reporter construct, CNBP repressed activity in a dosage-dependent manner, whereas repression was not observed with the shorter construct (CNBP). Cotransfection of a combination of CNBP and CNBP repressed reporter activity to an extent similar to cotransfection with CNBP alone, suggesting that CNBP is not translationally active under these conditions. The results of RNase protection assays and genomic sequencing indicated that the and 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 -MHC gene by interaction with a repressor region.

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.

()

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.

()

I. L. Flink and E. Morkin, manuscript in preparation.

ACKNOWLEDGEMENTS

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