Alternatively Processed Isoforms of Cellular Nucleic Acid-binding Protein Interact with a Suppressor Region of the Human β-Myosin Heavy Chain Gene

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