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Identification and Characterization of Single Strand DNA-Binding Protein That Represses Growth Hormone Receptor Gene Expression

Department of Pediatrics University of Pittsburgh School of Medicine Children’s Hospital of Pittsburgh Pittsburgh, Pennsylvania 15213

The GH receptor is essential for the actions of GH on growth and metabolism. Electromobility shift assay established that a 42-bp enhancer element in the promoter of the L1 transcript of the murine GH receptor bound nuclear proteins specific for the coding strand or the DNA duplex. Using methylation interference footprinting and electromobility shift assay with mutant oligonucleotides, the DNA-binding sites for the single-strand DNA-binding protein (SSBP) and the double-strand DNA-binding protein (DSBP) were mapped and shown to be contiguous with partial overlap. Shift-Western analysis indicated that the SSBP was a component of the DSBP complex. A functional interaction between SSBP and DSBP was suggested by the effect of the exclusion of SSBP on equilibrium binding and dissociation rate ("off rate") of the DSBP-DNA complex. Experiments using the anionic detergent deoxycholate provided evidence for a direct protein-protein interaction between SSBP and DSBP. Using lectin-affinity chromatography, discordance between the pattern of O-glycosylation of SSBP and DSBP was demonstrated. Transient transfection experiments support the role of SSBP as a repressor of DSBP’s activation of transcription of the GH receptor gene. Southwestern analysis indicated that a protein of molecular mass 23-kDa exhibited binding activity specific to the coding strand of the enhancer element. We conclude that single- and double-strand DNA-binding proteins conjointly regulate the expression of the murine GH receptor gene.

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