Negative regulation of bovine growth hormone gene by YY1 binding to NRE's

We have previously demonstrated the presence of three negative regulatory elements (NRE1, 2, and 3) in the upstream region of the bovine growth hormone (bGH) gene, whose sequences are similar to the binding elements of transcription factor YY1. The recombinant human YY1 protein indeed bound to these three NRE's in vitro, among which NRE1 is the strongest binding element. Both HeLa and rat pituitary GH3 nuclear extracts contained protein which caused the same retardation as YY1 binding in gel mobility shift assay. The specific band retarded by HeLa and GH3 nuclear extracts was competed out efficiently by a known YY1 binding element. Addition of antibodies against YY1 in the binding reaction produced a distinct supershifted band and/or caused reduction in the YY1-specific band. When the recombinant plasmids containing the chloramphenicol acetyltransferase (CAT) gene under the control of the bGH promoter were introduced together with the expression vector for YY1 into HeLa cells, the expression of the bGH promoter decreased with increasing amount of cotransfecting YY1 expression vector. These results demonstrate that YY1 or its very close homolog negatively regulates bGH expression via binding to NRE's.