Transcriptional regulation of two mammalian genes: Rat serum albumin and human cyclin D1

We used two mammalian genes as models to study two types of transcriptional regulation in the mammalian system. The first model is the rat serum albumin gene, whose transcription is highly tissue-specific and developmentally regulated. By deletion analysis of the albumin promoter, we found a positive regulatory element located between -207 and -153 by relative to transcription start site. Two putative HNF-3 binding sites located at -168 to -157 by (site X) and -145 to -134 by (site Y) were found. EMSAs using oligonucleotides X and Y demonstrated that both sites are capable of binding HNF-3alpha and HNF-3beta. Transient transfection of constructs containing monomeric and multimeric derivatives of the site X, and an albumin promoter luciferase construct containing the mutated X site into the HepG2 cells, and cotransfection of a luciferase construct containing site X along with a HNF-3 expression construct, further demonstrated the positive role of HNF-3 X site in activation of the albumin promoter. The second model gene is the human cyclin D1 gene. Overexpression of cyclin D1 is an important event leading to abnormal cell proliferation in early malignant breast lesions as well as in invasive breast adenocarcinoma, it is of interest to identify cis-acting element(s) within the cyclin D1 promoter that are required for promoter activity and transcription factors that bind to these elements in breast cancer cells. In addition, we used cyclopentenone to elucidate the molecular mechanism of the inhibitory effect of cyclopentenone prostaglandins on cyclin D1 expression. Cyclopentenone arrested MCF7 breast cancer cells in the G0/G1 phase and repressed cyclin D1 mRNA levels. Transient transfections showed that cyclopentenone inhibited the activity of the cyclin D1 and cyclin A promoters but not others. Furthermore, the cyclopentenone response element is located within cyclin D1 core promoter (-49 to +65 by relative to transcription start site). We also identified a positive acting element within +122 to +136 by (cycY) of the cyclin D1 promoter. Transfection experiments performed with cyclin D1 promoter 3'-deletion constructs, a construct in which cycY has been altered by site-directed mutagenesis, and luciferase constructs containing a monomeric or multimeric of cycY oligo demonstrated the functional role of cycY. EMSAs showed a protein present in MCF-7 nuclei that specifically bound to the cycY site but not to an Sp1 consensus sequence.