Specific nuclear protein binding to a negative regulatory element on the human CYP1A1 gene.
Employing reporter gene/CYP1A1 chimeric plasmids, we previously identified a 275-base pair (bp) cis-element (-833 to -558; NRE275) that down-regulated the CYP1A1 promoter. In the present study, this negative regulatory activity was further localized to two subfragments of 105 bp (-833 to -728; NRE105) and 170 bp (-728 to -558; NRE170), each of which exhibited activity with a heterologous promoter/enhancer. Co-transfection studies demonstrated a dependence on cellular trans-acting factors present at limiting concentrations. Electrophoretic mobility shift assays revealed the presence of protein(s) that specifically bound to NRE275, NRE105, and NRE170. Based upon competition studies, the protein(s) that bound to NRE105 appeared to recognize sites similar to those recognized by the NRE170-binding proteins. DNase I footprint analysis of NRE105 demonstrated nuclear protein binding to a 21-bp palindrome (-794 to -774). Protection was also observed along conserved guanine/cytosine-rich sequences that flank the palindrome, but in a strand-specific manner. Guanine residues involved in protein binding were identified by methylation interference experiments. Based on transient expression assays with each of the three NRE105 components, all three appear to be necessary for complete negative regulatory activity. However, it is clear the palindrome is the most important sequence with the guanine/cytosine-rich elements playing an ancillary role.
Boucher
P D
PD
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201.
Ruch
R J
RJ
Hines
R N
RN
D001483:Base Sequence; D001667:Binding, Competitive; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015536:Down-Regulation; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D003577:Cytochrome P-450 Enzyme System