SPECIFIC NUCLEAR-PROTEIN BINDING TO A NEGATIVE REGULATORY ELEMENT ON THE HUMAN CYP1A1 GENE

Citation
Pd. Boucher et al., SPECIFIC NUCLEAR-PROTEIN BINDING TO A NEGATIVE REGULATORY ELEMENT ON THE HUMAN CYP1A1 GENE, The Journal of biological chemistry, 268(23), 1993, pp. 17384-17391
Citations number
62
Categorie Soggetti
Biology
ISSN journal
00219258
Volume
268
Issue
23
Year of publication
1993
Pages
17384 - 17391
Database
ISI
SICI code
0021-9258(1993)268:23<17384:SNBTAN>2.0.ZU;2-W
Abstract
Employing reporter gene/CYP1A1 chimeric plasmids, we previously identi fied a 275-base pair (bp) cis-element (-833 to -558; NRE275) that down -regulated the CYP1A1 promoter. In the present study, this negative re gulatory activity was further localized to two subfragments of 105 bp (-833 to -728; NRE105) and 170 bp (-728 to -558; NRE,70), each of whic h exhibited activity with a heterologous promoter/enhancer. Co-transfe ction studies demonstrated a dependence on cellular trans-acting facto rs 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 protei n(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/c ytosine-rich sequences that flank the palindrome, but in a strand-spec ific manner. Guanine residues involved in protein binding were identif ied by methylation interference experiments. Based on transient expres sion assays with each of the three NRE105 components, all three appear to be necessary for complete negative regulatory activity. However, i t is clear the palindrome is the most important sequence with the guan ine/cytosine-rich elements playing an ancillary role.