IN-VITRO BINDING AND FUNCTIONAL-STUDIES COMPARING THE HUMAN CYP1A1 NEGATIVE REGULATORY ELEMENT WITH THE ORTHOLOGOUS SEQUENCES FROM RODENT GENES

In previous studies, we identified a 21 bp palindrome (-794 to -774) l ocated within the negative regulatory element of the human CYP1A1 gene consisting of an 8 bp inverted repeat and 5 bp spacer. This element s pecifically binds protein(s) present in HepG2 nuclear extract preparat ions and is capable of down-regulating heterologous promoters and enha ncers in transient expression assays. Conserved guanine/cytosine-rich regions which flank the palindrome also were implicated in this activi ty. In the present study, we examined similar regions from the rat (-8 81 to -746) and mouse (-822 to -683) CYP1A1 genes for their ability to bind nuclear protein and down-regulate heterologous promoters and enh ancers. These rodent DNA fragments contain the conserved guanine/cytos ine-rich sequences, as well as half-sites similar to those found in th e human CYP1A1 palindrome. However, each half-site is separated by sim ilar to 40 bp. DNase I footprint analyses revealed the presence of rat and mouse nuclear proteins which gave a similar protection pattern as that observed with nuclear proteins from the human cell line, HepG2. Electrophoretic mobility shift assays with the human negative regulato ry element demonstrated the formation of specific DNA - protein comple xes with rat and mouse nuclear protein(s). Interestingly, two specific DNA-protein complexes were observed with rodent extracts as compared to the single specific complex seen with human extract. Specific bindi ng was not observed with either the orthologous rat or mouse fragments using human or rodent extracts. In transient expression assays, the r at and mouse fragments were unable to down-regulate enhancer/promoter activity. This absence of negative regulatory activity occurred whethe r transfections were performed in human, rat or mouse hepatoma cell li nes. The human negative regulatory element, which was previously shown to down-regulate heterologous enhancers/promoters similar to 70% in h uman cells, did not exhibit this activity in rodent cell lines. UV cro ss-linking and southwestern blot analyses indicated a high degree of s imilarity between human and rodent NRE binding proteins, although some differences also were apparent. The possible implications of these fi ndings with regards to species differences in the regulation of CYP1A1 expression are discussed.