Study of the protein-DNA interactions in gene 33 promoter proximal region

The expression of gene 33, which encodes a protein of unknown function, is multihormonally regulated. It is regulated by glucocorticoids, agonists which elevate intracellular cAMP, insulin and phorbol esters. To confirm this response in the rat hepatoma KRC7 cells, the effect of various concentrations of insulin or various periods of exposure to insulin on the abundance of the gene 33 mRNA was investigated. The abundance of gene 33 mRNA in KRC7 cells was induced by insulin in a time and dose dependent manner. The maximal mRNA level was attained within 3 hours and half maximal expression occurred at an insulin concentration of about 1 nM. General basal expression and transcriptional activation of gene 33 by insulin in KRC7 cells required the presence of a 74 base pair element located at -129 to -56 relative to the mRNA cap site. A gel retardation assay was used to detect a protein(s) in KRC7 cell nuclear extracts that specifically binds to this promoter proximal region of gene 33. The specific protein recognition sequence(s) in this region was defined by the DNaseI footprint assay. These insulin-sensitive cells contained nuclear factors which bind to this promoter fragment of gene 33 in a concentration dependent manner. Insulin did not change the binding ability of these nuclear factors in KRC7 cells. Several protein protected regions included a sequence with homology to the CCAAT/enhance binding protein (C/EBP) binding site-CCAAT motif and a sequence with partially homology to serum response element—SRE. The affinity of those nuclear factors for the 74 bp fragment appeared to be distinct from that of C/EBP, SRF and hepatic nuclear factor-HNF-1.