CHARACTERIZATION OF DNA-PROTEIN INTERACTIONS WITHIN A DISTAL REGULATORY ELEMENT UPSTREAM OF A MAMMALIAN HOUSEKEEPING GENE PROMOTER

We have characterized a DNA-protein interaction within a sequence elem ent distal from the site of transcription initiation within the mouse housekeeping gene (HPRT) promoter region. This interaction occurs with in a 35-base pair regulatory element which confers cell type-specific gene transcription, designated as the HPRT cis-acting regulatory eleme nt (HCRE). Competition analysis by gel mobility shift electrophoresis indicates that this DNA-protein interaction is novel and not related t o many transcription factors previously reported. Cell cycle synchroni zation experiments and gel mobility shift assays have demonstrated tha t within the HCRE a specific DNA-protein complex responds to G1 activa tion of the cell cycle. Experiments to purify specific DNA-binding pro teins that interact with the HCRE has resulted in the purification of one sequence-specific DNA-binding protein of approximately 66 kDa. To determine the putative DNA-binding sequence, footprinting analysis has mapped the protection from DNase I hydrolysis which confers a core se quence of GTCTGGGT using both affinity purified protein and crude nucl ear extract. This DNA motif represents a novel protein-binding sequenc e. Interestingly, data base searches have identified the same or homol ogous sequences of this DNA motif in additional genes, potentially rel ated to cellular growth and proliferation. This consensus was most not able within a region 5' upstream of the ornithine decarboxylase gene. The unique cell type-specific regulation of the HPRT gene in the intes tinal mucosa is not completely understood at this time but because of the relationship of ornithine decarboxylase expression to cell prolife ration and more specifically, to mucosal cell renewal in the intestine , the function of DNA-protein interactions within the consensus sequen ce may prove analogous. This may account for the cell type-specific an d cell-cycle responsive gene regulation previously demonstrated with H PRT. Identification of one sequence-specific DNA-binding protein withi n the HCRE suggest that this protein contributes to the trans-activati on of specific genes during the immediate-early response of the cell c ycle.