title: Characterization of DNA-protein interactions within a distal regulatory element upstream of a mammalian housekeeping gene promoter.
abstract: We have characterized a DNA-protein interaction within a sequence element distal from the site of transcription initiation within the mouse housekeeping gene (HPRT) promoter region. This interaction occurs within a 35-base pair regulatory element which confers cell type-specific gene transcription, designated as the HPRT cis-acting regulatory element (HCRE). Competition analysis by gel mobility shift electrophoresis indicates that this DNA-protein interaction is novel and not related to many transcription factors previously reported. Cell cycle synchronization experiments and gel mobility shift assays have demonstrated that within the HCRE a specific DNA-protein complex responds to G1 activation of the cell cycle. Experiments to purify specific DNA-binding proteins 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 sequence of GTCTGGGT using both affinity purified protein and crude nuclear extract. This DNA motif represents a novel protein-binding sequence. Interestingly, data base searches have identified the same or homologous sequences of this DNA motif in additional genes, potentially related to cellular growth and proliferation. This consensus was most notable within a region 5' upstream of the ornithine decarboxylase gene. The unique cell type-specific regulation of the HPRT gene in the intestinal mucosa is not completely understood at this time but because of the relationship of ornithine decarboxylase expression to cell proliferation and more specifically, to mucosal cell renewal in the intestine, the function of DNA-protein interactions within the consensus sequence may prove analogous. This may account for the cell type-specific and cell-cycle responsive gene regulation previously demonstrated with HPRT. Identification of one sequence-specific DNA-binding protein within the HCRE suggest that this protein contributes to the trans-activation of specific genes during the immediate-early response of the cell cycle.
authors.affiliation: Department of Pediatrics, Mount Sinai School of Medicine, New York, New York 10029.
authors.forename: M J
authors.initials: MJ
authors.lastname: Walsh
authors.forename: K L
authors.initials: KL
authors.lastname: Tsao
authors.forename: N S
authors.initials: NS
authors.lastname: LeLeiko
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D016172:DNA Fingerprinting; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016193:G1 Phase; D005786:Gene Expression Regulation; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009955:Ornithine Decarboxylase / Q000235:genetics; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D013936:Thymidine / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D009955:Ornithine Decarboxylase; D013936:Thymidine