IDENTIFICATION OF A NOVEL SUPPRESSIVE VITAMIN-D RESPONSE SEQUENCE IN THE 5'-FLANKING REGION OF THE MURINE ID1 GENE

Vitamin D promotes differentiation of cells either by simply enhancing phenotypic gene expression and/or by suppressing expression of inhibi tors of differentiation. previously, we reported that expression of a gene encoding Idl, a negative type helix-loop-helix transcription fact or, was transcriptionally suppressed by 1,25-dihydroxyvitamin D-3 (1,2 5(OH)(2)D-3) (1). To identify the sequence required for the negative r egulation by 1,25(OH)(2)D-3, a 1,5-kilobase 5'-flanking region of muri ne Idl gene was examined by transiently transfecting luciferase report er constructs into ROS17/2.8 osteoblastic cells. The transcriptional a ctivity of this construct was repressed by 10(-8) hz 1,25(OH)(3)D-3. D eletion analysis revealed that a 57-base pair (bp) upstream response s equence (URS) (-1146/-1090) was required for the suppression by 1,25(O H)(2)D-3. This sequence conferred negative responsiveness to 1,25(OH)( 2)D-3 to a heterologous SV40 promoter. The 57-bp URS contained not onl y Egr-1 consensus sequence (2) but also four direct repeats of a hepta mer sequence (C/A)CAGCCC. Electrophoresis mobility shift assay reveale d that the 57-bp URS formed specific nuclear protein-DNA complexes, wh ich were neither competed by previously known positive and negative vi tamin D response elements nor supershifted by anti-vitamin D receptor antibody, suggesting the absence of vitamin D receptor in these comple xes. These results indicate the involvement of the novel 57-bp sequenc e in the vitamin D suppression of Idl gene transcription.