Characterization of a novel hexameric repeat DNA sequence in the promoter of the immediate early gene, IEX-1, that mediates 1alpha,25-dihydroxyvitamin D(3)-associated IEX-1 gene repression.
1alpha,25-Dihydroxyvitamin D(3)(1alpha,25(OH)(2)D(3)), the active metabolite of vitamin D(3), mediates anti-proliferative effects in cells by regulating the expression of 1alpha,25(OH)(2)D(3)-responsive genes. The expression of the proliferation-promoting Immediate Early gene X-1 (IEX-1) is reduced by 1alpha,25(OH)(2)D(3) through unknown mechanisms. Here we report the presence of a novel inhibitory hexameric repeat DNA response element in the promoter of the human IEX-1 gene that mediates 1alpha,25(OH)(2)D(3)-associated IEX-1 gene repression. To localize a vitamin D sensitive DNA response element we transfected the keratinocyte-like cell line, HaCaT, (referred as HaCaT) with a series of plasmids containing full-length and truncated IEX-1 promoter elements fused to the luciferase reporter gene in the absence or presence of 1alpha,25(OH)(2)D(3), and we performed electrophoretic gel mobility assays in the presence of receptors for 1alpha,25(OH)(2)D(3) (vitamin D receptor, VDR) and 9-cis-retinoic acid (RXRalpha). We mapped a negative response element between nt -405 and -391(15 bp) of theIEX-1 promoter (5'-TGAACC AGG GAGTCA-3') that mediates transcriptional inhibition in response to 1alpha,25(OH)(2)D(3) and which requires expression of both nuclear receptors for 1alpha,25(OH)(2)D(3) and 9-cis-retinoic acid. Our data indicate that the physiological repression of IEX-1 gene expression by 1alpha,25(OH)(2)D(3) is directly mediated by nuclear VDR/RXRalpha heterodimers through a specific transcriptional element.
Im
Hee-Jeong
HJ
Department of Internal Medicine, Mayo Clinic and Foundation, 200 First Street, SW, Rochester, Minnesota, MN 55905, USA.
Craig
Theodore A
TA
Pittelkow
Mark R
MR
Kumar
Rajiv
R
D051017:Apoptosis Regulatory Proteins; D001483:Base Sequence; D002460:Cell Line; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009363:Neoplasm Proteins; D011401:Promoter Regions, Genetic / Q000235:genetics*; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D047488:Retinoid X Receptors; D020079:Terminal Repeat Sequences; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014807:Vitamin D / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*
C116441:2-methyl-1,25-dihydroxyvitamin D3; D051017:Apoptosis Regulatory Proteins; C099815:IER3 protein, human; D017874:Immediate-Early Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014807:Vitamin D