Selective use of multiple vitamin D response elements underlies the 1 α,25-dihydroxyvitamin D3-mediated negative regulation of the human CYP27B1 gene

Mikko M. Turunen 0 Thomas W. Dunlop 0 Carsten Carlberg 0 Sami Va isa nen 0 0 Department of Biochemistry, University of Kuopio , FIN-70211 Kuopio, Finland The human 25-hydroxyvitamin D3 (25(OH)D3) 1a-hydroxylase, which is encoded by the CYP27B1 gene, catalyzes the metabolic activation of the 25(OH)D3 into 1a,25-dihydroxyvitamin D3 (1a,25(OH)2D3), the most biologically potent vitamin D3 metabolite. The most important regulator of CYP27B1 gene activity is 1a,25(OH)2D3 itself, which down-regulates the gene. The down-regulation of the CYP27B1 gene has been proposed to involve a negative vitamin D response element (nVDRE) that is located 500 bp upstream from transcription start site (TSS). In this study, we reveal the existence of two new VDR-binding regions in the distal promoter, 2.6 and 3.2 kb upstream from the TSS, that bind vitamin D receptor-retinoid X receptor complexes. Since the down regulation of the CYP27B1 gene is tissue- and cell-type selective, a comparative study was done for the new 1a,25(OH)2D3-responsive regions in HEK-293 human embryonic kidney and MCF-7 human breast cancer cells that reflect tissues that, respectively, are permissive and nonpermissive to the phenomenon of 1a,25(OH)2D3mediated down-regulation of this gene. We found significant differences in the composition of protein complexes associated with these CYP27B1 promoter regions in the different cell lines, some of which reflect the capability of transcriptional repression of the CYP27B1 gene in these different cells. In addition, chromatin architecture differed with respect to chromatin looping in the two cell lines, as the new distal regions were differentially connected with the proximal promoter. This data explains, in part, why the human CYP27B1 gene is repressed in HEK-293 but not in MCF-7 cells. - The biologically active form of vitamin D, 1a, 25-dihydroxyvitamin D3 (1a,25(OH)2D3)), is required for mineral homeostasis and skeletal integrity, as well as controlling cell growth and differentiation in several tissues (1). In the body, the amount of 1a,25(OH)2D3 is tightly controlled by several enzymes that are transcribed from genes belonging to the cytochrome P450 (CYP) family. This gene family encodes a wide variety of enzymes that are needed in the oxidative metabolism of a number of endogenous and exogenous compounds (2). One of the enzymes needed in the metabolism of 1a,25(OH)2D3 is the protein product of the CYP27B1 gene, 25-hydroxyvitamin D3 (25(OH)D3) 1a -hydroxylase. It has an important role in the synthesis of 1a,25(OH)2D3 because it catalyzes the metabolic activation of the main circulating form of vitamin D, 25(OH)D3, into 1a,25(OH)2D3 (3). CYP27B1 gene expression is negatively regulated by 1a,25(OH)2D3, and this has been proposed to occur via a negative vitamin D response element (nVDRE), located 500 bp upstream from transcription start site (TSS) (4). The down-regulation of this gene by 1a,25(OH)2D3 is a cell-type and tissue-restricted phenomenon. In the body, the major expression site of the CYP27B1 gene and its protein product, 25(OH)D3 1a-hydroxylase (CYP27B1), is the kidney. Within this organ, both the mRNA and the protein product have been observed to be repressed in the presence of 1a,25(OH)2D3 in the proximal tubules only (5). The CYP27B1 gene is also expressed in extra renal sites (6), however the suppression of the gene by 1a,25(OH)2D3 has been described only in a few other cell lines derived from other tissues, such as colon-derived cells (7). The effects of 1a,25(OH)2D3 are mediated via the vitamin D receptor (VDR), a member of the nuclear receptor superfamily, to which 1a,25(OH)2D3 binds with high affinity. Responsiveness of a given gene to 1a,25(OH)2D3 requires that its regulatory regions contain a VDRE. The VDREs of positively regulated genes are direct repeats of two hexameric-core-binding motifs spaced by 3 or 4 nt (DR3 or DR4, respectively) or everted repeats spaced by 69 nt (ER6 and ER9, respectively) (8,9). The hexameric sequences of the response elements of the primary 1a,25(OH)2D3 target genes usually have the consensus sequence RGKTSA (R A or G, K G or T, S C or G). The VDREs of most previously studied negatively regulated genes resemble those of positively regulated genes, although negative regulation may not necessarily require both VDRE half sites (1012). The reported negative VDRE of the CYP27B1 is an exception, because it does not contain a consensus sequence (4). In addition, the authors proposed that the regulation of the CYP27B1 gene involves an indirect binding of VDR to DNA, where VDR associates with the nVDRE liganddependently via another transcription factor, the VDR interacting repressor (VDIR). Binding of 1a,25(OH)2D3 causes a conformational change within the ligand-binding domain of the VDR, which modulates its interactions with nuclear proteins, such as coactivator (CoA) and corepressor (CoR) proteins (13). CoR proteins, such as NCoR1 (14) and SMRT/ NCoR2 (15), link non-liganded, DNA-bound VDR to enzymes with histone deacetylase activity that cause chromatin condensation (16). The conformational change within VDRs ligand-binding domain results in the replacement of a CoR by a CoA protein of the p160 family, such as SRC-1, SRC-2 or SRC-3 (17). These CoAs link the ligand-activated VDR to enzymes displaying histone acetyltransferase (HAT) activity, such as CBP, that cause chromatin relaxation by their action on histone tails and thereby reversing the action of unliganded VDR (18). Traditionally, VDREs are thought to locate relatively close to the TSS of 1a,25(OH)2D3 target genes. For example, both human and rat vitamin D 24-hydroxylase (CYP24) genes have a cluster of VDREs in their proximal promoters (approximate position 140 to 300) (1922). However, recently several promoter studies have revealed that the gene promoters may contain multiple response elements that locate not only within proximal promoters but also in more distal regions (23,24) and even within coding regions (25,26). These studies have so far concerned with only positively regulated genes. This raises a question, whether negatively regulated genes also have multiple response elements. Since the promoter studies of the human CYP27B1 gene have so far been limited to the first 1.7 kb upstream of the TSS (4,2729), in this study, we have extended the promoter analysis further upstream and examined the role of distal promoter regions to the regulation of the human CYP27B1 gene by 1a,25(OH)2D3. We analyzed 13 contiguous genomic regions spanning 5.4 kb of the CYP27B1 promoter by chromatin immunoprecipitation (ChIP) scanning. Our studies revealed two new 1a,25(OH)2D3-responsive regions in the distal promoter 2.6 and 3.2 kb upstream from the TSS. Interestingly, in contrast to the nVDREcontaining region, in silico screening revealed that both of the new 1a,25(OH)2D3-responsive regions contained classical VDRE sequences that were shown to directly bind VDRretinoid X receptor ( (...truncated)