The main regulator of the human tumor suppresser gene p21(waf1/cip1) is the transcription factor p53, but more recently it has been suggested to be a primary anti-proliferative target for the nuclear receptor VDR in the presence of its ligand 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). To identify VDR responding regions, we analyzed 20 overlapping regions covering the first 7.1 kb of the p21(waf1/cip1) promoter in MCF-7 human breast cancer cells using chromatin immuno-precipitation assays (ChIP) with antibodies against p53 and VDR. We confirmed two known p53 binding regions at approximate positions −1400 and −2300 and identified a novel site at position −4500. In addition, we found three VDR-associated promoter regions at positions −2300, −4500 and −6900, i.e. two regions showed binding for both p53 and VDR. In silico screening and in vitro binding assays using recombinant and in vitro translated proteins identified five p53 binding sites within the three p53-positive promoter regions and also five 1α,25(OH)2D3 response elements within the three VDR-positive regions. Reporter gene assays confirmed the expected responsiveness of the respective promoter regions to the p53 inducer 5-fluorouracil and 1α,25(OH)2D3. Moreover, re-ChIP assays confirmed the functionality of the three 1α,25(OH)2D3-reponsive promoter regions by monitoring simultaneous occupancy of VDR with the co-activator proteins CBP, SRC-1 and TRAP220. Taken together, we demonstrated that the human p21(waf1/cip1) gene is a primary 1α,25(OH)2D3-responding gene with at least three VDR binding promoter regions, in two of which also p53 co-localizes.

The nuclear receptor vitamin D receptor (VDR) is known to associate with three vitamin D response element (VDREs)-containing regions within the CDKN1A (p21) gene region. Here we show in MDA-MB453 breast cancer cells that the natural VDR ligand 1␣,25-dihydroxyvitamin D 3 causes cyclical transcription factor binding and chromatin looping of distal VDREs to the transcription start site (TSS) of the p21 gene, leading to cyclical accumulation of the p21 mRNA. At the chromatin level, association of the mediator protein MED1 precedes both the peaks of VDR binding to VDREs and phosphorylated RNA polymerase (p-Pol II) to the TSS. The loss of co-repressor NCoR1-histone deacetylase (HDAC) 3 complex and inhibitory chromatin looping from VDREs to the TSS are also initial events followed by increased acetylation of histone 3 at lysine 9 at the TSS prior to initiation of transcription. Simultaneous to VDR and p-Pol II peaks, chromatin loops between VDREs and the TSS are formed, and the lysine demethylase LSD1 and the histone acetyltransferase CBP are enriched in both regions. This is followed by a moderate peak in p21 transcript accumulation, repeated in cycles of 45-60 min. The transcript accumulation pattern is disturbed by siRNA inhibition of the mediator protein MED1, LSD1, NCoR1, or various HDACs, whereas CBP appears unnecessary for the response. Inhibition of MED1, HDAC4, or LSD1 by siRNA also attenuates ligand-induced chromatin looping. In conclusion, 1␣,25-dihydroxyvitamin D 3 regulates p21 transcription by inducing cyclical chromatin looping that depends on both histone deacetylation and demethylation.As a member of the nuclear receptor (NR) 2 superfamily the vitamin D receptor (VDR) acts as a transcription factor that binds to specific response elements (VDREs) within the regulatory regions of its primary target genes (1). The natural VDR ligand 1␣,25-dihydroxyvitamin D 3 (1␣,25(OH) 2 D 3 ) has an important role in the regulation of calcium and phosphate homeostasis and bone mineralization (2). In addition to this classical role, there is both epidemiological and preclinical evidence that 1␣,25(OH) 2 D 3 is an anti-proliferative agent (3). Vitamin D deficiency increases the risk of certain cancers, whereas the administration of 1␣,25(OH) 2 D 3 in cell culture or in animal models for cancer inhibits angiogenesis and induces G0/G1 arrest, differentiation, and apoptosis (4). These antiproliferative effects are exerted by various 1␣,25(OH) 2 D 3 target genes, among which the product of cyclin-dependent kinase inhibitor 1A (CDKN1A, also called p21) gene induces G0/G1 cell cycle arrest and differentiation (5). We have previously characterized three functional VDREs residing 2-7-kb upstream of the TSS of p21 gene (6).Non-liganded VDR is associated with primary co-repressors, such as the nuclear receptor co-repressor (NCoR1), that attenuate transcription via interaction with histone deacetylases (HDACs) that pose the chromatin modifications unfavorable for transcription or, at least in the case of NCoR1, are also able to rec...

No abstract

The anti-proliferative effects of histone deacetylase (HDAC) inhibitors and 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] converge via the interaction of un-liganded vitamin D receptor (VDR) with co-repressors recruiting multiprotein complexes containing HDACs and via the induction of cyclin-dependent kinase inhibitor (CDKI) genes of the INK4 and Cip/Kip family. We investigated the effects of the HDAC inhibitor Trichostatin A (TSA) and 1α,25(OH)2D3 on the proliferation and CDKI gene expression in malignant and non-malignant mammary epithelial cell lines. TSA induced the INK4-family genes p18 and p19, whereas the Cip/Kip family gene p21 was stimulated by 1α,25(OH)2D3. Chromatin immunoprecipitation and RNA inhibition assays showed that the co-repressor NCoR1 and some HDAC family members complexed un-liganded VDR and repressed the basal level of CDKI genes, but their role in regulating CDKI gene expression by TSA and 1α,25(OH)2D3 were contrary. HDAC3 and HDAC7 attenuated 1α,25(OH)2D3-dependent induction of the p21 gene, for which NCoR1 is essential. In contrast, TSA-mediated induction of the p18 gene was dependent on HDAC3 and HDAC4, but was opposed by NCoR1 and un-liganded VDR. This suggests that the attenuation of the response to TSA by NCoR1 or that to 1α,25(OH)2D3 by HDACs can be overcome by their combined application achieving maximal induction of anti-proliferative target genes.

No abstract