Cyclical Chromatin Looping and Transcription Factor Association on the Regulatory Regions of the p21 (CDKN1A) Gene in Response to 1α,25-Dihydroxyvitamin D3

Abstract

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...

DNA methylation-related vitamin D receptor insensitivity in breast cancer

“…41 p21 contains three VDREs, and is known to be regulated by Calcitriol-induced cyclical chromatin looping. 42 In agreement with these reports, we found decreased expression of the p21 tumor suppressor gene in our breast cancer samples, as would be expected in the context of an inactive Calcitriol pathway (see Fig. 6).…”

Dynamic Estrogen Receptor Interactomes Control Estrogen-Responsive Trefoil Factor (TFF) Locus Cell-Specific Activities

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The MEF2–HDAC axis controls proliferation of mammary epithelial cells and acini formation in vitro

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