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 1α,25-dihydroxyvitamin D3 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, 1α,25-dihydroxyvitamin D3 regulates p21 transcription by inducing cyclical chromatin looping that depends on both histone deacetylation and demethylation.
Highlights
At the time 0 point, this indicates that the region shows significantly higher association with the mentioned protein/looping to the TSS compared to the lowest point in treated cells, “Ϫ” indicates regions showing significantly decreased association with the mentioned protein/looping to the TSS compared to untreated cells, whereas blank entries represent no significant change
After a 24-h transfection response to 1␣,25(OH)2D3 treatment. period with siRNA oligonucleotides, the MDA-MB453 cells Histone Dimethylation and Deacetylation Are Indispensable were stimulated with 1␣,25(OH)2D3 and RNA was extracted. for Chromatin Looping—Because the proteins MED1, HDAC4, We focused on the period of 150 to 255 min after the onset of and LSD1 seem to have a major impact on the 1␣,25(OH)2D3
We observed the cyclic responses at high resolution over a time frame of up to 300 min and distinguished an early phase of dependent cyclical accumulation of p21 transcript levels (Fig. 4) minor transcriptional induction and a later phase of more proand are ligand-dependently enriched on the 1␣,25(OH)2D3-re- nounced activation, but still modest effects on mRNA inducsponsive regions and the TSS of the p21 gene (Fig. 2B), they tion
Summary
Introduction of a ligand results in a conformational change in the ligand-binding domain of the VDR, leading to enhanced binding to its heterodimerization partner retinoid X receptor [1] and an exchange of co-repressors to primary co-activators Those include the members of the p160 family that recruit secondary co-activators, such as the histone-modifying enzymes, the histone acetyltransferase CBP, and the lysine demethylase LSD1, and the mediator complex subunit MED1 that enables the contact to transcriptional machinery. Acetylated hismediator complex subunit 1; NCoR1, nuclear co-repressor 1; p-Pol II, serine 5-phosphorylated RNA polymerase II; RPLP0, acidic riboprotein P0; siRNA, small inhibitory RNA; TBL1, transducin -like protein 1; TSS, transcription start site; VDR, vitamin D receptor; VDRE, vitamin D response element. Both histone deacetylation and demethylation are essential for the ligand-dependent dynamic looping of chromatin and the increased transcription of p21 in response to 1␣,25(OH)2D3
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