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. 2009 Mar 20;284(12):8073-82.
doi: 10.1074/jbc.M808090200. Epub 2009 Jan 3.

Cyclical chromatin looping and transcription factor association on the regulatory regions of the p21 (CDKN1A) gene in response to 1alpha,25-dihydroxyvitamin D3

Anna Saramäki  1 , Sarah Diermeier, Ruth Kellner, Heidi Laitinen, Sami Vaïsänen, Carsten Carlberg
Affiliations

Cyclical chromatin looping and transcription factor association on the regulatory regions of the p21 (CDKN1A) gene in response to 1alpha,25-dihydroxyvitamin D3

Anna Saramäki et al. J Biol Chem. .

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 1alpha,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, 1alpha,25-dihydroxyvitamin D(3) regulates p21 transcription by inducing cyclical chromatin looping that depends on both histone deacetylation and demethylation.

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Figures

FIGURE 1.
FIGURE 1.
Cyclical induction of p21 transcription by 1α,25,(OH)2D3. Real-time quantitative PCR was performed to measure the time-dependent mRNA expression of the p21 gene in MDA-MB453 cells after treatment with 10 nm 1α,25(OH)2D3. The data were normalized to the expression of the housekeeping gene RPLP0, and fold inductions were calculated in reference to vehicle control. Data points indicate the means of at least three independent cell treatments, and the bars represent standard deviations. A two-tailed Student's t test was performed to determine the significance of the stimulation in reference to vehicle-treated control (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
FIGURE 2.
FIGURE 2.
Dynamic association of VDR, cofactors, and histone modifications to VDRE containing regions and the TSS of the p21 gene. Schematic overview on the human p21 promoter indicating the location of VDREs described previously (6) and of targeted genomic regions (A). On each of these four regions ChIP assays with indicated antibodies were performed on chromatin extracted from MDA-MB453 cells that had been treated with 10 nm 1α,25(OH)2D3 for the indicated times (B and C). Analysis was performed by real-time quantitative PCR using FAM-labeled probes, and association was calculated relative to input samples. Association of p53 was subtracted from relative association levels to control for nonspecific binding. Data points indicate the means of at least three independent cell treatments, and the bars represent standard deviations. A two-tailed Student's t test was performed to determine the significance of the stimulation in reference to vehicle-treated control (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
FIGURE 2.
FIGURE 2.
Dynamic association of VDR, cofactors, and histone modifications to VDRE containing regions and the TSS of the p21 gene. Schematic overview on the human p21 promoter indicating the location of VDREs described previously (6) and of targeted genomic regions (A). On each of these four regions ChIP assays with indicated antibodies were performed on chromatin extracted from MDA-MB453 cells that had been treated with 10 nm 1α,25(OH)2D3 for the indicated times (B and C). Analysis was performed by real-time quantitative PCR using FAM-labeled probes, and association was calculated relative to input samples. Association of p53 was subtracted from relative association levels to control for nonspecific binding. Data points indicate the means of at least three independent cell treatments, and the bars represent standard deviations. A two-tailed Student's t test was performed to determine the significance of the stimulation in reference to vehicle-treated control (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
FIGURE 3.
FIGURE 3.
1α,25(OH)2D3 induces dynamic looping of VDR binding regions to the TSS of the p21 gene. Schematic overview on the human p21 promoter indicating previously described VDREs (6), MvaI and Hpy81I restriction enzyme recognition sites and location of primers along with FAM-labeled probes used for quantification of the 3C products (A). 3C assays were performed with chromatin from MDA-MB453 cells that were treated for the indicated times with 10 nm 1α,25(OH)2D3 (B). Chromatin was extracted, cross-linked, and digested with either MvaI or Hpy8I. After ligation, the DNA was extracted and analyzed by PCR with primer A in combination with primers C, D, or E for Hpy8I-digested chromatin (left figure) or with primers F or G for MvaI-digested chromatin (right figure). Re-ligated digestions of subcloned p21 promoter fragments served as positive controls. Analysis was performed by real-time quantitative PCR using a FAM-labeled probe targeting the ligation site specific for the product. PCR efficacy was normalized to positive controls. Values indicate looping as relative values compared with basal looping of region p21-2 to the TSS. Data points indicate the means of at least three independent cell treatments, and the bars represent standard deviations. A two-tailed Student's t test was performed to determine the significance of the stimulation in reference to vehicle-treated control (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Targeted restriction fragments in A and quantitative results in B are shown in the same color.
FIGURE 4.
FIGURE 4.
Effect of cofactor silencing on the p21 mRNA response to 1α,25,(OH)2D3. MDA-MB453 cells were transfected for 24 h with siRNA oligonucleotides against the genes MED1, LSD1, HDAC4, NCoR1, HDAC3, CBP, or with a non-targeted siRNA (siCtrl), and subsequently either not stimulated (A) or stimulated (B) for the indicated times with 10 nm 1α,25(OH)2D3. Real-time quantitative PCR was used to determine the mRNA expression of indicated siRNA target genes (A). Remaining expressions were calculated in reference to samples transfected with a non-targeted siRNA (siCtrl). Real-time quantitative PCR was performed to measure the time-dependent mRNA expression of the p21 gene relative to the control gene RPLP0 (B). Fold inductions were calculated in reference to vehicle control that had been transfected with non-targeted siRNA (siCtrl). Data points indicate the means of at least three independent cell treatments, and the bars represent standard deviations. For B, a two-tailed Student's t test was performed to determine the significance of the effects of the specific siRNAs in reference to control siRNA (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
FIGURE 5.
FIGURE 5.
Effect of cofactor silencing on chromatin looping VDRE containing regions to the TSS of the p21 gene. MDA-MB453 cells were transfected for 24 h with siRNA oligonucleotides against HDAC4, MED1, or LSD1 and subsequently stimulated with 10 nm 1α,25(OH)2D3 for the indicated times. Chromatin was extracted, cross-linked, and digested with either Hpy8I (upper and center graphs representing regions p21-1 and p21-2, respectively) or MvaI (lower graph representing region p21-3). After ligation, the DNA was extracted and analyzed by PCR with primers and FAM-labeled probes as indicated in Fig. 3. Values indicate looping as percentage to that in untreated cells transfected with non-targeting siRNA from indicated region to the p21 TSS. Data points indicate the means of at least three independent cell treatments, and the bars represent standard deviations. A two-tailed Student's t test was performed to determine the significance of the effects of the specific siRNAs in reference to control siRNA (*, p < 0.05; **, p < 0.01; ***, p < 0.001).
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