Regulation of the human p21(waf1/cip1) gene promoter via multiple binding sites for p53 and the vitamin D3 receptor

Abstract

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.

Distinct p53 genomic binding patterns in normal and cancer-derived human cells

“…A fourth site (B, Fig. 1B) was reported at position -3,969 bp relative to the major TSS, 34 and a site at -11.7 kb from the major TSS (A, Fig. 1B) was identified and confirmed in the ChIP-PET study 23 and was later shown to regulate p53-dependent lincRNA-p21 transcription.…”

Loss of p21Sdi1 expression in senescent cells after DNA damage accompanied with increase of miR-93 expression and reduced p53 interaction with p21Sdi1 gene promoter

“…To evaluate regulation of p21 gene by p53 in senescent cells before and after DNA damage, the following primer pairs spanning À2478 to À2029 of p21 gene promoter were designed [24]; sense, 5 0 -CACCACTGAGCCTTCCTCAC-3 0 and anti-sense, 5 0 -CTGACTCCCAGCACACACTC-3 0 . Using the isolated chromatin templates, following PCR profile was applied: Preincubation for 5 min at 95°C, 34 cycles of 30 s at 95°C, 40 s at 54°C and 40 s at 72°C and one final incubation for 10 min at 72°C.…”

The vitamin D receptor in cancer

“…The common anti-proliferative VDR functions are associated with arrest at G 0 /G 1 of the cell cycle, coupled with up-regulation of a number of cell cycle inhibitors including p21 (waf1/cip1) and p27 (kip1) . Promoter characterization studies have demonstrated a series of vitamin D-responsive elements in the promoter-enhancer region of CDKN1A, a primary 1a,25(OH) 2 D 3 -responding gene (96,97) . By contrast, p27 (kip1) protein levels appear to be regulated by a range of post-transcriptional mechanisms, such as enhanced mRNA translation, and attenuating degradative mechanisms, often in a cell-type-specific manner (98)(99)(100) .…”