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

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.

Identification of single nucleotide polymorphisms in the p21 (CDKN1A) gene and correlations with longevity in the Italian population.

Longevity in humans is determined by multiple environmental and genetic factors. We have investigated possible associations between longevity and Single Nucleotide Polymorphisms (SNPs) in the p21 (CDKN1A) gene, a stress-inducible senescence-associated cell cycle inhibitor, expression of which upregulates genes implicated in several age-related diseases. By sequencing the promoter and exons of p21 in genomic DNA of ten individuals over 90 years old, we have identified 30 SNPs, many of which had not been previously characterized. A cluster of minor alleles within the -4547/-3489 bp region did not alter the basal activity or p53 responsiveness of the p21 promoter. We then compared the frequency of 41 p21 SNPs between 184 centenarians and 184 younger subjects in the Italian population. Rare alleles of two exon-derived SNPs, rs1801270 and rs1059234, were significantly under-represented among the centenarians; no significant differences were found for 39 non-exonic SNPs. SNP rs1801270 causes Ser to Arg substitution at amino acid 31 and SNP rs1059234 leads to a nucleotide change in the 3'-untranslated region. Previous studies showed that the rare alleles of these two SNPs may play a role in cancer. These p21 alleles may be potentially detrimental to longevity and therefore are rare in centenarians.

Distinct HDACs regulate the transcriptional response of human cyclin-dependent kinase inhibitor genes to Trichostatin A and 1alpha,25-dihydroxyvitamin D3.

The anti-proliferative effects of histone deacetylase (HDAC) inhibitors and 1alpha,25-dihydroxyvitamin D3 [1alpha,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 1alpha,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 1alpha,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 1alpha,25(OH)2D3 were contrary. HDAC3 and HDAC7 attenuated 1alpha,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 1alpha,25(OH)2D3 by HDACs can be overcome by their combined application achieving maximal induction of anti-proliferative target genes.

Three members of the human pyruvate dehydrogenase kinase gene family are direct targets of the peroxisome proliferator-activated receptor beta/delta.

The nuclear receptors peroxisome proliferator-activated receptors (PPARs) are known for their critical role in the metabolic syndrome. Here, we show that they are direct regulators of the family of pyruvate dehydrogenase kinase (PDK) genes, whose products act as metabolic homeostats in sensing hunger and satiety levels in key metabolic tissues by modulating the activity of the pyruvate dehydrogenase complex. Mis-regulation of this tightly controlled network may lead to hyperglycemia. In human embryonal kidney cells we found the mRNA expression of PDK2, PDK3 and PDK4 to be under direct primary control of PPAR ligands, and in normal mouse kidney tissue Pdk2 and Pdk4 are PPAR targets. Both, treatment of HEK cells with PPARbeta/delta-specific siRNA and the genetic disruption of the Pparbeta/delta gene in mouse fibroblasts resulted in reduced expression of Pdk genes and abolition of induction by PPARbeta/delta ligands. These findings suggest that PPARbeta/delta is a key regulator of PDK genes, in particular the PDK4/Pdk4 gene. In silico analysis of the human PDK genes revealed two candidate PPAR response elements in the PDK2 gene, five in the PDK3 gene and two in the PDK4 gene, but none in the PDK1 gene. For seven of these sites we could demonstrate both PPARbeta/delta ligand responsiveness in context of their chromatin region and simultaneous association of PPARbeta/delta with its functional partner proteins, such as retinoidXreceptor, co-activator and mediator proteins and phosphorylated RNA polymerase II. In conclusion, PDK2, PDK3 and PDK4 are primary PPARbeta/delta target genes in humans underlining the importance of the receptor in the control of metabolism.

Controlling the chromatin organization of vitamin D target genes by multiple vitamin D receptor binding sites.

An essential prerequisite for the direct modulation of transcription by 1alpha,25-dihydroxy vitamin D(3) (1alpha,25(OH)(2)D(3)) is the location of at least one activated vitamin D receptor (VDR) protein close to the transcription start site of the respective primary 1alpha,25(OH)(2)D(3) target gene. This is achieved through the specific binding of VDR to a 1alpha,25(OH)(2)D(3) response element (VDRE). Although these elements are well characterized in vitro, the function of VDREs in living cells in the context of chromatin is still largely unknown. To resolve this issue, approximately 8kB of the promoter regions of the primary 1alpha,25(OH)(2)D(3) target genes CYP24, cyclin C and p21((Waf1/Cip1)) were screened by chromatin immunoprecipitation (ChIP) assays for VDR binding sites using antibodies against VDR and its partner proteins. This approach identified three to four functional VDREs per gene promoter. In parallel, in silico screening of the extended gene areas (i.e. 10kB of promoter, introns, exons and 10kB of the downstream region) of all six members of the insulin-like growth factor binding protein (IGFBP) gene family was performed. Gel shift, reporter gene and ChIP assays identified in total 10 functional VDREs in the genes IGFBP1, IGFBP3 and IGFBP5. Taken together, both screening approaches suggest that a reasonable proportion of all VDR target genes, if not all, are under the control of multiple VDREs.

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

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 1alpha,25-dihydroxyvitamin D3 (1alpha,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 1alpha,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 1alpha,25(OH)2D3. Moreover, re-ChIP assays confirmed the functionality of the three 1alpha,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 1alpha,25(OH)2D3-responding gene with at least three VDR binding promoter regions, in two of which also p53 co-localizes.