## 5 Epigenetic Modulation of Vitamin D Effects
### VDR Expression Regulated by Epigenetics  
Concerning methylation, VDR seems to follow the classical model, according to which normal gene expression is linked with promoter and enhancers hypomethyllation and gene body hypermethylation. However, according to [PERSON] and colleagues, VDR expression is regulated by a non-classical methylation mechanism too. Indeed, the treatment of rat colonic mucosa with the established carcinogen DMH (dimethylhydrazinedihydrochloride) resulted in a gene-body hypermethylation and a consequent decreased expression of VDR, in conflict with the classical model [57].  
In many pathological conditions, especially cancer, VDR expression is altered due to epigenetic aberration. Breast cancer cell lines treated with the DNMT inhibitor 5'deoxy-azacytidine showed a reduced aberrant VDR promoter hypermethylation, an enhanced responsiveness to vitamin D and an increased expression of target genes, supporting the classic model [58]. Besides cancer, infectious diseases as HIV and tuberculosis are characterized by an aberrant VDR methylation profile too. For instance, HIV infected T cells show an increase of 2.5 fold in VDR gene body methylation compared to controls [59].  
Another mechanism involved in VDR transcriptional suppression in colon and breast cancer is the epigenetic silencing mediated by the methylation of CpG islands (CGI) within the VDR promoter region [58]. According to predictive model of CGIs made by [PERSON] and colleagues, VDR contains three bona fide CGIs at promoter regions (CGI 1065, 1062, and 1060); in particular the presence of TaqMan polymorphism influences the last CGI 1060 [60]. A change in methylation at 1060 CGI site results in a differential transcription of a long non-coding RNA present in this region that may potentially regulate VDR expression post-transcriptionally [43].  
Although the impact of specific histone modifications on VDR transcription is not characterized in detail, it is quite clear that histone acetylation induced by vitamin D gives an important contribution to the regulation of VDR expression [61]. In fact, the treatment of osteosarcoma cells with vitamin D was reported to induce a strong increase in H4 acetylation in VDR enhancers, compatible with an increase in VDR expression [62]. Moreover, the treatment of resistant malignant melanoma cell lines with the HDAC inhibitor trichostatin-A significantly restored VDR expression. In general, several modifications of VDR histones have known effects; for instance H3K4 me1 (monomethylation of lysine 4 on histone H3 protein) correlates with transcriptional elongation, while H3K4 me3 (trimethylation) is associated with active transcription and it is found near the transcription start sites, H3K27 ac (acetylation of lysine 27 of the histone H3 protein) correlates with active enhancers and H3K27 me3 (trimethylation of lysine 27 on histone H3 protein) is associated with transcriptional inhibition if found in VDR gene-body [63; 64; 65].  
Finally, also several microRNAs (miRNAs) are epigenetic modulators of VDR. miRNAs are a class of non-coding RNAs made of twenty two nucleotides, able to recognize and bind specific sequenceson mRNA. Usually mature miRNAs suppress gene expression by binding the 3'-UTR region of the target mRNA, although several studies have reported other interactions with 5'-UTR, coding sequence, and gene promoters [66]. miRNA are involved in the reversible silencing of VDR too, particularly in melanoma cells [67]. The mRNA of the receptor presents three experimentally verified MREs (miRNA response elements), located in the 3'-UTR of the transcript [68], which are recognized by miR-125b, miR-27b, and mmu-miR-298. Due to the characteristic reversibility of the epigenetic modifications, vitamin D resistance can be overcome with the administration of calcitriol and DNMT or miRNA inhibitors [58].