Senescence is a major developmental transition in plants that requires a massive reprogramming of gene expression and includes various layers of regulations. Senescence is either an age-dependent or a stress-induced process, and is under the control of complex regulatory networks that interact with each other. It has been shown that besides genetic reprogramming, which is an important aspect of plant senescence, transcription factors and higher-level mechanisms, such as epigenetic and small RNA-mediated regulators, are also key factors of senescence-related genes. Epigenetic mechanisms are an important layer of this multilevel regulatory system that change the activity of transcription factors (TFs) and play an important role in modulating the expression of senescence-related gene. They include chromatin remodeling, DNA methylation, histone modification, and the RNA-mediated control of transcription factors and genes. This review provides an overview of the known epigenetic regulation of plant senescence, which has mostly been studied in the form of leaf senescence, and it also covers what has been reported about whole-plant senescence. Arabidopsis leaves have shown changes in the expression of up to 16% of genes during senescence [Senescence  is the final, main developmental phase transition in plants. It occurs at different levels, including at the level of cells, tissues, organs, and the whole plant . Senescenescence ,5. Furthnescence . Genes tnescence ,6.RD20 (responsive to dehydration-20) and RAP2.4 (related to AP2.4) are Arabidopsis drought stress-responsive genes which are also upregulated during leaf senescence, and their expression is controlled via differential methylation patterns [Arabidopsis leaves.In addition, epigenetic mechanisms that are known to play an important role in regulating the transition of plants to the next developmental stages also have a key impact on senescence . They dypatterns  during spatterns , and up Arabidopsis leaves, which have a very short lifetime, and their senescence may not necessarily—or at least entirely—be linked to the whole-plant senescence. Whole-plant senescence leads to death, and it depends on both chronological age and environmental cues, and identifying the changes in gene expression and epigenetics during leaf senescence may or may not be directly relevant to what happens at the time of whole-plant senescence [It should be noted that most senescence studies of plants have focused on nescence . This reMET1), or CHG , which is mainly modulated by CHROMOMETHYLASE 3 (CMT3), or asymmetric, as with CHH DNA-methylation, which is usually controlled by DOMAINS-REARRANGED METHYLTRANSFERASEs (DRMs) [ROS1), DEMETER (DME), and DEMETER-LIKE proteins (DML2/3) that contain DNA glycosylase domains [DNA methylation in plant genomes is more extensive than in animals , and it s (DRMs) . DNA met domains ,13. DNA  domains . In addi domains .MET1, CMT3, ROS1, DME and DML2/3, but the specific mechanisms are not clear yet [MET1 transgenic Arabidopsis plants have reported that when MET1 activity is reduced, for example in met1 mutants or with the constant expression of the MET1 antisense gene, hypomethylation in genomic DNA and developmental abnormalities happen. When studying transgenic plants with a MET1 antisense gene fused to the DEMETER (DME) promoter (DME:MET1 a/s), Kim et al. [MET1 expression is suppressed, major delays in senescence and other developmental deficiencies occur [Global DNA methylation changes that occur across developmental life events are reported in plants, such as the giant redwood tree . The efflear yet . In a stlear yet . Studiesm et al.  showed tes occur .Arabidopsis and barley have shown the release of TEs during leaf senescence [Arabidopsis thaliana ecotypes. A class that they named NMR19-4 appeared to be a naturally occurring epiallele that controlled leaf senescence. They found that the DNA methylation of NMR19-4 negatively regulates the transcription of the gene “pheophytin pheophorbide hydrolase (PPH)”, which encodes an enzyme involved in chlorophyll breakdown during leaf senescence. The levels of DNA methylation and TEs were also shown to be related [Arabidopsis’ dark-induced senescent leaves and reported that, upon senescence, chromatin-silencing genes were downregulated, which led to the interruption of TE silencing and the reactivation of young TEs. They also found that although heterochromatin at chromocenters was decondensed, the global DNA methylation pattern was maintained, with only localized changes in CHH methylation. They concluded that senescence is associated with global chromatin reorganization but is only limited to changes in localized DNA methylation. Vatov et al. [ros1 and the triple dmr1/2 cmt3 knockout). They showed that as senescence progressed, wide-type plants showed a moderate decrease in DNA methylation, mostly in a CG context, and the most senescent leaf was mainly associated with CHH de novo methylation.In addition, the involvement of DNA methylation in maintaining genome integrity by silencing TEs and repetitive sequences during senescence has been reported before ,21. Studnescence ,23. He enescence  detected related . Changes related . In anot related  investigv et al.  observedPinus radiata trees and found significant DNA methylation differences between the meristematic tissues of juvenile and mature trees, while they found little differences in DNA methylation between their differentiated tissues. They reported a gradual reduction in the extent of genomic DNA methylation in meristematic areas as the level of reinvigoration increased and, therefore, they suggested that the level of DNA methylation can serve as a marker of aging and reinvigoration.In some plants, aging has shown to cause the loss of morphogenic ability to the point that mature plants do not have the ability to propagate vegetatively . It has A. thaliana plants as they aged, Ogneva et al. [CMT3 and METI, and consequently decreased the cytosine methylation of DNA regions with aging, while the transcription of demethylase genes, ROS1, DME, DML2 and DML3 increased. They concluded that plants experience the demethylation of DNA during aging through a reduction of DNA methyltransferase and an increase in levels of demethylase enzymes [DML3 controls leaf senescence as the dml3 knockout mutants showed enriched DNA methylation in the promoters of many SAGs that suppressed their expression, resulting in delayed leaf senescence. They suggested that DML3-mediated DNA demethylation may control leaf senescence by regulating the expression of some SAGs [The de-methylation of DNA has also been reported to be accompanied by aging. Studying a et al.  reported enzymes . Yuan et enzymes  also shoome SAGs .Studies have shown that besides DNA methylation, various types of histone modifications also change the dynamic expression of genes when plants move through their developmental phases, such as senescence, or as they respond to the environmental signals. The DNA of eukaryote organisms is wrapped around eight histone molecules (a histone octamer), forming the nucleosome, which is the element of the chromatin structure . NucleosSUVH2) histone methyltransferase in H3 lysine methylation and its role in the delay of leaf senescence. They showed that in plants with overexpressing SUVH2 histone methyltransferase, which is involved in RNA-directed DNA methylation and transcriptional gene silencing by keeping the chromatin structure compact, leaf senescence is delayed. The delay in senescence was because of the repression of key senescence regulators, such as SIRK (senescence-induced receptor-like serine/threonine-protein kinase) or SAG101 (senescence-associated carboxylesterase 101) and was connected to the inhibition of WRKY53, a main transcription factor that promotes leaf senescence. The levels of H3K27me2 and H3K27me3 at the 5′-end region of WRKY53 was elevated and therefore caused the suppressed transcription of WRKY53 along with some SAGs [WRKY53 and some of the SAGs through H3K27me2/3 modifications. The overexpression of SUVH2 histone methyltransferase represses almost half of the senescence-related regulatory factors (SRRFs). It was reported that in plants with overexpressing SUVH2, leaf senescence is delayed by about two weeks and, therefore, SAGs are either not expressed or repressed [WRKY53 locus showed that complex epigenetic processes are involved in senescence-related gene expression reprogramming. The induced expression of WRKY53 during senescence was associated with an increasing number of active histone marks (H3K4me2 and H3K4me3) at the 5′ end and coding regions of WRKY53 [Arabidopsis leaf senescence, mostly within WRKY53, regulating the expression of many SAGs [One of the first studies that showed a direct connection between histone modification and leaf senescence regulation was when Ay et al.  reportedome SAGs ,41. Jingome SAGs  also repepressed . Epigenef WRKY53 , similarf WRKY53 . The levany SAGs .Arabidopsis leaves, and found SAGs with higher H3K4me3 signals in the older leaves, while for genes that downregulate during senescence (SDGs), H3K4me3 was higher in the younger leaves. Likewise, the silencing histone mark, H3K27me3, was lost at some SAGs in the older leaves, and established at some SDGs. This again indicated the role of epigenetic regulation in the expression of senescence-related genes during leaf senescence [Brusslan et al.  studied nescence . Next, inescence . They agnescence .Arabidopsis. They found an increase in the number of H3K4me3 marks after three days of darkness, and the genes with dark-increased H3K4me3 were mainly involved in senescence. The upregulated genes after dark treatment were highly expressed in senescent leaves as opposed to younger leaves and, likewise, the downregulated genes after dark treatment showed lower expression in senescing leaves than in younger leaves [WRKY6, SAG113, and SAG101, that are known to promote senescence, and some were involved in dark-induced leaf senescence  Insensitive 5 (ABI5), ETHYLENE INSENSITIVE 3 (EIN3), and ORESARA 1 (ORE1)) [JMJ16 is an Arabidopsis JmjC-domain-containing protein and acts as an H3K4 demethylase. A decrease in JMJ16 is reported to be associated with the increase in the amount of H3K4me3 during senescence [JMJ16 negatively affects age-dependent leaf senescence by repressing WRKY53 and SAG201 via its demethylase activity and reducing their level of H3K4me3. They found the overexpression of various SAGs associated with the hypermethylation of H3K4me3 in loss-of-function jmj16 mutants [A continuous lack of light causes changes in the expression of many genes, which leads to various developmental disruptions, including early leaf senescence . To inver leaves . They alr leaves . GO enri (ORE1)) . Therefonescence . Liu et nescence  reported mutants .An important role of leaf senescence is known to be facilitating nutrient remobilization to younger leaves and reproductive organs to maximize fitness, and many SAGs are upregulated during the process. It is interesting to know how SAGs remain transcriptionally inactive before the onset of leaf senescence to ensure photosynthesis. Wang et al. , identified an epigenetic mechanism that prevents the premature expression of these genes. They reported that RELATIVE OF EARLY FLOWERING 6 (REF6) promotes H3K27me3 demethylation at the promoter and coding regions of ten target senescence genes to activate them. The number of H3K27me3 marks decreases during senescence, as they repress the expression of senescence genes. This is caused by genes, such as REF6 . REF6 diArabidopsis leaves at different time points during developmental senescence and reported that many upregulated senescence-related genes were also marked with H3K9ac, which activates TFs, such as WRKY53. They found that the H3K9ac levels around SAGs were high at the early stages of senescence and decreased gradually as senescence progressed. This was the opposite of what happens in the case of the H3K4me3 level, which it is usually low at the early stage of senescence and increases drastically towards the end of the senescence stage. The histone marks were highly convergent throughout aging; however, the average number of H3K4me3 marks around the SAGs covered the gene area almost two times more than what was covered by H3K9ac marks.Another histone mark that is reported to have a role in regulating leaf senescence is histone acetylation (H3K9ac). The coordinated activities of histone acetylation and deacetylation play important roles in gene expression and are organized by HATs and HDAs enzymes. While histone acetylation correlates with active transcription, histone deacetylation is often associated with the repressing and silencing of the gene by removing acetylation and inducing chromatin compaction ,51. The Arabidopsis as well. Huang et al. [HDA15 interacts with the single-stranded DNA-binding protein WHIRLY1 and increases H3K9ac in the promoter of WRKY53 to suppress its expression and therefore delay leaf senescence. Furthermore, histone acetyltransferase HAC1 is known to positively regulate leaf senescence [HDA9, HDA15, HD2C, and AtSRT1 (from SILENT INFORMATION REGULATOR2 (SIR2) family proteins), have been shown to have a negative effect on stress-triggered senescence in Arabidopsis [HDA19, a histone deacetylases also known as AtHD1 that belongs to the RPD3 (reduced potassium dependency 3) class of histone deacetylases [loss-of-function mutants of HDA6 show increases in the acetylation of histone H3 and the downregulation of SAG12 and SEN4, which delayed the senescence. However, the expression of RPS17 , which is normally downregulated at the time of senescence, was maintained at high level in the mutants. HDA9 is known to promote leaf senescence and regulate the genes involved in the onset of both developmental and dark-induced senescence. In HDA9 mutants, leaf senescence is reported to be delayed [HDA9 makes a complex with a SANT domain-containing protein POWERDRESS (PWR) and the transcription factor WRKY53. The WRKY53 then directs POWERDRESS and HDA9 to the promoters of negative senescence regulators, such as AUTOPHAGY 9 (ATG9), NUCLEAR PROTEIN X 1 (NPX1), and WRKY57 [Arabidopsis leaf senescence, which has a short life and can be easily experimentally manipulated. Future studies focusing on whole-plant senescence are necessary to obtain a better understanding of the role of histone modifications in plant senescence. One study has focused on the epigenetic and transcriptional mechanisms of fruit senescence in longan trees, which showed that histone deacetylase HD2 interacts with ethylene response factors (ERFs) and is involved in longan plant fruit senescence [DlHD2 and two ethylene-responsive factor-like genes, DlERF1 and DlERF2, during fruit senescence, and showed that DlHD2 might cooperate with DlERF1 to regulate the transcription of fruit senescence genes [Histone deacetylation has been reported as a leaf senescence regulator in g et al.  found thnescence , whereasbidopsis ,56. Analbidopsis  studied etylases , and repetylases  showed t delayed ,59. Chen delayed  reportedd WRKY57 . As mentnescence . They ance genes .In addition to histone modifications and DNA methylation, ATP-dependent chromatin reorganization modulates the DNA accessibility for transcription factors and RNA polymerase II, and therefore regulates the transcription of the genes within the chromatin structure ,62,63. THeterochromatic and euchromatic areas bear different histone marks. The heterochromatin region at the pericentric area is characterized by typical heterochromatic histone marks, such as H3K9me2, and the euchromatic regions bear marks, such as H3K4me2 and H3K4me3. Therefore, it is feasible to study the changes in distribution of eu- and heterochromatic marks in the nuclei during developmental transitions using electron microscopy. For example, Ay et al.  showed tORE7) has been reported to cause a strong delay in leaf senescence [SNF2 (SUCROSE NONFERMENTING) family genes, such as CHR10 (ALTERED SEED GERMINATION 3 (ASG3)) and CHR19 (ETL1), which are shown to be upregulated during senescence [REF6 facilitates the recruitment of an SWI/SNF chromatin remodeling ATPase BRAHMA (BRM), which directly targets many senescence-related genes [BRM can also colocalize senescence genes with REF6, meaning BRM-containing SWI/SNF complexes might act along with REF6 to epigenetically control the leaf senescence by controlling the chromatin structure of senescence genes [DRD1: DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1) that is reported to be involved in the regulation of leaf senescence genes [DRD1 and DDM1: DECREASED DNA METHYLATION 1 (ATP-dependent DNA helicase DDM1 from the same group of proteins as DRD1), Cho et al. [drd1-6 mutants, and the expression of SAGs was significantly inhibited in the drd1-6 mutant even after 5 days of dark-induced senescence [CEN) and transcriptionally silent information (TSI) repeats in both wild type (WT) and the drd1-6 mutant, which showed a much lower increase in the number of mutants. They concluded that SWI2/SNF2 chromatin-remodeling proteins play a key role in the regulation of leaf senescence through epigenetic regulation. The histone deacetylase and ore7 mutants have also been reported to delay flowering, which might suggest that they cause delays in the entire developmental program [The overexpression of a chromatin remodeler AT-hook protein ORESARA 7 , a key NAC TF that promotes developmental leaf senescence, is also implicated in salt-triggered senescence [Many of senescence-associated genes also play a role in other biological processes. This suggests that genes that have evolved for senescence may have been recruited in other biological networks as well . For exanescence ,75. Darknescence . Howevernescence  studied WRKY70, which encodes a TF involved in the response to salicylic acid and jasmonic acid, and also in leaf senescence [ATX1 histone methylase) [WRKY6 and WRKY53 also are known to be involved in the regulation of both senescence and responses to pathogens [hda19 mutants, genes that are in charge of both plant developmental processes, including flowering and senescence, and stress responses are differentially expressed. This shows that the regulatory pathways of the main developmental phases, such as flowering and senescence, and of biotic and abiotic stress responses, are connected, and together orchestrate a network controlling environment-sensitive plant development, in which epigenetic mechanisms seem to be involved in forming a higher-order control level [Heterochromatic patterns are shown to change during senescence as they do in response to stress, too . Scientinescence , is showthylase) . The traathogens ,81. Studathogens ,83,84,85WHIRLY1 is a chloroplast–nucleus protein that has a role in regulating leaf senescence through binding to the promoters of SAGs, such as HvS40. Studying the expression of WHIRLY1 in premature senescence triggered by drought stress, Janack et al. [HvS40 gene, under drought stress, indicating the role of epigenetic mechanisms in response to drought stress, no significant increase in H3K9ac was seen in RNAi-W1 transgenic plants (which are impaired in their accumulation of WHIRLY1). They suggested that WHIRLY1 knockdown results in delayed senescence that involves changes in gene expression, by means such as altering the chromatin structure via histone modification [k et al.  reportedfication .Abscisic acid (ABA) is a stress- and senescence-associated phytohormone which is reported to trigger the signaling involved in changes in histone acetylation levels ,87. On tATM (ATAXIA TELANGIECTASIA MUTATED) and ATR (ATM AND RAD3-RELATED), two main protein kinase activated in response of DSBs and SSBs (single-strand breaks), respectively [DNA damage, either caused internally, for example from replication errors, or externally from genotoxic stresses , abioticectively , but theectively .Arabidopsis, the abundance of acetylation and methylation in histone H3 is shown to respond to X-ray radiation [loss-of-function Arabidopsis mutants for key members of the DNA repair pathway and reported that the deficiency in ATM, which is the main transducer of the DSB signal, results in the age-dependent accumulation of DSBs and leads to premature senescence. This is epigenetically regulated through the inhibiting of the transcription of senescence-associated TFs, such as ANAC016 (NAC domain-containing protein 16), WRKY6, WRKY53, and WRKY75, by ATM, which then manifests as leaf senescence when SAGs are upregulated by the modulation of H3K4me3/H3K27me3 marks [Arabidopsis [SAGs during DSB-induced leaf senescence.Chromatin remodeling, such as histone modification, is reported to be involved in the repair of DSBs in animals . In Arabadiation . Therefoadiation , is not adiation  providedadiation . In addie3 marks . They albidopsis . They suSmall RNAs (sRNAs) consist of about 20–30 nucleotides and have been reported to regulate the expression of the genes involved in plant development and stress responses ,101. TheORE1, miR164, and EIN2 seems to robustly regulate age-induced cell death in Arabidopsis leaves. It has been reported that miR164 in Arabidopsis negatively regulates the expression of ORE1, which is a positive regulator of leaf senescence [EIN2, it leads to the increased expression of ORE1, which promotes age-induced cell death in Arabidopsis leaves. Therefore, in the miR164 mutant, early senescence occurs. miR319 and miR396 were also identified as positive regulators of senescence because they repress members of signaling pathways (miR319—DNA-binding transcription factor) and induce a local limitation of regulator activity  [Some miRNAs and siRNAs are reported to regulate the progress of plant senescence by controlling the expression of TFs or phytohormone response factors ,7. For enescence . Kim et nescence  showed tor, GRF) ,105,106.Arabidopsis arf2 (Auxin response factor 2) mutants, leaf senescence is delayed [ARF2 is involved in auxin-dependent senescence, and it can be knocked out by the miR390-mediated production of a transcriptional silencing complex protein (TAS3). Phased, secondary, small interfering RNA (phasiRNA) is a class of plant-specific non-coding siRNA with a phase alignment structure, mediated by miRNAs at protein coding sites and non-coding sites  [trans-acting siRNAs (tasiRNAs). TAS3 (TRANS-ACTING SIRNA LOCUS 3) encodes trans-acting siRNAs and suppresses the transcription of the auxin response genes, such as ARF2 [ARF2 repression.It has been shown that in  delayed ,108. ARFAS loci) . A group as ARF2 , which imiR319 targets where the overexpression of miR319 led to the generation of a stay-green phenotype [miR319-regulated clade of TCP (TEOSINTE BRANCHED/CYCLOIDEA/PCF) TF genes by studying the downstream events regulated by five TCP-TFs that are controlled by the miR319 in A. thaliana. They analyzed leaf extracts from plants with increased activity of miR319 and found that the expression changes of the biosynthetic genes result in changed jasmonic acid levels and that changes in senescence behavior return to normal after treating with jasmonate. They concluded that miR319-controlled TCP transcription factors may negatively regulate leaf growth and positively regulate leaf senescence. Luan et al. [Solanum lycopersicum) can improve the plant’s tolerance to various stresses by changing the expression and interaction of some miRNAs, and phasiRNAs and their target genes. As ABA is known to accelerate senescence as well, future studies investigating the potential role of small RNAs in this process could expand our understanding of the importance of small RNAs in senescence.In another study, Schommer et al.  reportedhenotype . They idn et al.  reportedcis- and trans-acting factors that activate the transcription of SAGs, it has been reported that epigenetic mechanisms can regulate plant senescence via DNA methylation, histone modification, ATP-dependent chromatin remodeling, and non-coding RNAs [Arabidopsis, by studying other plant species, one could investigate (4) if epigenetic mechanisms of senescence may be conserved among plant species.Senescence is a very important developmental phase in plants which has to be in coordination with other developmental phases, and it also needs to synchronize with environmental signals . In planing RNAs , and difing RNAs ,114,115.


Oncogenic human papillomavirus (HPV) genomes are often integrated into host chromosomes in HPV-associated cancers. HPV genomes are integrated either as a single copy or as tandem repeats of viral DNA interspersed with, or without, host DNA. Integration occurs frequently in common fragile sites susceptible to tandem repeat formation and the flanking or interspersed host DNA often contains transcriptional enhancer elements. When co-amplified with the viral genome, these enhancers can form super-enhancer-like elements that drive high viral oncogene expression. Here we compiled highly curated datasets of HPV integration sites in cervical (CESC) and head and neck squamous cell carcinoma (HNSCC) cancers, and assessed the number of breakpoints, viral transcriptional activity, and host genome copy number at each insertion site. Tumors frequently contained multiple distinct HPV integration sites but often only one “driver” site that expressed viral RNA. As common fragile sites and active enhancer elements are cell-type-specific, we mapped these regions in cervical cell lines using FANCD2 and Brd4/H3K27ac ChIP-seq, respectively. Large enhancer clusters, or super-enhancers, were also defined using the Brd4/H3K27ac ChIP-seq dataset. HPV integration breakpoints were enriched at both FANCD2-associated fragile sites and enhancer-rich regions, and frequently showed adjacent focal DNA amplification in CESC samples. We identified recurrent integration “hotspots” that were enriched for super-enhancers, some of which function as regulatory hubs for cell-identity genes. We propose that during persistent infection, extrachromosomal HPV minichromosomes associate with these transcriptional epicenters and accidental integration could promote viral oncogene expression and carcinogenesis. One factor that can contribute to oncogenic progression of HPV-positive lesions is integration of the viral genome into host chromatin. Integration is associated with increased genetic instability in high-grade cervical intraepithelial neoplasia, and cervical and oropharyngeal carcinomas9 due to dysregulated expression of the viral oncoproteins, E6 and E7. Many studies have compared the human genomic regions associated with HPV integration sites to elucidate the mechanisms that might promote integration and carcinogenesis. Here we have curated datasets from The Cancer Genome Atlas and other published sources to define a rigorous database of integration breakpoints and correlated these with “in-house” datasets of common fragile sites and enhancer elements defined in cervical carcinoma cells.Persistent infection with high-oncogenic risk human papillomavirus (HPV) types is responsible for almost all cervical and ~70% oropharyngeal carcinomas13. Common fragile sites are regions of the genome that have difficulty completing replication and, as such, are susceptible to chromosome breakage in mitosis. They are prone to replication stress that can be due to a shortage of replication origins or clashes between replication and transcriptional processes. Therefore, they vary in fragility depending on cell type and disease state14. Most previous studies that documented an association of HPV integration sites with common fragile sites used the classical FRA (fragile) regions that were defined cytogenetically in lymphocytes15. As such, these fragile sites are not cell-type-specific and are often large, poorly defined regions that cover a large proportion of the human genome. FANCD2 is required for resolution of these genetically unstable sites and, as such, is a marker of common fragile sites18. Fragile sites are often still undergoing DNA synthesis during mitosis and novel datasets have recently been generated by analysis of nascent DNA synthesis in mitotic cells20. Here we use published datasets of mitotic DNA synthesis (MDS) in HeLa cells, as well as our own “in-house” datasets to define common fragile sites in cervical cancer cells.Integration of HPV DNA occurs in all human chromosomes; however, integration sites are often found within or in close proximity to common fragile sites15. Co-amplification of the viral genome and flanking cellular sequences can result from unlicensed initiation of replication at the viral origin resulting in endoreduplication23. Subsequent recombination can result in amplified tandem repeats. Genome amplification can also occur at common fragile sites by breakage-fusion-bridge cycles24.HPV integration sites occur frequently in amplified regions of the host genome and focal amplification of cellular flanking sequences at sites of viral integration are frequently observed in HPV-positive tumors25. We previously identified an HPV16 integration site in the W12 20861 cervical cell line that was adjacent to a cell-type-specific enhancer. Co-amplification of this regulatory element and the viral genome to ~25 copies resulted in the formation of a super-enhancer-like element to drive high viral oncogene expression27. This “enhancer-hijacking” is a novel mechanism by which HPV integration can promote oncogenesis; however, it is unclear how common this mechanism is in HPV-associated cancers.HPV integration is also enriched at transcriptionally active regions of the host genome28 to generate a common fragile-site dataset in cervical carcinoma cell lines using higher resolution mapping of FANCD2 binding and have mapped cellular enhancers and super-enhancers in an HPV16-positive cell line derived from a cervical lesion29 using H3K27ac and Brd4 chromatin immunoprecipitation sequencing (ChIP-seq)  in our CESC dataset and compared them to previously defined hotspots from the literaturep = 0.004). Higher numbers of clustered breakpoints at sites of recurrent integration suggests that these regions are susceptible to genomic instability.The distribution of clustered breakpoints at each integration locus and across each chromosome is shown in Fig. 37. To identify driver versus passenger integrations, the transcriptional activity of each integration locus was determined for the subset of samples that had matched RNA sequencing data . The transcription status of each integration locus is indicated in Supplementary Data Tables 2 and 3. Integration loci in which no viral–host chimeric junctions were detected by RNA sequencing (RNA-seq) analysis were classified as inactive or passenger loci. In CESC, all samples with a single integration locus (n = 86) were transcriptionally active , more than one transcriptionally active integration locus was observed in 35 (60.3%) CESC and 4 (30.8%) HNSCC tumors. Three HNSCC samples had no driver integrations; one sample (4.5%) had a single integration locus and two samples (15.4%) had multiple integration loci . In total, we defined 513 FANCD2-enriched regions between the two cervical carcinoma cell lines and they are listed in Supplementary Data Table Genomic instability occurs frequently at common fragile sites. We previously used FANCD2 ChIP-chip to map fragile sites in an aphidicolin-treated cervical carcinoma cell line (C33-A)p = 0.0634), which reflects differences in replication stress at these regions in different cell types.We compared our cervical carcinoma cell line derived FANCD2 mapped fragile sites (genomic coverage 7.9%) to the 77 aphidicolin-induced common fragile sites (FRA regions) defined cytogenetically in lymphocytes and reported in the HGNC (HUGO Gene Nomenclature Committee) database (genomic coverage 48.3%) Fig. . A total20 and show that they colocalize with FRA regions and FANCD2 foci in cells treated with aphidicolin. We compared the overlap between our dataset of FANCD2-enriched regions and the mitotic DNA synthesis regions profiled in HeLa cells  of mitotic DNA synthesis regions . Mitotic DNA synthesis regions that overlapped with FANCD2-associated fragile sites are indicated in Supplementary Data Table Recent studies used high-resolution MiDA-seq (next-generation sequencing of EdU incorporation at sites of mitotic DNA synthesis) to map fragile sites in HeLa cellsons Fig. . Permuta44. We and others have previously shown that FANCD2-enriched regions overlap with transcriptionally active long genes in C33-A28 and U2OS cells45. Here we extended that association to include genes that are >0.3 Mb in length46. A total of 184/513 (35.9%) FANCD2-enriched regions overlapped with protein-coding genes that were ≥0.3 Mb, which corresponded to 185/782 (23.7%) long genes. A Fisher’s exact test was used to determine the significance in overlap between FANCD2-enriched regions and long genes . Of the long genes that overlapped with FANCD2-enriched regions, 121/185 (65.4%) were expressed in C33-A and/or HeLa cells . Example alignments of our FANCD2-associated fragile sites relative to common FRA regions, mitotic DNA synthesis regions and long genes are shown in Fig. The instability of fragile sites is often due to transcription-replication conflicts that frequently occur at long geneslls Fig. , further9 of the C33-A and HeLa FANCD2-enriched regions  W12 Brd4/H3K27ac enriched peaks overlapped ENCODE defined enhancers  were common across CESC and HNSCC. Gene Ontology analysis of our enhancer regions identified epithelium development, epithelial cell differentiation, and negative regulation of keratinocyte differentiation to be significantly enriched biological processes associated with CESC integration loci ots Fig. . Twelve nes Fig. . For HNS55. We defined 338 super-enhancers in W12 cervical keratinocytes (Supplementary Data Table p < 0.0001). For HNSCC, 8/57 (14%) integration loci were associated with W12 super-enhancers and 62.5% of these loci overlapped integration hotspots profiled in CESC, including the KLF5/KLF12, MYC, ERBB2, and VMP1 gene loci. Permutation testing showed that HNSCC integration loci were significantly associated with super-enhancers profiled in W12 cells (p < 0.0001). Thus, keratinocyte-specific super-enhancers are enriched at integration loci in HPV-associated tumors and are frequently found at integration hotspots.Large enhancer clusters were characteristic of the integration targets identified through GREAT analysis. We therefore defined super-enhancers in our Brd4-defined W12 enhancer dataset based on relative peak height of the H3K27ac and Brd4 ChIP-seq datasets using the Rank Ordering of Super-Enhancers (ROSE) toolASXL1, CACNA1A, IRF6, KANSL1, KLF5, KRT222, MYC, PPM1D, PTCH1, and PTPDC156 were located within 1 Mb of super-enhancers that overlapped with integration hotspots  relative to those that had either a normal genomic profile  or deletions within the host DNA flanking sequences  Fig. . Of the 25. Here we combined and curated DNA sequencing and RNA-seq datasets from HPV-positive CESC and HNSCC tumors and compared them with novel “in-house” datasets of common fragile sites defined by FANCD2 ChIP-seq, and enhancers and super-enhancers defined by Brd4 and H3K27ac ChIP-seq in cervical carcinoma-derived cells. We show that viral integration sites in CESC are enriched at FANCD2-associated fragile sites in cervical cells. We also show that cervical cell enhancers are overrepresented at HPV integration sites and that HPV integration is often associated with super-enhancers, particularly at integration hotspots enriched for cell-identity genes. Furthermore, we show that the flanking host DNA that is enriched for enhancers and super-enhancers is frequently amplified in CESC tumors.Many studies have documented the “landscape” of HPV integration sites with respect to traditional common fragile sites, host genome amplification, and transcription and related regulatory elements57. At different stages of infection, the viral DNA associates with nuclear ND10 bodies and interphase and mitotic host chromatin, and highjacks the DNA damage repair processes to amplify viral DNA59. Concomitantly, the viral E6 and E7 proteins induce cell proliferation and replication stress, abrogate cell cycle checkpoints, and inhibit the innate immune response60. E6 and E7 proteins also modify the epigenetic landscape of the host genome by changing the levels of different histone-modifying enzymes61. Together, these activities could promote the accidental integration of viral DNA that is closely associated with host chromatin.HPV genomes replicate as extrachromosomal nuclear minichromosomes at every stage of the infectious cycle. The virus relies on the host replication and transcriptional machinery and it is thought that the HPV genome localizes to regions of the nucleus that facilitate these processes63. The formation of tandem repeats at integration sites could be related to these processes and over-replication of viral and host sequences could result from repeated initiation of replication at the viral replication origin, especially if the HPV E1 and E2 proteins are expressed64. In fact, unscheduled firing of replication origins and increased replication fork stalling has been shown to occur in both viral and host sequences at HPV integration sites in the MYC locus22, which is frequently amplified in HPV-associated cancers. Tandem repeating units of co-amplified viral and cellular DNA could result from this endoreduplication, replication fork arrest, and homologous recombination. Highly rearranged integrations are also consistent with the breakage-fusion-bridge–type model of genome amplification21. At fragile sites, perturbed replication dynamics could also generate focal amplifications and/or rearrangements of viral–host sequences.HPV genomes replicate using two different modes: in maintenance replication the genomes replicate bidirectionally at low copy number, but this switches to a unidirectional recombination-directed mechanism in the amplification stage66 likely due to replication stress that arises from perturbed replication dynamics in conflict with transcription of long genes67. Accordingly, our C33-A and HeLa common fragile sites were overrepresented at long genes expressed in these cells. We did not observe an enrichment of HPV integration sites in HNSCC samples with our FANCD2-associated common fragile sites, although an association was previously noted between traditional FRA regions and integration sites in oropharyngeal squamous cell carcinomas10. This difference could reflect the larger genomic coverage of FRA regions used in the previous study, or the limited number of samples in our HNSCC dataset. Moreover, common fragile sites are likely distinct in cervical and oropharyngeal derived keratinocytes, or alternatively these findings could reflect differences in the biology of HPV infection and mechanisms of oncogenic progression in the different tissue types.In this study, we generated a dataset of aphidicolin-induced common fragile sites in two cervical carcinoma cell lines, C33-A and HeLa, and found a significant association between these sites and integration breakpoints in CESC, particularly at those loci with clustered breakpoints. Common fragile sites are susceptible to somatic copy number alterations68 and we previously described an example of enhancer-hijacking and co-amplification of cellular and viral regulatory sequences at an HPV integration site in cervical lesion derived cells27. The association of viral integration breakpoints with putative enhancer regions in HPV-associated cancers has been reported15, but the enhancer regions used were based on ENCODE histone modifications and therefore did not reflect the specific enhancer profiles of HPV-positive cervical cells. Here, we defined keratinocyte enhancers in HPV16-positive W12 cervical keratinocytes by H3K27ac and Brd4 enrichment and show that these specific enhancers are significantly overrepresented at HPV integration loci. In some cases, these loci were associated with focal amplification of host DNA, providing evidence for potential enhancer-capture. A recent study showed enrichment of active histone marks at HPV integration loci in cervical tumors, which correlated with upregulation of local gene expression, and increased gene expression levels at loci with increased breakpoints69. Kamal et al.38 also found increased local host gene expression at loci with multiple junction copies . Furthermore, integration loci with associated somatic copy number alterations have also been shown to have increased gene expression31. These observations could represent enhancer-hijacking. The three-dimensional interactions between host and viral DNA at integration loci can also perturb local and long-range cellular gene expression70, and can also result in enhancer-hijacking in HPV-associated cancer71. Integration hotspots often contain genes that drive cancer56 and accidental integration at these regions could result in disruption of proximal and distal regulatory regions, clonal expansion and selection due to perturbation of these oncogenic pathways. Thus, enhancer-hijacking can drive expression of both viral and cellular genes.HPV integration often occurs in transcriptionally active chromatin within the host genome55. We defined super-enhancers in our W12 cervical cell line datasets and showed that they were strongly associated with integration hotspots, including the MYC, KLF5/KLF12, and ERBB2 gene loci, which are important regulators of cell cycle, proliferation, and apoptosis74; TP63, which is a master regulator of epidermal keratinocyte proliferation and differentiation75; and RAD51B, which is a key regulator of homologous recombination repair76. We propose that, during persistent infection, extrachromosomal HPV genomes specifically localize at key transcriptional regulatory hubs within the host genome, several of which are important for keratinocyte biology.Super-enhancers are large clusters of enhancers, rich in Brd4 binding and H3K27ac modification, which often control cell-identity genes and are coopted in tumorigenesis77. Brd4 is also important at multiple stages of the HPV infectious cycle78, binds to common fragile sites in C33-A cells28, and is important for tethering HPV genomes to mitotic chromatin80. Brd4 is enriched at the HPV16 integration site/super-enhancer in W12 cells and inhibition of Brd4 binding reduces E6/E7 transcription and cell growth26. As such, inhibitors against Brd4 have great therapeutic potential in HPV-associated cancers. Therefore, Brd4 is an example of a factor that is crucial for key cell and viral chromatin-related processes and the juxtaposition of these processes could promote integration of viral DNA and oncogenic progression.The cellular Brd4 protein is involved in many of the cellular and viral processes described in this study. Brd4 is a chromatin scaffold protein that modulates transcriptional initiation and elongation and is a major component of super-enhancers37). In CESC-derived cells, expression of the viral E6 and E7 oncoproteins is necessary for cell proliferation, survival, and maintenance of the tumor phenotype81. Therefore, integration is common, but requires the right genomic location for constitutive viral oncogene expression. For example, the viral oncogenes are usually expressed from a viral–host fusion transcript that requires a splice acceptor and polyadenylation signal in the flanking host DNA83. Most likely, most integration events do not lead to dysregulated viral oncogene expression and many that do are silenced by DNA methylation84. Therefore, HPV integrants require a combination of events and processes that are dependent on the genetic and/or epigenetic landscape of the flanking host chromatin to drive oncogenesis.In conclusion, many factors contribute to the integration of a viral genome that eventually drives oncogenesis. Cancer genomes often contain multiple HPV integration sites but usually only one is transcriptionally active , DIPS (detection of integrated papillomavirus sequences), and next-generation sequencing technologies. The use of hybrid-capture technologies for detection of viral integration sites has been reported to give high rates of false positives36 and so insertion breakpoints identified by this method were only included if they were validated by other means, such as Sanger sequencing. The methodology used to identify each integration breakpoint is referenced in Supplementary Data Tables 2 and 3. RNA-based sequencing methods give an approximation of the insertion site based on the closest splice acceptor site within the host genome; therefore, integration breakpoints identified by RNA-seq and/or APOT were only used to determine the viral transcription status of an integration site for samples with matched DNA sequencing data. For The Cancer Genome Atlas (TCGA) CESC and HNSCC samples, unmapped reads were extracted from RNA-seq, whole genome sequencing (WGS), and whole exome sequencing BAM files  and pre-processed with prinseq-lite.pl version 0.20.2 to remove low-quality reads85. Pre-processed reads were mapped with Bowtie 2 using the very-sensitive preset option against the Viral Refseq database87. All unmapped reads were subjected to BLASTN with default parameters against the Viral Refseq database88. All aligned reads were then subjected to BLASTN against the human hg19 reference assembly. Bowtie 2 and BLASTN reports were passed into SummonChimera using a 1000 bp deletion size for integration detection89. The SummonChimera reports were manually parsed to remove chimeric junctions with lower than 20 read coverage, chimeric junctions with no cross-analysis verification, and ambiguously reported integration predictions. Finally, a unique ID was provided to all uniquely detected chimeric junctions. For analysis of the association of integration breakpoints with different genomic features of interest, only integration breakpoints identified by DNA sequencing methods were used. The characteristics of samples included in this study, categorized by histology type, HPV type, tumor location, and sequencing methods are summarized in Supplementary Fig. A systematic literature review identified genomic datasets from HPV-positive CESC and HNSCC that contained information on HPV type and integration breakpoints within the host and viral genomes, which were identified by sequencing  were used to define sites of recurrent integration. Integration hotspots defined from our CESC dataset and previously in the literature are listed in Supplementary Data Tables 30. Matched SNP6 copy number segment data for TCGA CESC and HNSCC tumor samples were downloaded from the Broad Institute on 04/07/2020, http://firebrowse.org/ (genome_wide_snp_6-segmented_scna_minus_germline_cnv_ hg19)91. Positive and negative mean segment values above 0.3 and below −0.3 represented copy number gains and losses, respectively, and mean segment values between 0.3 and −0.3 were considered noise91. Deletions that occurred within 50 Kb of an integration breakpoint were included in this analysis. Deletions that directly overlapped an integration breakpoint were excluded; these likely reflected the alternative chromosome as sequencing of the chimeric viral–host junction was available for the associated HPV insertion sites. Integration loci defined as having associated somatic copy number alterations in CESC and HNSCC are detailed in Supplementary Data Tables The amplification status of the cellular sequences flanking integration breakpoints had been assessed in a subset of samples by comparative genomic hybridization (CGH) or SNP array datasets. For CGH array data, we defined twofold or more focal amplification or deletion of the host genome at an integration locus as having an associated somatic copy number alteration92  were maintained in F-medium . All cells were grown in the presence of irradiated 3T3-J2 feeder cells. C33-A and HeLa cervical carcinoma-derived cell lines were purchased from ATCC and maintained in Dulbecco’s modified Eagle’s medium, supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. To induce replication stress, C33-A and HeLa cells were treated for 24 h with 0.2 µM aphidicolin (Sigma A0781) prior to collecting for FANCD2 ChIP-seq experiments, described below.Subclones  derived from HPV16-positive W12 cervical keratinocytes26. Briefly, cells were crossed-linked with 1% formaldehyde and chromatin was isolated and sheared to 100–500 bp DNA fragments using a Bioruptor sonicator (Diagonode) on high power settings. Chromatin samples (25 μg per ChIP) were incubated overnight at 4 °C with an antibody against FANCD2 . Rabbit IgG  was used to determine nonspecific binding to control regions . Chromatin immunocomplexes were precipitated for 1 h at 4 °C with blocked Dynabeads Protein G (Invitrogen), subjected to multiple wash steps and the chromatin eluted in elution buffer . Chromatin was reverse cross-linked overnight at 65 °C in 0.2 M NaCl, followed by RNase A and proteinase K treatment, and the DNA purified using the ChIP DNA Clean & Concentrator kit (Zymo Research). ChIP DNA from two biological replicates were pooled and subjected to 2 × 150 bp paired-end read sequencing on the Illumina HiSeq-4000 platform  to a sequencing depth of >14 million reads per sample. FANCD2 ChIP-seq datasets are accessible through GEO Series accession number GSE183048.Aphidicolin-treated C33-A and HeLa cells were processed for ChIP as previously described27. However, only the sequence data flanking the HPV16 integration sites was previously analyzed and published. Here we analyze the same dataset but for the entire human genome. Antibodies used were Brd4  or H3K27ac . No antibody controls were included to monitor nonspecific binding. Brd4 and H3K27ac ChIP-seq datasets are accessible through GEO Series accession number GSE183048.W12 chromatin samples were isolated as described above, and have been described previously93. All reads aligning to the ENCODE hg19 v1 blacklist regions94 were identified by alignment with BWA version 0.7.1795 and removed with Picard SamToFastq, https://broadinstitute.github.io/picard/. Remaining reads were aligned to an hg19 reference genome using BWA. Reads with a mapQ score less than 6 were removed with SAMtools version 1.696 and PCR duplicates were removed with Picard MarkDuplicates. Peaks were called by comparing each ChIP sample to its matching input sample. For FANCD2, the mean fragment size was estimated by Phantompeakqualtools version 2.097. Peaks were called using SICER version 1.198 with the following parameters: redundancy threshold of 100, effective genome fraction of 0.75, window size of 25,000 bp, and gap size of 50,000 bp. H3K27ac and Brd4 peaks were called using macsBroad (macs version 2.1.1 from 09/03/2016)99 with the following parameters: -broad-cutoff 0.01 -f “BAMPE”. Data was converted into bigwigs for viewing and normalized by reads per genomic content (RPGC) using deepTools version 3.0.1100 using the following parameters: -binSize 25 -smoothLength 75 -effectiveGenomeSize 2700000000 -centerReads -normalizeUsing RPGC. RPGC-normalized input values were subtracted from RPGC-normalized ChIP values of matching cell-type genome-wide using Deeptools with -binSize 25.Reads were trimmed with Cutadapt version 1.18q-value of 10 or above, to remove low-confidence calls. Filtered C33-A ChIP-seq peaks were combined with previously mapped aphidicolin-induced FANCD2 peaks identified by ChIP-chip in these cells28. C33-A  database (https://www.genenames.org/download/custom/) on 27/08/2020 using advanced filtering: gd_locus_type = ‘fragile site’.FANCD2 ChIP-seq peaks were filtered by a −log10 The Gencode Release 19 human reference genome (GRCh37) was filtered for protein-coding genes greater than or equal to 0.3 Mb in length, including untranslated regions 52. ENCODE GRCh38 enhancer files were converted to hg19 using the UCSC Genome Browser LiftOver tool, http://genome.ucsc.edu/cgi-bin/hgLiftOver. W12 enhancers were compared to ENCODE NHEK enhancers using regioneR102.Consensus peak sets for H3K27ac were defined as overlapping regions found in at least four out of eight W12 samples using DiffBind9) with enhancers . Samples with multiple reported breakpoints within the same chromosome were classified as a single integration locus if the 5′ and 3′ most breakpoints were within 3 Mb of each other. This 3 Mb cutoff was based on manual analysis of the distance between clustered breakpoints identified by WGS and/or hybrid-capture technologies for the CESC and HNSCC datasets. Each integration locus was assigned a unique integration ID so that the number of breakpoints per integration could be categorized as a cluster. Each integration breakpoint was analyzed independently for their association with the genomic feature of interest, as well as with adjacent HPV breakpoints, which were classified as belonging to the same integration locus/cluster. For significance testing, the data were permutated 10,000 times to create an expected distribution of the overlap between integration breakpoints and loci with FANCD2-enriched regions or enhancers using regioneR102.The intersect between the genomic coordinates of HPV integration breakpoints  were analyzed using the GREAT using default parameters, accessed July 2021, Further information on research design is available in the Supplementary InformationSupplementary Data 1-17Reporting Summary


This is not true for other functions of VDR such as intestinal calcium transport. Tumor formation in the skin after UVB radiation or the application of chemical carcinogens also appears to be at least partially independent of 1,25(OH)2D in that Vdr null mice develop such tumors after these challenges, but mice lacking Cyp27b1, the enzyme producing 1,25(OH)2D, do not. Examples in other tissues emerge when studies comparing Vdr null and Cyp27b1 null mice are compared, demonstrating a more severe phenotype with respect to bone mineral homeostasis in the Cyp27b1 null mouse, suggesting a repressor function for VDR. This review will examine potential mechanisms for these ligand‐independent actions of VDR, but as the title indicates, there are more questions than answers with respect to this role of VDR. © 2021 The Author. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.Our predominant understanding of the actions of vitamin D involve binding of its ligand, 1,25(OH)D, to the vitamin D receptor (VDR), which for its genomic actions binds to discrete regions of its target genes called vitamin D response elements. However, chromatin immunoprecipitation‐sequencing (ChIP‐seq) studies have observed that the VDR can bind to many sites in the genome without its ligand. The number of such sites and how much they coincide with sites that also bind the liganded VDR vary from cell to cell, with the keratinocyte from the skin having the greatest overlap and the intestinal epithelial cell having the least. What is the purpose of the unliganded VDR? In this review, I will focus on two clear examples in which the unliganded VDR plays a role. The best example is that of hair follicle cycling. Hair follicle cycling does not need 1,25(OH) 2D, where it binds to a VDRE. When ligand bound, the RXR/VDR heterodimer activates genes promoting calcium transport, hormone secretion, and immune regulation, among other functions. In the absence of 1,25(OH)2D, the VDR/RXR heterodimer activates genes regulating hair follicle cycling and skin tumor suppression. This simplified model omits many of the processes underlying the mechanisms by which these events occur.This model depicts the movement of the RXR/VDR from the cytoplasm into the nucleus, a step that can occur with or without 1,25(OH) This translocation of the VDR from the cytoplasm into the nucleus is highly regulated at least in part by retinoid X receptors (RXR) and is not necessarily ligand dependent. In particular, RXR, which forms a heterodimer with VDR in the cytoplasm, is capable of translocating VDR into the nucleus even in the absence of 1,25(OH)2D as long as the nuclear localization signal (NLS) of the RXR is intact. However, this translocation of the unliganded heterodimer is lost when the NLS of RXR is mutated but is regained with the addition of 1,25(OH)2D, which promotes the translocation of the RXR/VDR heterodimer regardless of whether the RXR NLS was intact as long as the NLS of the VDR is functional. But translocation into the nucleus is only half the story as the VDR can cycle in out of the nucleus again regulated by RXR, which reduces the export of the unliganded VDR from the nucleus but accelerates it in the presence of 1,25(OH)2D. That said, a significant number of sites in the genome appear to bind VDR in a persistent rather than transitory fashion.Nearly all known actions of the vitamin D receptor (VDR) involve its partnership with the active metabolite of vitamin D, 1,25(OH)ependent.1 In p 1,25(OH)2D increases VDR binding on average about ×2.5, but a substantial number of sites to which VDR bound do not require ligand. Of the ligand‐dependent VDR binding sites, most were direct repeats of two RGKTSA motifs  separated by three nucleotides , whereas the sites to which ligand was not required for VDR binding were predominantly non‐classical motifs. Although the VDR is associated with a variety of complexes that alter its function, it is not clear that these complexes are distinctly different with respect to the type  binding site involved. I will refer to both the classical and non‐classical VDR binding sites as VDREs. However, it should not be inferred that all VDREs so identified are regulatory in a given cellular context as well demonstrated when the VDREs of theTnfsf11 (Rankl) gene in cells of the mesenchymal and hematopoietic lineages are compared, showing that the VDREs regulating Tnfsf11 expression differ between the two cell types. The extent to which ligand‐independent VDR binding occurs in a given genome is highly cell specific. For example, from chromatin immunoprecipitation studies of THP‐1 human monocytic leukemia cells, we learn that of the 1318 VDR binding sites, 789 showed an increase in VDR binding with 1,25(OH)2D administration, but 364 did not, and only 165 of these sites overlapped. In MC3T3E1 cells, VDR binding sites increased from 1325 to 8241 after 3 hours of 1,25(OH)2D administration. Even more extreme is the example of LS180 colon cancer cells in which VDR binding sites with vehicle alone numbered 262 but increased to 2209 with partial overlap after the addition of 1,25(OH)2D. On the other hand, in preliminary observations, we (Oda and colleagues) have found that 1,25(OH)2D has much less effect on VDR binding in the keratinocyte genome with 878 binding sites noted with vehicle, 929 sites after 1,25(OH)2D administration, and almost complete overlap. Although these numbers are dependent to a large extent on the experimental conditions of the analyses, they illustrate variability of ligand dependence/independence in different cell types. This raises the question of what function VDR is having on the genome in the absence of 1,25(OH)2D. Moreover, does the much greater percentage of ligand‐independent binding of VDR in the keratinocyte genome relative to the intestinal epithelial cell, osteoblast, or monocyte provide clues to the best characterized ligand‐independent actions of VDR—namely hair follicle cycling and resistance to UVB or chemically induced skin cancer? In this review, I will examine the role of VDR in hair follicle cycling and cancer resistance along with the concept that the unliganded VDR may regulate gene transcription in a manner different from what the liganded receptor would do. An important qualification is that throughout this review I will use the term unliganded VDR to mean independent of 1,25(OH)2D. There are other VDR ligands that may substitute for 1,25(OH)2D at least in some of its functions. Moreover, a full discussion of coregulators of VDR, both coactivators and corepressors, is outside the scope of this review, albeit quite important for the function of VDR.Analysis of data sets from several different cell types indicates more than 23,000 unique VDR binding loci in the human genome.4 1,25 After the developmental cycle, which leads to the initial coat of hair, the follicle undergoes repetitive cycling until senescence. Growth of the follicle occurs during anagen. The length of the hair is dependent on the duration of anagen. During this stage, the follicle grows through the dermis into the subcutaneous tissue. As the follicle develops, different cell layers appear. The outer root sheath (ORS) is a direct extension of the stratum basale, the basal layer of the epidermis, and separates the hair follicle from the surrounding connective tissue sheath (CTS). From outside in are found the companion layer, the three layers of the inner root sheath (IRS)—Henle's layer, Huxley's layer, cuticle of the IRS—and the hair shaft itself, including the cuticle of the shaft, shaft cortex, and shaft medulla. Stem cells in the bulge are capable of generating all cells in the hair follicle and epidermis, although stem cells reside in the stratum basale and infundibulum, where under normal circumstances they regulate growth and regeneration of the epidermis and sebaceous gland, respectively. These stem cells all express VDR. The keratins produced by the cells of the IRS and hair shaft differ from those expressed by epidermal keratinocytes. Of particular interest is these hair keratins have β‐catenin/lef1 binding sites in their promoters that regulate their expression. After anagen, the follicle enters catagen, during which massive apoptosis occurs primarily in the cells of the proximal follicle , and the hair shaft produced during anagen is generally shed. The distal portion of the follicle  remains intact during hair follicle cycling. At the end of catagen, the follicle enters telogen, the resting phase. A new cycle then begins with anagen. The juxtaposition of the dermal papilla to the bulge is critical for this process to begin, and it is associated with increased proliferation of stem cells in the bulge with migration of cells from the bulge into the hair bulb to restart the growth of the hair follicle. The regulatory elements that control the transition from one stage to the next are not well understood.The hair follicle cycle is divided into three main stages: anagen, catagen, and telogen. The duration of these stages in a given species varies from location to location on the body and between sexes. Furthermore, there are two types of cycles: developmental and postnatal. The developmental cycle is initiated during embryogenesis. The follicle develops from specific regions of the epidermis called placodes. The development, number, and placement of these placodes are under the control of a number of factors but not VDR. The follicle is induced to grow by its interaction with a collection of specialized mesenchymal cells in the dermis called the dermal papilla. Wnt signaling (β‐catenin) appears to be necessary to maintain the ability of the dermal papilla to stimulate hair follicle growth.10 AftVDR, a syndrome currently known as hereditary vitamin D‐resistant rickets (HVDRR). In that vitamin D deficiency per se is not associated with alopecia, the explanation for this phenomenon has remained obscure. Exploration of the link between alopecia and VDR received a major boost with the development of the Vdr null mouse by several groups. These mice develop their first coat of hair normally, but reinitiation of anagen after the first cycle or after depilation is impaired. Reconstitution of the Vdr to the Vdr null mouse skin using a keratinocyte‐specific promoter reverses the defect in hair growth without reversing the metabolic defects of skeletal growth retardation, hypocalcemia, and rickets otherwise associated with the Vdr null condition. Moreover, hair follicle cycling can even be restored when the Vdr is mutated in the ligand binding domain (L233S) or the AF2 domain (L417S) such that it no longer binds 1,25(OH)2D or is activated by it. Furthermore, inactivating mutations in Cyp27b1, the enzyme producing 1,25(OH)2D, do not result in alopecia, although the other metabolic defects  found in Vdr knockout (KO) occur. On the other hand, correction of the metabolic abnormalities with a high calcium rescue diet prevents the rickets and hyperparathyroidism but does not prevent the alopecia. Furthermore, it is the lack of VDR in the keratinocyte as opposed to the dermal papilla that is critical. Dermal papilla cells obtained from either Vdr null or wild‐type mice can initially induce hair growth in a hair reconstitution assay when mixed with epidermal keratinocytes obtained from wild‐type or Vdr null mice, but if the hair grown with keratinocytes from Vdr null mice is then depilated, anagen will not be reinitiated regardless of the source of dermal papilla cells.Alopecia is a well‐known part of the phenotype of many patients with mutations in their VDR,15 a sVdr null mice (and humans) is essentially identical to that found in mice and humans with mutations in or null for hairless (Hr), deletion of Rxrα and Rxrβ, transcriptional partners of Vdr, mutations in Ctnnb1 (β‐catenin) and its transcriptional partner Lef1, or overexpression of c‐myc, which is downstream of the β‐catenin pathway and inhibited by 1,25(OH)2D/VDR. In general, these conditions show disruption of hair follicle cycling during catagen of the first adult hair cycle, show a separation of dermal papillae from the receding hair follicle during this time, show a loss of ability to reinitiate anagen after the first hair follicle cycle, and show a disruption of sonic hedgehog (Shh) signaling in the bulge, which appears critical for the reinitiation of anagen. The dissociation of the dermal papilla from the hair bulb by the end of catagen is thought to account for the failure to initiate the subsequent anagen in both Hr mutant and Vdr null mice. Additional clues come from RNA‐seq data of keratinocyte stem cells from Vdr wild‐type and KO mice, which showed a number of genes that were upregulated in the KO cells, one of which was peroxisome proliferator‐activated receptor γ (Pparg). Haploinsufficiency of Pparg reduced the mRNA levels of this gene in Vdr null skin and restored hair growth in the Vdr KO. The concept here is VDR acts as a repressor blocking the expression of Pparg and other genes involved with the regulation of hair follicle cycling. A similar situation will be described for the hedgehog pathway and its role in skin cancer. The distal  portion of the hair follicle including the sebaceous gland as well as the interfollicular epidermis are likewise impacted. The large dermal cysts that develop with time contain markers of the differentiated interfollicular epidermis and sebaceous gland,) suggesting their origin from the distal portion of the hair follicle or epidermis, features similar to that found in Hr and Ctnnb1 mutant animals. We interpret these changes as due to altered cell fates in the stem cells that otherwise regulate not only the stem cells in the bulge controlling hair follicle cycling but also in the infundibulum of the hair follicle regulating sebaceous gland development and in the stratum basale regulating epidermal regeneration.The mechanism mediating this ligand‐independent action of VDR is not fully understood. Although we know many genes and pathways that are ligand‐dependent actions of VDR in the skin and elsewhere, we know almost nothing about this ligand‐independent action. Clues come from the observation that the abnormality in hair follicle cycling in ess (Hr),31 del and a large number of factors are implicated in this process. Disruption of canonical wnt signaling (Ctnnb1 or Lef1 mutations) disrupts both developmental and postnatal hair follicle cycling and is associated with loss of Shh expression. Both Hr null and Vdr null mice show loss of Shh expression in the hair follicle. As will be discussed, this differs from the effect of Vdr deletion from the epidermis or keratinocytes in vitro. The Hr null mouse shows an increase in expression of the wnt inhibitor WISE, whereas we have shown a reduction of Wnt4 in the Vdr null hair follicle. Wnt4 is normally expressed in the matrix and precortex and would be expected to have an important role in interactions with the dermal papilla. Although we have not observed an obvious difference in β‐catenin mRNA levels, its protein levels and nuclear localization are reduced in the ORS keratinocytes and bulb of the hair follicle in the Vdr null mouse compared with wild‐type at the end of catagen. In other cells, VDR has been found to bind to β‐catenin directly, reducing its interaction with Tcf4 or Lef1 and so reducing the transcriptional activity of β‐catenin. On the other hand, Lef1 binds to VDR in its N terminal region independent of β‐catenin but nevertheless important for wnt signaling. Therefore, function of β‐catenin rather than expression may be altered in Vdr null hair follicles. As mentioned, VDR binds to β‐catenin, repressing its transcriptional activity, although this binding is stimulated by 1,25(OH)2D3. However, some binding of VDR to β‐catenin occurs in the absence of ligand, and in cells such as keratinocytes with a lot of β‐catenin, this may suffice to regulate transcription. The C terminal region of β‐catenin (aas 671–781) and the AF‐2 (C terminus) domain of VDR are required for this binding. Mutation at L417S of the Vdr in mice blocks this binding as well as Vdr transcriptional activity. Surprisingly, the mutation E420Q, which also prevents transcriptional activity, is still capable of binding β‐catenin, and in the presence of high levels of β‐catenin, some transcriptional activity can be achieved. This mutation causes rickets but not alopecia, suggesting that the ability of VDR to bind β‐catenin possibly in the absence of 1,25(OH)2D may enable it to support hair follicle cycling.The control of hair follicle development and cycling is complex,40 and Hr has characteristics of a coregulator in that it resides in the nucleus; its structure contains a nuclear localization signal, a putative zinc finger, and three LXXLL motifs like that found in coactivators that interact with nuclear hormone receptors such as VDR in the presence of ligand as well as ΦXXΦΦ motifs (Φ = hydrophobic amino acid) similar to regions in corepressors like SMRT and NCoR responsible for the binding of these corepressors to nuclear hormone receptors in the absence of ligand. In the brain, Hr has been suggested as a corepressor of the thyroid receptor (THRb) in that Hr can bind to THRb and inhibit its transcriptional activity. However, Hr does not appear to regulate thyroid hormone action in the keratinocyte. Rather, VDR appears to be the target in keratinocytes. Hsieh and colleagues demonstrated that Hr could bind to VDR in COS cells. They noted that Hr bound to VDR in the same region predicted for corepressor binding, and different from the C‐terminal region to which coactivators bind. The region of Hr responsible for VDR binding contains one LXXLL motif but also a ΦXXΦΦ motif, and only mutations in the ΦXXΦΦ motif altered binding to VDR. However, when we tested both motifs separately for their binding to VDR, both did with comparable affinity. Neither motif was substantially affected by 1,25(OH)2D, consistent with the findings of others that Hr/VDR binding is ligand independent. Binding of Hr to VDR correlated with inhibition of 1,25(OH)2D3 stimulation of a Cyp24a1 (24‐hydroxylase) promoter construct containing the VDRE of this vitamin D target gene. We have shown that the endogenous VDR binds to endogenous Hr in keratinocytes. Overexpression of Hr blocks the ability of 1,25(OH)2D3 to induce differentiation markers in keratinocytes, whereas inhibition of Hr expression enhances the stimulation by 1,25(OH)2D3 of these markers. The Hr null animal demonstrates upregulation of differentiation markers in the epidermis  consistent with a corepressor role for Hr in vitamin D‐regulated epidermal differentiation. This difference between VDR and Hr in the epidermis emphasizes that the signaling pathways in epidermal differentiation are not the same as in hair follicle cycling and can be just the opposite. Antibodies to Hr enhance the binding of VDR to VDREs in vitamin D target genes in gel retardation assays, suggesting that Hr binding to VDR blocks its binding to VDREs. Using the chromatin immunoprecipitation assay (ChIP), we noted that 1,25(OH)2D3 displaced Hr from the VDREs tested, whereas 1,25(OH)2D3 recruited the coactivators DRIP205 (aka Med 1) and SRC3 to these same VDREs.We and others have found VDR and Hr expressed in the nuclei of keratinocytes in the stratum basale, ORS, and matrix of the hair follicle.53 Hr 2D3. That question remains.These data indicate that Hr and β‐catenin can alter VDR transcriptional activity. But the question remains how this translates into regulation of hair follicle cycling or epidermal differentiation that is independent of 1,25(OH)Vdr null mice develop skin tumors after UVB radiation or administration of the chemical carcinogen 7,12 dimethylbenzanthracene (DMBA) but mice lacking Cyp27b1 and so lacking 1,25(OH)2D production do not. That said, discerning mechanisms that are not also influenced by 1,25(OH)2D have not been performed in a consistent fashion.The ligand‐independent actions of VDR acting as a tumor suppressor in skin are less clear than that in hair follicle cycling. However, two publications demonstrate that n do not.60 Tha observed that 85% of the Vdr null mice but none of the controls developed skin tumors within 2 months of DMBA administration. These were primarily papillomas. These results have been confirmed using topical administration of DMBA/TPA. However, although only papillomas were observed in the Vdr null mice, Rxrα null mice developed both basal cell carcinomas (BCC) and squamous cell carcinomas (SCC). Subsequently, Ellison and colleagues and our own groupdemonstrated that Vdr null mice were also more susceptible to tumor formation after UVB, and many of the tumors were SCC and BCC, but Cyp27b1 KO mice were not. The appearance of BCC in these studies was initially surprising because the typical malignancy induced in mouse skin by UVR, ionizing radiation, or chemical carcinogens is SCC, not BCC. Given that BCC generally result from increased hedgehog (Hh) signaling and that lack of VDR results in BCC when β‐catenin signaling is increased, we became interested in the relationship between vitamin D, Hh, and β‐catenin signaling in tumor suppression. We also discovered that VDR regulated the expression of long non‐coding RNAs (lncRNAs) such that in the Vdr null mouse epidermis the balance between oncogenic and tumor suppressor lncRNAs was shifted to oncogenic species. Additionally, we noted a reduction in clearance in cyclobutane pyrimidine dimers (CPD) after UVB exposure of the skin of Vdr null mice, suggesting that disruption of DNA damage repair was playing a role in tumor susceptibility in these mice. Hr may also play a role in epidermal carcinogenesis as it does in hair follicle cycling in that mice with mutations in Hr are also quite susceptible to UVB‐induced skin cancer, but Hr has not been studied in its relationship to VDR during skin cancer protection. In what follows, I will examine potential mechanisms and pathways within those mechanisms for their contribution to the role of VDR as a tumor suppressor, including regulation of proliferation and differentiation with particular attention to the Hh and wnt/β‐catenin pathways, long non‐coding RNAs, and DNA damage repair.The potential for vitamin D signaling as protection against epidermal tumor formation was demonstrated when Zinser and colleagues61 obs2D increases essentially every step of the differentiation process in the epidermis, while inhibiting proliferation at least at concentrations above 1 nM. These actions complement those of calcium, but calcium at least in vitro does not require 1,25(OH)2D as keratinocyte differentiation is readily induced by calcium in serum‐free media. However, the calcium response is enhanced by 1,25(OH)2D via its induction of the calcium‐sensing receptor (CaSR), which along with induction of the phospholipase C enzymes regulates intracellular calcium and other signaling molecules critical for the differentiation process. On the other hand, at least some of these 1,25(OH)2D‐independent actions of calcium require VDR. An example of this is the effect of calcium on the translocation of E‐cadherin to the membrane after calcium administration, which is blunted in keratinocytes lacking the VDR. Thus, calcium stimulation of keratinocyte differentiation does not appear to depend on 1,25(OH)2D, but at least some of its actions require VDR in the absence of 1,25(OH)2D.1,25(OH)pidermis,75 whiVdr null mouse to tumor formation and that are at least partially regulated by VDR in a 1,25(OH)2D‐independent fashion are the Hh and wnt/β‐catenin pathways, pathways likely not coincidentally also playing important roles in hair follicle cycling.Two pathways important in vitamin D signaling in the epidermis with respect to proliferation and differentiation that we believe underlie the predisposition of the  When SHH binds to PTCH 1, the inhibition of SMO is relaxed, GLI1 and 2 are released from SUFU, and they move into the nucleus, where they initiate transcription of a number of factors, including each other as well as PTCH 1, the anti‐apoptotic factor BCL2, cyclins D1 and D2, E2F1, and CDC45 , while suppressing genes associated with keratinocyte differentiation, such as the keratins K1and K10, involucrin, loricrin, and the VDR.In the skin, SHH is the ligand for patched (PTCH) 1, a 12‐transmembrane domain protein that in the absence of SHH inhibits the function of another membrane protein smoothened (SMO). SMO in turn maintains a family of transcription factors, GLI1 and GLI2 in particular, in the cytoplasm bound to suppressor of fused (SUFU).84 Whe although activation of Hh signaling also predisposes to UVR‐induced SCC formation.Vdr null animals overexpress elements of the Hh signaling pathway in their epidermis and the epidermal portion (utricles) of the hair follicles. Moreover, 1,25(OH)2D suppresses the expression of all elements of the Hh pathway in a dose‐dependent fashion that requires the VDR and reduces tumor growth in Ptch 1 null mice. On the other hand, VDR in the absence of 1,25(OH)2D also has a suppressor effect on this pathway, in that keratinocytes in which the VDR is knocked down overexpress both SHH and GLI1. The promoters of SHH and GLI1 have binding sites for VDR suggesting that the effects of VDR on these genes is direct.The appearance of BCC is characteristic of tumors formed when Hh signaling is activated,90 alt important for proliferation. β‐catenin also forms part of the adherens junction complex with E‐cadherin, where it plays an important role in keratinocyte differentiation. Tyrosine phosphorylation of E‐cadherin, as occurs after calcium administration to keratinocytes, promotes the binding of β‐catenin and other catenins to the adherens junction complex making it less available for transcriptional activity. As noted earlier, calcium and 1,25(OH)2D increase E‐cadherin expression and its membrane localization in a VDR‐dependent fashion, although as noted this action of calcium does not require 1,25(OH)2D. Overexpression and/or activating mutations in the β‐catenin pathway lead to skin tumors, in this case pilomatricomas or trichofolliculomas (hair follicle tumors). VDR binds to β‐catenin and reduces the transcriptional activity of β‐catenin in a 1,25(OH)2D‐dependent fashion. On the other hand, binding of β‐catenin to VDR in its AF‐2 domain enhances the 1,25(OH)2D‐dependent transcriptional activity of VDR. Palmer and colleagues evaluated the interaction between VDR and β‐catenin in transcriptional regulation in keratinocytes and identified putative response elements for VDR and β‐catenin/LEF in a number of genes. These interactions were either positive or negative, depending on the gene being evaluated. The hypothesis put forward is that genes in which the interaction was positive  benefited from β‐catenin acting as a coactivator for VDR on VDREs, whereas in situations where the interaction was negative  VDR prevented β‐catenin from binding to TCF/LEF required for transcription in those genes. We have found in keratinocytes that knockdown of VDR reduces E‐cadherin expression and formation of the β‐catenin/E‐cadherin membrane complex, resulting in increased β‐catenin transcriptional activity, whereas 1,25(OH)2D administration has the opposite effect. This was associated with increased (with VDR knockdown) or decreased 2D administration) keratinocyte proliferation and cyclin D1 expression, providing evidence that VDR in the absence of 1,25(OH)2D might be acting to suppress this β‐catenin responsive gene.Wnt signaling via activation of β‐catenin has a complex role in VDR function. In the canonical pathway, the receptor for Wnt ligands is a family of seven‐transmembrane Frizzled receptors and an LRP5 or LRP6 coreceptor. When Wnt binds to this complex, disheveled (Dvl) is phosphorylated, resulting in disruption of the axin/APC complex and inhibition of glycogen synthase kinase 3β (GSK‐3β). In the basal state, GSK‐3β phosphorylates the serine(s) within exon 3 of β‐catenin, resulting in its degradation by the E3 ubiquitin ligase. Wnt signaling, by blocking this phosphorylation, increases the availability of β‐catenin in the nucleus, where it binds to transcription factors of the T‐cell factor (TCF) and lymphoid enhancer factor (LEF) families to promote expression of genes such as cyclin D1 and c‐myc,93 imp These non‐coding transcripts can be broadly categorized into short and long non‐coding RNAs. The arbitrary size delineation is at 200 bases in length: small non‐coding RNAs are less than 200 bases, whereas lncRNAs are endogenous cellular RNAs larger than 200 bases and can even be greater than 100 kb in length. LncRNAs account for 80% of the transcriptome; they are spliced and contain polyadenylation signals, much like messenger RNAs. LncRNAs are expressed across all mammalian genomes and have emerged as master regulators of embryonic pluripotency, differentiation, and body axis patterning, promoting developmental transitions and regulating histone modifications, hence influencing the epigenetic programs of the transcriptome. A number of these lncRNAs when aberrantly expressed are associated with cancers. We explored the potential role of lncRNAs in VDR protection against skin tumor formation by profiling 90 well‐annotated mouse lncRNAs from mouse keratinocytes cultured in vitro and mouse epidermis from epidermal‐specific VDR null mice and their normal littermates. We found that several well‐known oncogenes, including H19, HOTTIP, and Nespas, are significantly increased, whereas tumor suppressor lncRNAs  were attenuated in VDR deleted keratinocytes. These were serum‐free cultures indicating a 1,25(OH)2D independent action of VDR on these lncRNAs. A similar pattern of lncRNA‐expression profiling was observed in the epidermis of epidermal‐specific Vdr null mice versus control littermates. In addition to the altered lncRNAs  in VDR deleted human cultured keratinocytes, there was an increase in other oncogenes  and a decrease in other tumor suppressors  in Vdr null mouse epidermis. However, we have not documented direct effects of VDR on the genes expressing these lncRNAs or demonstrated their regulation by 1,25(OH)2D.Only about 2% of the genome is actively transcribed and translated into proteins, while a much larger percentage of the genome is actively transcribed without protein coding potential.100 Th DDR involves a cascade of damage recognition, repair, and signal transduction that coordinates the response of the cell to DNA damage. DDR activates checkpoints that delay the cell cycle, provides time for repair, and directs damaged cells into senescent or apoptotic pathways. DDR involves a number of components, is well orchestrated, tightly controlled, and highly accurate in normal primary cells such that the spontaneous mutation rate is very low, and changes in copy number are negligible. As noted earlier, UVB causes CPD and 6‐4PP formation, which are bulky adducts that block the movement of replicative DNA polymerase, a high‐fidelity enzyme, with a shift to trans lesion synthesis by lower‐fidelity DNA polymerases. Moreover, CPDs, if they occur in promoter regions, can block the binding of transcription factors. With malignant transformation, DDR becomes less controlled, and mutation rates and copy number abnormalities increase by orders of magnitude. Nucleotide excision repair (NER) is the principal means by which UVR damage is repaired, enabling repair before DNA replication begins. This is important as NER plays a major role in reducing the amount of damage that becomes fixed as mutations during replication. During NER, the DNA damage is recognized by XPC acting in a complex with hRAD23B supported in some cases by the DNA damage‐binding protein DDB1 and 2, the DNA is unwound around the lesion, and 30 base pair portions of DNA containing the lesion are excised by endonucleases such as XPF and XPG followed by fill‐in with DNA polymerases such as Pol δ,ε,κ.DNA damage response (DDR) is the means by which UVR and chemical‐induced DNA damage is prevented from producing fixed DNA mutations.106 DD: transcription coupled repair (TCR), during which DNA polymerases stop replication at the site of the lesion until it is repaired, and global genomic repair (GGR), during which non‐transcribed regions of the genome are repaired. UVB increases VDR levels in keratinocytes. Keratinocytes in the epidermis of mice lacking Vdr are deficient in DDR as demonstrated by a reduced rate of clearing CPDs and 6,4PPs after UVB. Although 1,25(OH)2D can promote DNA damage repair at least in part through non‐genomic mechanisms, this action appears to be at least partially 1,25(OH)2D independent as epidermal explants from mice lacking Vdr likewise show this defect when evaluated in vitro in serum‐free media.The NER process has two main branches involving different mechanisms for the initial recognition of DNA damage119: tVdr has been mutated to block 1,25(OH)2D binding in tissues other than the skin. These studies demonstrate that VDR, like other nuclear hormone receptors, can act as a regulator of gene transcription in the absence of its ligand. Lee and colleagues developed a knock‐in version of HVDRR in which the Vdr lacked the ability to bind 1,25(OH)2D (L233S mutation). In comparison to Vdr null mice, the mutant mice had higher PTH levels, comparable to Cyp27b1 null mice, which they also examined. The Cyp27b1 null mice also showed greater reductions in the intestinal expression of calcium transport genes, including Trpv6 and S100g  as well as Atp2b1  and Slc30a10 (zinc transporter) than did the Vdr null mice, but other target genes did not differ in their expression between the two knockout models. In a somewhat different model in which the Vdr mutation in the ligand binding domain (L307H) eliminated 1,25(OH)2D binding but maintained binding to the 1,25(OH)2D analog Gemini (Vdr gem), Huet and colleagues found a more severe bone mineral phenotype in the Vdr gem mouse than in the Vdr null mouse, including a greater reduction in serum calcium and phosphate, increased alkaline phosphatase, and decreased bone mineral. They also observed more downregulated genes in microarray studies, including Trpv6, Slc30a10, and Cyp24a1, comparable to the results by Lee and colleagues in their model. Earlier studies comparing Cyp27b1 and Vdr null mice on different calcium diets by Panda and colleagues likewise showed increased PTH and alkaline phosphatase levels and a more disorganized growth plate in the Cyp27b1 null mouse compared with the Vdr null mouse. VDR can interact with other transcription factors that regulate its activity, including p53 and Ets‐1, the latter showing ligand‐independent VDR stimulation of prolactin expression. The activation of FoxO requires its deacetylation and dephosphorylation, processes carried out by a complex of VDR/RXR in combination with Sirt1 and the catalytic subunit of the protein phosphatase 1. The recruitment of each to VDR is ligand independent. Thus, like other nuclear hormone receptors, ligand‐independent actions of VDR comprise a significant part of its mechanisms of action.A number of studies have been performed comparing the phenotypes of mice lacking Cyp27b1 to those lacking Vdr or to mice in which the s ligand.128 Le2D, the unliganded receptor is also active 2D for VDR binding to occur. This too shows remarkable cell specificity with the keratinocyte having the most overlap between VDR binding with and without 1,25(OH)2D and the intestine showing the least. There remains much to learn about the ligand‐independent actions of VDR, but as the field progresses, these actions must be considered to appreciate the full understanding of VDR mechanisms of action.Although most known actions of VDR require its ligand, 1,25(OH)https://publons.com/publon/10.1002/jbm4.10578.The peer review history for this article is available at