Many of the transcriptional and growth regulating activities of 1α,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] in the intestine and colon are recapitulated in the human colorectal cancer cell LS180. We therefore used this line together with chromatin immunoprecipitation-seq and gene expression analyses to identify the vitamin D receptor (VDR)/retinoid X receptor (RXR) and transcription factor 7-like 2 (TCF7L2/TCF4)/β-catenin cistromes and the genes that they regulate. VDR and RXR colocalized to predominantly promoter distal, vitamin D response element-containing sites in a largely ligand-dependent manner. These regulatory sites control the expression of both known as well as novel 1,25-(OH)(2)D(3) target genes. TCF4 and β-catenin cistromes partially overlapped, contained TCF/lymphoid enhancer-binding factor consensus elements, and were only modestly influenced by 1,25-(OH)(2)D(3). However, the two heterodimer complexes colocalized at sites near a limited set of genes that included c-FOS and c-MYC; the expression of both genes was modulated by 1,25-(OH)(2)D(3). At the c-FOS gene, both VDR/RXR and TCF4/β-catenin bound to a single distal enhancer located 24 kb upstream of the transcriptional start site. At the c-MYC locus, however, binding was noted at a cluster of sites between -139 and -165 kb and at a site located -335 kb upstream. Examined as isolated enhancer fragments, these regions exhibited basal and 1,25-(OH)(2)D(3)-inducible activities that were interlinked to both VDR and β-catenin activation. These data reveal additional complexity in the regulation of target genes by 1,25-(OH)(2)D(3) and support a direct action of both VDR and the TCF4/β-catenin regulatory complex at c-FOS and c-MYC.

The maintenance of active levels of hormone is a dynamic process responsive to a multitude of systemic signals that monitor the mineral status of the organism. 1,25(OH) 2 D 3 production in the kidney is regulated positively by many factors, including parathyroid hormone (3) and regulated negatively by others (4), including the phosphate hormone FGF23 (5, 6). CYP27B1 is also down-regulated by 1,25(OH) 2 D 3 itself, which exerts strong negative feedback control on CYP27B1 transcription to curb 1,25(OH) 2 D 3 synthesis (3, 7-9). Interestingly, this negative feedback mechanism does not appear to be operable in the many extrarenal tissues that are also known to express CYP27B1, albeit at more modest levels (10). 1,25(OH) 2 D 3 also regulates its own degradation as well, because the hormone potently induces CYP24A1 expression in all cells in which it acts (2). Because the magnitude of this induction is generally cell-specific, the regulation of CYP24A1 expression likely represents a mechanism whereby cellular response can be precisely tailored to individual cellular requirements in the face of changing circulating 1,25(OH) 2 D 3 levels. Not surprisingly, reductions in CYP27B1 expression, enhanced synthesis of CYP24A1, or both can lead to a decrease in biologically active 1,25(OH) 2 D 3 levels that results in altered mineral homeostasis and increased disease potential (11). In summary, the collective activity of both renal CYP27B1 and target cell CYP24A1 results in the maintenance of highly dynamic steady-state levels of biologically active 1,25(OH) 2 D 3 in all tissues in which the hormonal ligand is active.The cloning of Cyp24a1 in 1993 revealed Cyp24a1 as a bone fide member of a large family composed of all the cytochrome

The vitamin D receptor (VDR) mediates the actions of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) in target cells and tissues by orchestrating the expression of gene networks responsible for vitamin D-induced phenotypes. The molecular mechanisms of these regulatory systems have been studied for decades under the principle that transcriptional regulation occurs near the transcriptional start site of the gene. However, this now appears to be an outdated view of transcriptional control. In this study, we examined the genome-wide chromatin immunoprecipitation on microarray (ChIPchip) across pre-osteoblastic cells for VDR, retinoid X receptor (RXR), RNA polymerase II, and histone H4 acetylation (H4ac). We uncovered potential regulatory mechanisms for genes important to osteoblast biology as well as skeletal formation under the control of 1,25(OH) 2 D 3 . We found that VDR, along with RXR and H4ac, binds to distal regions 43% of the time; and within gene introns and exons 44%, leaving only 13% of activation at traditional promoter regions. Here, we briefly summarize our findings for all the VDR/RXR cis-acting transcriptional elements (VDR/RXR cistrome) in pre-osteoblastic cells, MC3T3-E1, provide a few examples of this dynamic control by VDR and 1,25(OH) 2 D 3 , and demonstrate that distal transcriptional control contributes to the majority of vitamin D 3 -mediated transcription.

The DREAM (Dp/Retinoblastoma(Rb)-like/E2F/MuvB) transcriptional repressor complex acts as a gatekeeper of the mammalian cell cycle by establishing and maintaining cellular quiescence. How DREAM’s three functional components, the E2F-DP heterodimer, the Rb-like pocket protein, and the MuvB subcomplex, form and function at target gene promoters remains unknown. The current model invokes that the pocket protein links E2F-DP and MuvB and is essential for gene repression. We tested this model by assessing how the conserved yet less redundant DREAM system in Caenorhabditis elegans is affected by absence of the sole C. elegans pocket protein LIN-35. Using a LIN-35 protein null mutant, we analyzed the assembly of E2F-DP and MuvB at promoters that are bound by DREAM and the level of expression of those "DREAM target genes" in embryos. We report that LIN-35 indeed mediates the association of E2F-DP and MuvB, a function that stabilizes DREAM subunit occupancy at target genes. In the absence of LIN-35, the occupancy of E2F-DP and MuvB at most DREAM target genes decreases dramatically and many of those genes become upregulated. The retention of E2F-DP and MuvB at some target gene promoters in lin-35 null embryos allowed us to test their contribution to DREAM target gene repression. Depletion of MuvB, but not E2F-DP, in the sensitized lin-35 null background caused further upregulation of DREAM target genes. We conclude that the pocket protein functions primarily to support MuvB-mediated repression of DREAM targets and that transcriptional repression is the innate function of the evolutionarily conserved MuvB complex. Our findings provide important insights into how mammalian DREAM assembly and disassembly may regulate gene expression and the cell cycle.

The MuvB complex recruits transcription factors to activate or repress genes with cell cycle-dependent expression patterns. MuvB contains the DNA-binding protein LIN54, which directs the complex to promoter cell cycle genes homology region (CHR) elements. Here we characterize the DNA-binding properties of LIN54 and describe the structural basis for recognition of a CHR sequence. We biochemically define the CHR consensus as TTYRAA and determine that two tandem cysteine rich regions are required for high-affinity DNA association. A crystal structure of the LIN54 DNA-binding domain in complex with a CHR sequence reveals that sequence specificity is conferred by two tyrosine residues, which insert into the minor groove of the DNA duplex. We demonstrate that this unique tyrosine-mediated DNA binding is necessary for MuvB recruitment to target promoters. Our results suggest a model in which MuvB binds near transcription start sites and plays a role in positioning downstream nucleosomes.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) functions as a steroid hormone to modulate the expression of genes. Its actions are mediated by the vitamin D receptor (VDR) which binds to target genes and functions to recruit coregulatory complexes that are essential for transcriptional modulation. ChIP analysis coupled to tiled DNA microarray hybridization (ChIP-chip) or massively parallel DNA sequencing (ChIP-seq) is now providing critical new insight into how genes are regulated. In studies herein, we utilized these techniques as well as gene expression analysis to explore the actions of 1,25(OH)2D3 at the genome-wide and individual target gene levels in cells. We identify a series of overarching principles that likely define the actions of 1,25(OH)2D3 at most target genes. We discover that while VDR binding to target sites is ligand-dependent, RXR binding is ligand-independent. We also show that while VDR/RXR binding can localize to promoters, it occurs more frequently at multiple sites many kilobases from target gene promoters. We then describe a new method whereby the regulatory regions of complex genes can be evaluated using large recombineered bacterial artificial chromosomes. We conclude that these new approaches are likely to replace many of the traditional methods used to explore the regulation of transcription.

The five-protein MuvB core complex is highly conserved in animals. This nuclear complex interacts with RB family tumor suppressor proteins and E2F-DP transcription factors to form DREAM complexes that repress genes that regulate cell cycle progression and cell fate. The MuvB core complex also interacts with proteins Myb family oncoproteins to form the Myb-MuvB complexes that activate many of the same genes. We show that animaltype Myb genes are present in Bilateria, Cnidaria, and Placozoa, the latter including the simplest known animal species. However, bilaterian nematode worms lost their animal-type Myb genes hundreds of millions of years ago. Nevertheless, amino acids in the LIN9 and LIN52 proteins that directly interact with the MuvB-binding domains of human B-Myb and Drosophila Myb are conserved in C. elegans. Here we show that, despite greater than 500 million years since their last common ancestor, the Drosophila melanogaster Myb protein can bind to the nematode LIN9-LIN52 proteins in vitro and can cause a synthetic multivulval (synMuv) phenotype in vivo. This phenotype is similar to that caused by loss-of-function mutations in C. elegans synMuvB class genes including those that encode homologs of the MuvB core, RB, E2F, and DP. Furthermore, amino acid substitutions in the MuvB-binding domain of Drosophila Myb that disrupt its functions in vitro and in vivo also disrupt these activities in C. elegans. We speculate that nematodes and other animals may contain another protein that can bind to LIN9 and LIN52 in order to activate transcription of genes repressed by DREAM complexes.

47The mammalian pocket protein family, which includes the Retinoblastoma protein (pRb) 48 and Rb-like pocket proteins p107 and p130, regulates entry into and exit from the cell 49 cycle by repressing cell cycle gene expression. Although pRb plays a dominant role in 50 mammalian systems, p107 and p130 represent the ancestral pocket proteins. The Rb-51 like pocket proteins interact with the highly conserved 5-subunit MuvB complex and an 52 E2F-DP transcription factor heterodimer, forming the DREAM (for Dp, Rb-like, E2F, and 53 MuvB) complex. DREAM complex formation on chromatin culminates in direct 54 repression of target genes mediated by the MuvB subcomplex. Here, we examined how 55 the Rb-like pocket protein contributes to DREAM formation by disrupting the interaction 56 between the sole Caenorhabditis elegans pocket protein LIN-35 and the MuvB subunit 57 LIN-52 using CRISPR/Cas9 targeted mutagenesis. Disrupting the LIN-35-MuvB 58 association did not affect DREAM chromatin occupancy but did cause a highly 59 penetrant synthetic multivulval (SynMuv) phenotype, indicating that blocking DREAM 60 assembly impairs MuvB function. Some DREAM target genes became derepressed, 61 indicating that for those genes MuvB chromatin binding alone is not sufficient for gene 62 repression and that direct LIN-35-MuvB association potentiates MuvB's innate 63 repressive activity. In a previous study we showed that in worms lacking LIN-35, E2F-64 DP and MuvB chromatin occupancy is reduced genome-wide. With LIN-35 present, this 65 study demonstrates that the E2F-DP-LIN-35 interaction promotes E2F-DP's chromatin 66 localization, which we hypothesize supports MuvB chromatin occupancy indirectly 67 through DNA. Altogether, this study highlights how the pocket protein family may recruit 68 4 regulatory factors like MuvB to chromatin through E2F-DP to facilitate their 69 transcriptional activity.