Fundamentals of vitamin D hormone-regulated gene expression

Abstract

Initial research focused upon several known genetic targets provided early insight into the mechanism of action of the vitamin D hormone (1,25-dihydroxyvitamin D3 (1,25(OH)2D3)). Recently, however, a series of technical advances involving the coupling of chromatin immunoprecipitation (ChIP) to unbiased methodologies that initially involved tiled DNA microarrays (ChIP-chip analysis) and now Next Generation DNA Sequencing techniques (ChIP-seq analysis) has opened new avenues of research into the mechanisms through which 1,25(OH)2D3 regulates gene expression. In this review, we summarize briefly the results of this early work and then focus on more recent studies in which ChIP-chip and ChIP-seq analyses have been used to explore the mechanisms of 1,25(OH)2D3 action on a genome-wide scale providing specific target genes as examples. The results of this work have advanced our understanding of the mechanisms involved at both genetic and epigenetic levels and have revealed a series of new principles through which the vitamin D hormone functions to control the expression of genes. This article is part of a Special Issue entitled '16th Vitamin D Workshop'.

Keywords: 1,25(OH)(2)D(3); ChIP-seq methods; Epigenetics; Transcription; Unbiased genome-wide analysis; VDR.

Figure 1

ChIP-seq profiles at specific gene loci. Mouse MC3T3-E1 cells were treated for 3 hrs with either vehicle or 1,25(OH)₂D₃ (10⁻⁷M) and then subjected to ChIP-seq analysis using antibodies to VDR, RXR, RUNX2, C/EBPβ and histone H3K4me1 or H3K4me2. ChIP-seq tag densities (normalized to 10⁷ reads) were quantified and mapped to the mouse MM9 genome using MACS (Model-based Analysis for ChIP-seq), HOMER (Hypergeometric Optimization of Motif Enrichment) and Cistrome Data Analyses. The genomic loci (chromosome number and nucleotide interval are indicated) contain the Vdr (A), Tnfsf11 (B), and Spp1 (C) genes and their respective neighbors. Genomic tracks (read scales are indicated on the Y-axis) for VDR, RXR, C/EBPβ, RUNX2, H3K4me1 and H3K4me2 each contain two mapped data sets derived from vehicle- and 1,25(OH)₂D₃-treated cells (red and blue, respectively with overlap in purple). The transcriptional start sites and direction of transcription for each gene is indicated by an arrow; exons are indicated by vertical boxes within the gene. Shaded vertical columns highlight the locations of several known regulatory regions for each target gene, as explored in specific detail in references 25 and 40 (Vdr), 24 and 54 (Tnfsf11), and 3 and 57 (Spp1); numbers/letters below the data sets in (A) and (B) represent our CRM designation.

Figure 2

The classic VDRE motif defined via genome-wide ChIP-seq analysis in human colorectal LS180 cells (23). This motif logo describes the relative nucleotide sequence of the most enriched 15 bp DNA segment located within the collection of VDR/RXR binding sites found across the LS180 genome (compared to 50,000 GC-matched random 15 bp DNA sequences).