Regulation of gene expression by 1,25-dihydroxyvitamin D3 in bone cells: exploiting new approaches and defining new mechanisms

Abstract

The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) serve both to orchestrate calcium and phosphorus homeostasis in higher vertebrates and to regulate a diverse set of cellular functions unrelated to control of mineral metabolism. With regard to bone, mesenchymal lineage cells, including both early and late osteoblasts as well as osteocytes represent classic targets of the vitamin D hormone. Accordingly, much of the early information regarding our current understanding of the mechanism of action of 1,25(OH)2D3, of which gene regulation is central, derives from a broad array of studies in these cell types. Indeed, a gene that provided both the earliest and perhaps the most extensive information regarding this and additional mechanisms was that of osteoblast-specific osteocalcin. Subsequent work has provided much additional detail as to how 1,25(OH)2D3, through the vitamin D receptor (VDR), mediates the modulation of many bone cell genes. In recent years, however, a series of technical advances involving the coupling of chromatin immunoprecipitation (ChIP) to unbiased methodologies that involve next-generation DNA sequencing techniques (ChIP-seq) have opened new avenues in the study of gene regulation. In this review, we summarize early work and then focus on more recent studies that have used ChIP-seq analysis and other approaches to provide insight into not only the regulation of specific genes such as the VDR, TNFSF11 (RANKL), LRP5, CBS and CYP24a1, but overarching genome-wide principles of gene regulation as well. The results of these studies highlight the value of these new approaches and the increased insight that can be gained.

Figure 1

Site-specific and genome-wide methodologies associated with chromatin immunoprecipitation (ChIP) methods. ChIP-chip, ChIP linked to tiled microarray analysis; ChIP-seq, ChIP linked to DNA deep sequencing methods.

Figure 2

ChIP-seq profiles at specific gene loci. Mouse MC3T3-E1 cells were treated for 3 h with either vehicle or 1,25(OH)₂D₃ (10⁻⁷ M) and then subjected to ChIP-seq analysis using antibodies to VDR, RXR, histone H3K4me1 or H3K4me2. ChIP-seq tag densities (normalized to 10⁷ reads) were quantified and mapped to the mouse MM9 genome using MACS, HOMER and cistrome data analyses. The genomic loci (chromosome number and nucleotide interval are indicated) contain the Vdr (a), Tnfsf11 (b), Lrp5 (c), Cbs (d) and Cyp24a1 (e) genes and their respective neighbors. Each data track (scales are indicated on the Y axis) contains two mapped data sets derived from vehicle- and 1,25(OH)₂D₃-treated cells (red and blue, respectively). The TSS and direction of transcription for each gene is indicated by an arrow; exons are indicated by boxes. Shaded vertical columns highlight the locations and number/letter designation for each of the regulatory regions of the target genes. 1,25(OH)₂D₃, 1,25-dihydroxyvitamin D₃; CBS, cystathionine β-synthase; ChIP, chromatin immunoprecipitation; HOMER, hypergeometric optimization of motif enrichment; LPR5, lipoprotein receptor-related protein 5; MACS, model-based analysis for ChIP-seq; TSS, transcriptional start site; VDR, vitamin D receptor; PP, promoter proximal.

Figure 3

Methodology associated with the preparation and use of bacterial artificial chromosomes (BACs). Chromatin immunoprecipitation (ChIP)-seq analysis is used to define the regulatory locus of a specific gene. A BAC clone containing this segment of DNA is modified through recombineering methods and then introduced via stable transfection into culture cells or through transgenic methods into mice. Orientation of the target transcription unit (5′ and 3′) together with representative exons (E) is indicated. The recombineering cassette contains an internal ribosome entry site (IRES)/luciferase and a thymidine kinase promoter-neomycin gene (TK-NEO) component. Direction of transcription is indicated by the arrow.