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. 2009 Nov 30;285(4):2227–2231. doi: 10.1074/jbc.C109.071225

FIGURE 1.

FIGURE 1.

Hormonal vitamin D induces expression of CD susceptibility gene NOD2/CARD15/IBD1. A, quantitative PCR analyses are shown of reverse transcribed RNA from primary human keratinocytes, immortalized HIEC cells, and differentiated human THP-1 cells treated with 100 nm 1,25D, as indicated. B, 1,25D induces NOD2 expression in cultures of human monocytes treated with 1,25D (100 nm) for 24 h prior to RNA isolation for quantitative PCR analysis. CYP24 mRNA levels were also monitored as a positive control for 1,25D action. C, Western analysis of NOD2 protein expression or β-actin internal control in primary cultures of human keratinocytes or differentiated THP-1 monocytic cells treated with 100 nm 1,25D, as indicated. D, induction of 1,25D target genes in LPS-primed differentiated THP-1 cells incubated with 25D analyzed by qPCR. Cells were incubated with LPS as indicated for 24 h prior to treatment with 25D for 12 h at the concentrations indicated. Expression of 1,25D target gene CAMP was monitored as a positive control, along with expression of NOD2. Error bars in A, B, and D indicate S.E. E, schematic representation of the human NOD2 gene, with VDREs indicated by black boxes, and exons (gray) are numbered. Putative VDREs were identified as described (38) using the NCBI Build 35 Human May 2004 (hg17) genome assembly (University of California Santa Cruz (UCSC) Genome Browser data base). Regions encompassing putative VDREs or the TSS amplified by PCR in ChIP assays are indicated by inverted arrows. F, ChIP analysis using primary human keratinocytes of 1,25D-dependent binding of the VDR to putative VDREs, along with ChIP analysis of pol II binding to the TSS. Cells were treated with 100 nm 1,25D for 2 h prior to cross-linking for ChIP assay. G, 3C analysis using primary human keratinocytes of 1,25D-dependent association of genomic regions encompassing VDREs 1, 2 and 3 with the NOD2 TSS. No chromatin looping between VDRE2 and the TSS was detected under any conditions. Controls (BAC) are provided, showing that expected PCR products (arrowheads) were generated when primer sets for 3C detection were used for amplification digestion and religation of BAC clones encompassing the NOD2 gene.