Activation of the chicken Ig-beta locus by the collaboration of scattered regulatory regions through changes in chromatin structure.

A total of 10 B-lymphocyte-specific DNase I hypersensitive sites located in the chicken Ig-beta locus were divided into four regions and combinations of deletions of these regions were carried out. A decrease in transcription of the Ig-beta gene to <3% was demonstrated in cells with deletions in all four regions. The Ig-beta chromatin was resistant to DNase I digestion in these cells.

Effect of deletion of the DNase I hypersensitive sites on the transcription of chicken Ig-beta gene and on the maintenance of active chromatin state in the Ig-beta locus.

The role of DNase I hypersensitive sites (DHSs) in transcription of the B cell-specific Ig-beta gene and in maintenance of active chromatin state in the Ig-beta locus were examined. A total of 10 DHSs were divided into four regions, and each region was deleted separately in chicken B lymphocyte-derived DT40 cells. Deletion of three DHSs located between the Ig-beta promoter and its upstream Na channelgene, resulted in the absence of Ig-beta mRNA.

DNase I hypersensitive sites and histone acetylation status in the chicken Ig-beta locus.

DNase I hypersensitive sites (DHSs) and histone acetylation status were examined in the Ig-beta locus of chicken B lymphocyte-derived DT40 cells and liver-derived LMH cells. Twelve DT40-specific DHSs were identified: one in the Ig-beta promoter, one in the first intron of the Ig-beta gene, three in the sodium channel gene located upstream of the Ig-beta gene, two between the sodium channel gene and the Ig-beta gene, four between the Ig-beta gene and a downstream growth hormone (GH) gene, and one in the downstream region of the GH gene. Transient transfection studies show that the DHS in the intron of Ig-beta gene enhances the activity of the Ig-beta promoter fourfold.

State of chromatin sensitivity to DNase I in the rat Ig-beta/growth hormone locus determined by real-time PCR.

Using Ig-beta and growth hormone producing cells with liver-derived cells for controls, sensitivity of chromatin to DNase I was measured by real-time PCR at eleven targets in rat Ig-beta/growth hormone locus where four cell type-specific genes and two ubiquitously expressed genes are present in a compact 88-kb region. Chromatin situated at the promoter of actively-transcribed gene and placed at cell type-specific DNase I hypersensitive sites with enhancer activity was sensitive to DNase I. In the case of inactive gene, chromatin located in these regions was resistant to DNase I.

State of histone modification in the rat Ig-beta/growth hormone locus.

The state of acetylation in H3 and H4 histones and dimethylation in the H3 histone Lys4 residue were examined by chromatin immunoprecipitation (ChIP) at 11 targets in the rat Ig-beta/growth hormone locus. Marked enhancement of the acetylation of histones H3 and H4 and the dimethylation of H3 Lys4 was observed in the chromatin situated close to the promoter of an actively transcribed gene. Chromatin positioned near a cell-type-specific DNase I-hypersensitive site with enhancer activity had the same histone modifications as the active promoter.