| |
[摘要]：Previously, we used homologous recombination to delete six groups of cell-type-specific DNase I hypersensitive sites (DHSs), potential transcriptional and epigenetic regulators, scattered in and around the Ig-beta gene from their natural context in B-lymphocyte-derived chicken DT40 cells. Simultaneous deletion of all six groups completely shut down transcription and epigenetic regulation of the Ig-beta gene; therefore, the cooperation of the scattered regulatory regions was essential for transcription and epigenetic regulation. In this study, we regrouped the cell-type-specific DHSs of Ig-beta, those in the original six deletions and three additional ones, into three larger regional groups the long upstream region, the intron, and the long downstream region-and deleted these groups individually or in combination. Combinatorial deletion of all three regional groups decreased Ig-beta mRNA levels to 0.4% of the control, which was significantly higher than <0.1%, the level resulting from deletion of all six smaller groups. Histone H3 and H4 acetylation and H3K4 dimethylation levels at the Ig-beta promoter were low in cells carrying deletions of all six smaller groups, but intermediate levels of acetylation and enhanced H3K4 dimethylation were observed in cells carrying deletions of all three larger groups. While CG methylation was definitely present at the Ig-beta promoter in cells carrying all six smaller deletions, it was nearly absent from the Ig-beta promoter in cells carrying all three larger deletions. Thus, combinatorial deletion of larger regulatory regions had less effect on transcription and epigenetic regulation at the chicken Ig-beta gene than combinatorial deletion of shorter ones. Analysis of several combinatorial deletions, where combinations included two larger deletions and one smaller deletion, revealed the relative effects of each deletion on transcription of the Ig-beta gene. Investigation of the CG methylation status at the Ig-beta promoter in one combinatorial deletion demonstrated that USI was involved in the maintenance of CG methylation. (C) 2011 Elsevier B.V. All rights reserved. |
|
