During fiscal year 2007 we accomplished the following:? (1) Completed analysis of the chromatin structure and transcriptional status of the DH-C domain of the IgH locus in pro-B cells. These studies led to the proposal of repeat-induced transcriptional gene silencing as a mechanism that suppresses recombination of intervening DH segments at the first step of IgH recombination.? ? (2) Completed analysis of chromatin structural and transcriptional alterations that occur with a 220bp deletion of the intronic enhancer, E. We found that histone H3 and H4 acetylation, RNA polymerase II loading and transcription were significantly reduced. In contrast, positive and negative histone methylation marks were not significantly affected. These observations suggest that E-dependent and E-independent steps establish the pre-rearrangement chromatin state at IgH. ? ? (3) Extended DNA methylation studies to DJH rearranged alleles in primary pro-B cells, pre-B cells and thymocytes. Our studies show highly localized CpG demethylation accompanies DH to JH recombination in the B, but not the T, lineage. ? ? (4) We generated cell lines that contain specific DJH recombined alleles to probe chromatin structural and transcriptional changes that accompany DH to JH recombination. We examined five forms of histone modifications, nuclease sensitivity and transcription in six different cell lines. We found highly localized alterations of histone modifications and DNase 1 sensitivity and increased transcription after recombined junction. Cumulatively, we suggest that the several differences between DJH junction and 5' unrearranged DH may target VH recombination only to the DJH junction.? ? (5) We developed BAC engineering technology in our lab and used it to introduce several changes in the IgH locus. These modified BACs have been sent to make transgenic mice that will be used to study the effects of these changes in IgH chromatin structure and recombination.? ? (6) We completed the first phase of regulating V(D)J recombination in-vitro. We used nucleosome-assembled plasmids that contain Gal4 binding sites to direct the chromatin re-modeling complex SWI/SNF to sites close to recombination signal sequences (RSS). We found that SWI/SNF recruitment to both RSSs (12 and 23) was needed to increase RAG1/2-dependent DNA cleavage, which occurred in the absence of RNA polymerase II-dependent transcription. Our observations distinguish the effects of purely chromatin remodeling changes from the effects of transcription on the first step of V(D)J recombination.
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