Chromosome movements that regulate tissue-specific gene expression

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During FY17 we accomplished the following: 1) Continued with studies to localize fluorescent RAG1 and RAG2 derivatives in nuclei of pro-B cell lines. RAG2 derivatives were expressed in RAG2-deficient pro-B cell lines. To reduce background of endogenous RAG1 we had previously transduced siRNA against WT RAG1 into cells that expressed fluorescent RAG1 derivatives. To further reduce background effects we obtained a RAG1-deficient pro-B cell line in which further studies of RAG1 will be carried out. 2) Investigated the conformational state of the IgH locus in CD4+CD8+ (DP) thymocytes, in which the locus undergoes partial DH to JH rearrangements. We found that 3D structure of the distal 1Mb of the VH locus was disrupted in DP thymocytes compared to pro-B cells. This observation is consistent with our earlier proposal that distal VH locus structure requires Pax5, which is not expressed in DP thymocytes. Within the 1Mb domain, smaller CTCF-dependent domains of a few hundred kilobases were also disrupted, despite normal levels of CTCF binding. We used anti-Rad21 ChIP to show that these sites lacked Rad21, providing a plausible explanation for lack of VH compaction. These observations raised the interesting possibility that Rad21 recruitment to CTCF-bound sites in the genome is tissue-restricted. The basis for this will be examined in the future. 3) The IgH enhancer was found to be partially occupied by transcription factors in DP cells. Specifically, E47 and Els-1 were absent from the enhancer in DP cells, while HEB (a E2A-related bHLM factor) was found to the enhancer. PU.1 is another enhancer binding protein that is not expressed in DP thymocytes. In contrast, binding of the architectural protein YY1 was comparable in pro-B cells and DP cells. Thus, the constellation of DNA binding proteins that bind to the IgH intronic enhancer is different between pro-B and DP cells. These differences presumably lead to partial enhancer function in DP cells.