Gene Regulation in Lymphocytes

National Institute On Aging

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Abstract

During fiscal year 2010 we accomplished the following: 1) We extended studies of epigenetic changes that accompany DH to JH recombination by analyzing DJH junctions in Eμ-deficient pro-B cell lines. Both H3K9ac and H3K4me3 levels were significantly reduced at DJH junctions on Eμ-deficient alleles. Analyses of H3K4me2 will be completed soon. Our observations indicate that Eμselectively activates the DJH junction with epigenetic marks that favor recombination. 2) We used chromatin immunoprecipitation to determine that RAG1 and RAG2 proteins are also selectively recruited to the DJH junction. This recruitment is very precise since RAG1/2 cannot be detected at unrearranged DSP gene segments located within 4 kb of the DJH junction. 3) 4C assays using Eμanchor primers identified Eμ-dependent chromatin looping to DFL16.1 (56 kb upstream) and to the 3'regulatory region (250 kb downstream). Using chromatin immunoprecipitation we found that all three looping sites bind the transcription factor YY1. YY1 binding to DFL and the 3'RR was not affected on Eμ-deficient alleles, however, the DFL-3'RR chromatin loop was disrupted. We conclude that Eμnucleates a chromatin domain at the 3'end of the IgH locus that contains all DH and JH gene segments and all constant region exons. We surmise that this structure is, in part, responsible for DH recombination occurring before VH recombination during B cell ontogeny. 4) 4C assays using Eμanchor primers also identified two sites within the VH region that are in close proximity to Eμin pro-B cells. These regions also bind YY1. We propose that Eμinteraction with these sites may lead to locus contraction of the IgH locus to promote VH recombination. 5) We used primary pro-B cells and pro-T cells from mice with defined deletions of IgH-associated cis-regulatory sequences in 3C assay to determine which interactions were Eμ-dependent. We found that Eμassociated loops to 5'and 3'sites within the IgH locus were disrupted on Eμ-deficient alleles. Thus, these loops are Eμ-dependent. 6) We used ChIP -loop assays to identify parts of the IgH locus that underwent transcription factor CTCF-dependent compaction. Using anchor primers located within proximal VH genes we found looping to DFL16.1. This loop was independent of Eμ. Additional anchor primers within the distal VHJ558 genes also isolated sequences within 500 kb. These loops were also Eμ-independent. These observations indicate that CTCF may be involved in short-range (within a few hundred kbs) looping that is Eμ-independent. We conclude that the IgH locus is compacted by Eμ-dependent and Eμ-independent chromosome loops.