Chromosome movements that regulate tissue-specific gene expression

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During FY15, we accomplished the following: 1) We published studies of a structural hierarchy of chromosome conformation of the IgH locus mediated by three different transcription factors. Briefly, we found that the first level of folding is mediated by the transcription factor CTCF. This mechanism folds the IgH locus into multiple 180-250 kb domains. Second, the transcription factor Pax5 compacts 2Mb of the VH part of the locus. Finally, the transcription factor YY1 brings together the 5 and 3 ends of the IgH locus. We proposed that stepwise generation of chromatin conformation could apply generally for the establishment of 3D-genome architecture. 2) We followed up studies on the disruption of IgH chromatin structure in bone marrow pro-B cells from aged mice. Specifically, we tested the involvement of the histone methyl transferase Ezh2 and the transcription factor PU.1 by altering levels of these proteins in primary pro-B cells. RNA analyses were consistent with the proposed hypothesis being tested. Fluorescent in situ hybridization (FISH) studies are in progress to determine whether manipulating levels of these factors imparts an older phenotype to IgH locus conformation and function. 3) We collaborated with Dr. Michael Atchison (U. Pennsylvania) to test the structural role of the transcription factor YY1 during class switch recombination (CSR). FISH studies showed disrupted looping between E and the 3RR in YY1-deficient B cells activated to undergo CSR ex vivo. Looping was re-established by ectopic retroviral expression of YY1. We used several truncated versions of YY1 in this assay to identify the domain of the protein required for this interaction.