During fiscal year 2011, we accomplished the following: 1. Completed the analyses of chromatin structural changes that accompany the first step of IgH gene rearrangements. 2. Completed the first level of chromosome conformation capture (3C) studies on the pre-rearrangement IgH locus using a combination of 3C, 4C, and ChIP-loop studies Our results indicate that the conformation of the IgH locus is established by two kinds of loops;one that involves the intronic enhancer, Em, and a second form of loop that does not involve Em. We proposed that the first kind of loops utilize the transcription factor YY1 and the second kind utilizes the transcription factor CTCF. 3. Carried out directed 3C assays with DJ recombined cell lines to identify looping changes that occur after the first step of recombination. 4. Initiated collaborative studies with Dr. Amy Kenter (University of Illinois) to analyze IgH locus conformation by 5C assay. 5. Initiated collaborative studies with Drs. Sam John and Gordon Hager (NCI) to map DNase1 hypersensitive sites genome-wide. This assay was carried out with pro-B cells that contain WT or Em-deleted IgH alleles to determine whether Em interacts with other loci in trans . 6. Developed a micro-ChIP procedure to determine the genome-wide epigenetic state of hematopoietic precursors leading up to committed B and T lymphocyte lineages. We isolated hematopoietic stem cells (HSC), lymphoid-myeloid progenitor cells (LMPP), early thymic progenitors (ETP) and double negative 2 and 3 cells (DN2, DN3) and used anti-H3K4me3, anti-H3K27me3 and anti-H3K36me3 for antibodies for immunoprecipitation. Immunoprecipitated DNA was subjected to high throughput sequencing. Data analysis is ongoing.
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