During FY12, we accomplished the following: 1. Identified new CTCF-involving chromosomal loops using anti-CTCF-ChIP loop assays. 2. Used CTCF knock-down (KD) primary pro-B cells and 3D-FISH to determine the importance of CTCF for these loops. Quantitative distance measurements revealed 1.6-2-fold decompaction of these loops in CTCF KD cells. 3. Used YY1 shRNA virus developed in previous FY to generate YY1-knock down pro-B cell lines. We carried out 3C and 3D-FISH assays in these cells to study the role of YY1 in IgH locus conformation. Loops between the distal VH genes (3558) and the proximal VH genes (57183) were disrupted in YY1-KD cells in both assays. 4. We initiated studies of inter-chromosomal interactions using IgH and c-Myc loci since these loci are particularly susceptible to chromosomal translocations. We found that the 3-prime end of the IgH locus was close to c-Myc gene in approximately 25% nuclei. This putative interaction was lost in CTCF-KD cells indicating a role for CTCF in this inter-chromosomal interaction. Biochemical assays confirmed this interaction in WT pro-B cells and are being repeated in CTCF-KD cells. 5. We initiated studies to map the 5-prime end of the IgH locus using 3D-FISH in pro-B cells. 6. We collaborated with Dr. Amy Kenter to study chromosomal loops at the 3-prime end of the IgH locus in mature splenic B cells. We found that conditions that activated class switch recombination resulted in inducible looping of the IgH locus that resulted in juxtaposition of E to the 3 regulatory region.
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|Subrahmanyam, Ramesh; Sen, Ranjan (2012) Epigenetic features that regulate IgH locus recombination and expression. Curr Top Microbiol Immunol 356:39-63|
|Guo, Changying; Gerasimova, Tatiana; Hao, Haiping et al. (2011) Two forms of loops generate the chromatin conformation of the immunoglobulin heavy-chain gene locus. Cell 147:332-43|