During FY12, we accomplished the following: 1. We obtained two kinds of mutant mouse strains with modified IgH alleles. a. ΔE mice contain tandem TetO and Gal4 binding sites instead of E and b) Site 4 mice contain multimerized TetO sites inserted 3 of the IgH locus. Both strains still contain the neomycin resistant gene (Neo) flanked by loxP sites, and are being bred to β-actin-cre transgenic mice to delete Neo. Additional breeding to RAG-deficient mice is needed before we can proceed with analyses of chromosome structure and conformation. 2. Studies of the chromatin structure of DJH recombined alleles led to a unified model for regulated activation of VH recombination. To move forward, we further explored whether chromosome conformational changes accompanied DH recombination to further facilitate VH recombination. For this we generated Abelson murine leukemia virus transformed cell lines that contain defined DJH recombined alleles. Unique DJH junction sequences were used to anchor chromosome conformation capture assays. In two different cell lines that contain different DJH recombinant alleles we found increased looping of VH gene segments to DFL16.1 indicative of recombination-induced alterations in IgH locus conformation. These observations are currently being further substantiated by 3D-FISH. 3. We used Carbon Copy Chromosome Conformation Capture assay (5C) in collaboration with Dr. Amy Kenter (University of Illinois) to study the conformation of un-rearranged and DJH rearranged IgH alleles. These studies revealed novel chromosome folding patterns in the 2Mb VH domain of un-rearranged IgH alleles and DJH recombination-induced changes in this structure. We hypothesize that the latter changes may facilitate VH gene recombination. We are currently using 3D-FISH to corroborate these observations.
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