During FY15 we accomplished the following: 1) Continued studies with DeltaEμ mice. In this strain Eμ is replaced by TetO and Gal4 binding sites. Flow cytometric analyses demonstrated that these mice recapitulate the phenotype of Eμ deficiency. We are ready to commence protein recruitment studies to investigate function of individual, and combinations of, transcription factors that activate Eμ. 2) μE1 mutated mice that were generated by gene targeting in ES cells were bred to delete the Neo gene from the locus, and then crossed with RAG2-deficient mice to maintain IgH in unrearranged configuration. This strain will be ready for experiments in 6 to 8 weeks. 3) We generated a second μE1 mutation using CRISPR/Cas9. This strain will be on a C57Bl6 background, and will complement the results obtained with μE1 mutation created in ES cells of 129 strain background. 4) We generated μE2+μE5 mutated IgH alleles using CRISPR/Cas9. These mice are being bred for preliminary phenotyping. 5) We generated μA+μB mutated IgH alleles using CRISPR/Cas9. These mice are being bred for preliminary phenotyping. Cumulatively, studies with μE1, μE2+μE5 and μA+μB mutated IgH alleles will provide the first comprehensive analysis of the function of individual motifs in a complex mammalian enhancer. 6) We studied the epigenetic and transcriptional state of IgH alleles in CD4+ CD8+ (DP) thymocytes. IgH alleles undergo partial rearrangements in DP cells, the basis for which remains a mystery. We hypothesized that the nuclear milieu of DP cells may be deficient in a subset of factors required for complete activation of IgH alleles, and thereby serve as a natural mutation for the identification of such factors. We made two striking observations. First, the VH domain does not undergo CTCF-dependent compaction in DP cells despite normal levels of CTCF binding. Second, anti-sense transcripts arising at Eμ are substantially reduced in DP cells. We are investigating the molecular bases for these differences.
Showing the most recent 10 out of 20 publications
