During FY14 we accomplished the following: 1. We re-derived the delta Emu mouse strain and bred it to a RAG2-deficient background. To test the function of different E-binding proteins we generated Gal4-DBD- and Tet DBD-fusion proteins with E2A, YY1 and TFE3. Deletion mutants of each of these transcription factors fused to respective DBDs are in the process of being generated. 2. We almost completed analyses of 6 strains of bacterial artificial chromosome (BAC) transgenic mice. In these strains we test the hypothesis whether enhancer function is distance-independent by moving Emu to two different locations within the DH -CH domain of the IgH locus. Pro-B cells obtained from transgenic mice were assayed for transcription, epigenetic and recombination function. Our results indicate that Emu location is essential for appropriate enhancer function, thereby directly contradicting the text book dogma that enhancer function is distance- and orientation-independent. 3. We made a targeted mutation in the E1 site of Emu to test the hypothesis that YY1-bound to E1 is required for IgH locus compaction. Embryonic stem cells (ES) were transfected with a targeting construct, and one ES cell clone with correct homologous recombination (as assayed by Southern blots) was injected into mouse blastocysts to generate chimeric mice. These mice are currently being bred to WT mice to identify chimeras that transmit the mutant allele. 4. We carried out genome-wide DNAase I hypersensitive site mapping in pro-B cells with WT and E-deficient IgH alleles. Our experimental design should distinguish between weak and strong DNAase I hypersensitive sites, and determine whether Emu-deficiency affects other regulatory elements in pro-B cell nuclei. 5. We carried out RNA-Seq and ChIP-Seq and analyses of hematopoietic precursors in the T cell developmental pathway. These studies extended earlier analysis through the DP and SP stages of T cell development to examine the dynamics of bivalent chromatin. Additionally, the RNA-Seq analysis provides better quantitation to gauge changes in transcriptional status that accompany the generation and resolution of bivalent chromatin
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