Chromosome movements that regulate tissue-specific gene expression

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Our progress during the present (2008) year was: (1) Dr. Tatiana Gerasimova established fluorescence in-situ hybridization (FISH) technology in our laboratory. Using it we confirmed nuclear periphery to center migration of the IgH locus and locus contraction in pro-B cells. (2) We applied this method to pro-B cells isolated from the bone marrow of mice in which two tissue-specific DNase 1 hypersensitive sites that mark the un-rearranged IgH locus have been deleted. We found that the germline IgH locus does not migrate to the center of the nucleus, demonstrating that Eμ and a promoter 5' of Dq52 (Pq52) are required for chromosome migration. This effect was attributed mostly to Eμ-deficiency because Pq52+ -deficient alleles moved normally to the nuclear center. (3) We standardized immunohistochemical localization of RNA polymerase II to punctuate "transcription factories" in primary pro-B cells. (4) We combined anti-Pol II fluorescence with FISH to demonstrate co-localization of IgH alleles in Pol II foci in pro-B cells. (5) We mapped CTCF binding sites in the intervening genomic region between the 3'-most VH gene segment (VH7183.lb) and the 5'-most DH gene segment (DFL16.1). CTFC binding was confirmed by EMSA using fragments of CTCF expressed and purified from bacteria and in-vitro translated protein. (6) We standardized conditions for anti-CTCF and anti-cohesin chromatin immunoprecipitation.