Chromosome movements that regulate tissue-specific gene expression

National Institute On Aging

Investigators

Abstract

Our progress during the present (2008) year was:[unreadable] [unreadable] (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.[unreadable] [unreadable] (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.[unreadable] [unreadable] (3) We standardized immunohistochemical localization of RNA polymerase II to punctuate "transcription factories" in primary pro-B cells.[unreadable] [unreadable] (4) We combined anti-Pol II fluorescence with FISH to demonstrate co-localization of IgH alleles in Pol II foci in pro-B cells.[unreadable] [unreadable] (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.[unreadable] [unreadable] (6) We standardized conditions for anti-CTCF and anti-cohesin chromatin immunoprecipitation.