Chromosome movements that regulate tissue-specific gene expression

我们在本(2008)年的进展如下： (1)Tatiana Gerasimova博士在实验室建立了荧光原位杂交(FISH)技术。利用它，我们证实了前B细胞中IgH基因位点向中心的迁移和基因位点的收缩。 (2)我们将这种方法应用于从小鼠骨髓中分离的Pro-B细胞，其中两个标记未重排的IgH基因的组织特异性DNase1超敏位点已经缺失。我们发现生殖系IgH基因没有迁移到核的中心，这表明染色体迁移需要E&#956；和DQ52(Pq52)的5‘启动子。这种影响主要归因于E&-缺乏，因为缺乏Pq52+的等位基因通常会移动到核中心。 (3)我们标准化了RNA聚合酶II的免疫组织化学定位，以标示原代Pro-B细胞中的“转录工厂”。 (4)我们将抗POL II荧光标记与FISH技术相结合，证实了前B细胞POL II病灶中IgH等位基因的共定位。 (5)在3‘-Most VH基因片段(VH7183.1b)和5’-Most-IH基因片段(DFL16.1)之间的基因组区域定位了CTCF结合位点。利用从细菌中表达和纯化的CTCF片段和体外翻译的蛋白，EMSA证实了CTFC的结合。 (6)规范了抗CTCF和抗粘连蛋白染色质免疫沉淀的条件。

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.