Properties of the genome in normal and SV40 transformed WI38 human diploid fibroblasts. II. Metabolism and binding of histones.

Composition, metabolism and extractability of histone fractions from WI38 human diploid fibroblasts and SV40 transformed WI38 fibroblasts are compared. Two alternate procedures were used for isolation of nuclei which allow for either optimal recovery of arginine-rich histones F3 (III) and F2a1 (IV) or for optimal retention of lysine-rich F1 (I) and slightly lysine rich F2b (II b2). While the relative amount of each histone fraction was found to be similar in normal and SV40 transformed cells, substantial increases in the levels of F 3 acetylation and F1 and F2a2 phosphorylation are reported for the histones of SV40 transformed cells. Differences in extractability of arginine-rich histones with 0.25 M HCl are also reported. While F 3 is extracted more rapidly than F 2a1 from nuclei of normal WI38 fibroblasts, the reverse is true in SV40 transformed WI38 cells. These differences are discussed in relation to modification reactions, binding of histones to DNA and SV40-induced alterations in gene readout.     

通过系统分析揭示组蛋白修饰模式与转录因子结合之间的关联

转录因子对顺势调控元件的选择性结合,在哺乳动物细胞类型特异的基因表达中扮演重要的角色.这个过程受到染色质表观遗传状态的潜在调控.近期,染色质免疫共沉淀结合测序的研究提供了大量泛基因组水平的数据,阐述转录因子结合以及组蛋白修饰的位点,这为系统地研究转录因子和表观遗传标记之间的空间及调控关系提供了基础.该研究对公共数据库中的染色质免疫共沉淀结合测序数据进行整合分析,涉及5个细胞系中的85种转录因子、9种组蛋白修饰,目的是研究转录因子结合位点与组蛋白修饰模式以及基因表达在泛基因组水平上的关联.作者发现,不同转录因子与组蛋白修饰的共定位模式高度一致,并且组蛋白修饰在距离转录因子结合位点约500碱基对的位置富集.作者还发现,转录因子结合位点的占有率与活性组蛋白修饰的水平和双峰模式正相关,并且启动子区域组蛋白修饰的双峰和共定位模式和基因的高转录水平相一致.组蛋白修饰模式、转录因子结合位点的占有率与基因转录之间的关联暗示了细胞可能利用的基因表达调控机制.     

Differential ASF/SF2 activity in extracts from normal WI38 and transformed WI38VA13 cells.

The normal human fibroblast cell line WI38 and a transformed derivative, WI38VA13, differentially splice fibronectin pre-mRNA in vivo. As a first step to understand the molecular basis for this regulation of splicing, we examined the ability of WI38 and WI38VA13 nuclear extracts to splice model adenovirus and globin pre-mRNAs. Adenovirus RNA splicing was detected in WI38VA13 but not in WI38 extracts. Likewise, when supplemented with a HeLa post-nuclear supernatant (S100), human beta-globin RNA splicing was detected in WI38VA13 but not in WI38 extracts. The splicing defect in WI38 extracts was associated with a reduced ability to form splicing complexes and with a corresponding decrease in the interaction of U2 small nuclear ribonucleoprotein (snRNP) with the branchsite. These defects did not correlate with a decrease in 65 kD U2AF binding since equivalent U2AF level and activity were detected in WI38 and WI38VA13 extracts. Rather, WI38 extracts displayed reduced ASF/SF2 activity and contained a low level of 30 and 40 kD SR phosphoproteins. Moreover, addition of purified ASF/SF2 dramatically increased splicing complex formation in WI38 extracts. These results raise the possibility that variations in the level and activity of ASF/SF2 and other SR proteins play a role in the regulation of fibronectin splicing.     

Overexpression of ORC subunits and increased ORC-chromatin association in transformed mammalian cells

The origin recognition complex (ORC) is a conserved heterohexamer required for the formation of pre-replication (pre-RC) complexes at origins of DNA replication. Many studies of ORC subunits have been carried out in transformed human cell lines but the properties of ORC in primary cells have not been addressed. Here, we compare the expression levels and chromatin-association of ORC subunits in HeLa cells to the primary human cell line, WI38, and a virally transformed derivative of WI38, VA13. ORC subunits 2 and 4 were highly overexpressed in both HeLa and VA13, whereas ORC1 levels were elevated in VA13 but considerably higher in HeLa cells. Cellular extraction revealed that the proportion of ORC2 and ORC4 subunits bound to chromatin was similar in all three cell lines throughout the cell-cycle. In contrast, very little ORC1 was associated with chromatin after extraction of primary WI38 cells, whereas the majority of overexpressed ORC1 in both HeLa and VA13 co-fractionated with chromatin throughout the cell-cycle. Although none of the cell lines displayed significant changes in the levels or chromatin-association of ORC during the cell-cycle, the chromatin-associated fraction of ORC1 displayed an increase in apparent molecular weight during S-phase. Similar experiments comparing immortalized CHO cells to an isogenic virally transformed derivative revealed no changes in levels of ORC subunits but an increase in the proportion of all three ORC subunits associated with chromatin. These results demonstrate a complex influence of cellular immortalization and transformation properties on the expression and regulation of ORC subunits. These results extend the potential link between cancer and deregulation of pre-RC proteins, and underscore the importance of considering the transformation status of cell lines when working with these proteins.     

组蛋白与转录因子在hAMFR基因启动子序列上的结合及相互作用

采用从鸡红细胞中分离纯化的组蛋白Ｈ１,核心组蛋白Ｈ２Ａ＋Ｈ２Ｂ和Ｈ３＋Ｈ４,以及从ＨｅＬａ细胞中萃取的含有ＲＮＡ聚合酶Ⅱ和多种Ⅱ类基因转录因子的可溶性ＨｅＬａ细胞核抽提物,通过凝胶迟滞电泳,对组蛋白和ＨｅＬａ细胞核抽提物中的转录因子在人自泌移动因子受体（Ｈｕｍａｎａｕｔｏｃｒｉｎｅｍｏｔｉｌｉｔｙｆａｃｔｏｒ,简称ｈＡＭＦＲ）基因上游启动子序列的相互作用关系进行了初步研究,得到以下结论,组蛋白Ｈ１