Functional Analysis of H1 and Core Histone Variants by Gene Targeting Technique

在44个鸡H1和核心组蛋白基因中，有39个位于一个110 kb的主要组蛋白基因簇中。利用基因打靶技术，我们分别产生了几个DT40突变体，它们分别缺乏一个或两个特定的组蛋白基因，一个110kb的主基因簇的等位基因，大约57kb的携带21个基因的簇片段，以及12个H1基因拷贝中的11个。此外，我们还产生了四个dt40突变体，分别缺失chHDAC-1、2、3和4。对产生的突变体的系统分析使我们得到了一些值得注意的结论，如下：1)组蛋白基因家族H1、H2 A、H2 B、H3和H4具有先天的能力，可以补偿大约一半自身成分的缺失，并基于其余成员表达的增加来维持每个组蛋白亚型的数量，而不管主要基因簇的一个等位基因完全中断或大约…的中断两个等位基因的一半以上片段。因此，主要组蛋白基因簇的一个等位基因足以促进细胞增殖。2)12个H1基因拷贝中的11个拷贝的缺失对细胞功能的影响不显著，即生长速度和全局染色质结构，表明只有一个拷贝的H1基因足以用于细胞增殖。3)2D-PAGE上的蛋白质图谱在该簇的一个等位基因缺失的情况下没有改变，这是由于任何组蛋白变体的组成没有改变，但在该簇的大约一半片段缺失的情况下发生变化，而后者加上更多的H1基因(S)，4)突变体中的蛋白质模式发生了变化，分别缺少特定的H1和H2B变体。这些可变蛋白是相应突变体所特有的，这表明H1和核心突变体应该在特定基因的表达调控中发挥单独的作用，可能是通过改变特定基因组区域的染色质结构。5)chHDAC-2在其基因转录和其前mRNA的交替处理步骤中控制IgM H链的数量。6)chHDAC-3对于DT40细胞的生存是必不可少的。较少

Thirty-nine of the 44 chicken H1 and core histone genes are located in a major histone gene cluster of 110 kb. Using gene targeting techniques, we generated several DT40 mutants, respectively, which are devoid of one or two particular histone genes, one allele of the major gene cluster of 110 kb, an approximately 57 kb segment of the cluster carrying 21 genes, and 11 of the 12 H1 gene copies. In addition, we generated four DT40 mutants, respectively, devoid of chHDAC-1, 2, 3 and 4. Systematic analyses of the resultant mutants led us to some noticeable conclusions, as follows.1) Histone gene families, H1, H2A, H2B, H3 and H4, have the inherent ability to compensate for the deletion of approximately half of their own constituents and to maintain the amount of each of the histone subtypes in stoichiometric balance, based on increases in the expression of the remaining members, regardless of the complete disruption of one allele of the major gene cluster or the disruption of approximately … More half segments of the two alleles. Therefore, one allele of the major histone gene cluster is enough for cell proliferation.2) The deletion of 11 of the 12 H1 gene copies resulted in insignificant influence on the cell functions, i.e. the growth rate and global chromatin structure, indicating that only one copy of the H1 genes is enough for cell proliferation.3) The protein patterns on 2D-PAGE are not altered in the case of the deletion of one allele of the cluster, due to no changes in the composition of any histone variant, but vary in the case of the deletion of approximately half segments of the cluster, and the latter plus more H1 gene(s), due to changes in the quality of the H1 and core variants.4) The protein patterns are altered in the mutants, respectively, lacking particular H1 and H2B variants. The variable proteins are specific for the corresponding mutants, suggesting that the H1 and core variants should play individual roles in regulation of the expression of specific genes, probably through alterations in the chromatin structure localized in specific genomic regions.5) chHDAC-2 controls the amount of the IgM H-chain at the steps of both transcription of its gene and the alternative processing of its pre-mRNA.6) chHDAC-3 is essential for the viability of DT40 cells. Less