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

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。利用基因靶向技术，我们分别生成了几个DT40突变体，它们缺乏一个或两个特定的组蛋白基因，110 kb的主要基因簇中的一个等位基因，大约57 kb的集群片段中携带21个基因，以及12个H1基因拷贝中的11个。此外，我们产生了四个DT40突变体，分别不含chHDAC-1、2、3和4。对结果突变体的系统分析使我们得出了一些值得注意的结论，如下所示。1)组蛋白基因家族，H1、H2A、H2B、H3和H4，无论主要基因簇的一个等位基因被完全破坏，还是两个等位基因的大约一半以上片段被破坏，都具有补偿大约一半自身成分缺失的内在能力，并基于剩余成员表达的增加来维持每种组蛋白亚型的化学计量平衡。因此，一个主要组蛋白基因簇的等位基因就足以使细胞增殖。2) 12个H1基因拷贝中有11个拷贝的缺失对细胞功能，即生长速度和整体染色质结构的影响不显著，表明只要一个H1基因拷贝就足以实现细胞增殖。3)由于组蛋白变体的组成没有变化，当集群的一个等位基因被删除时，2D-PAGE上的蛋白质模式没有改变，但当集群的大约一半片段被删除时，2D-PAGE上的蛋白质模式会发生变化，后者加上更多的H1基因(s)，这是由于H1和核心变体的质量变化。4)突变体中的蛋白质模式分别发生改变，缺乏特定的H1和H2B变体。可变蛋白对相应的突变体是特异性的，这表明H1和核心变异可能通过改变特定基因组区域的染色质结构，在调节特定基因的表达中发挥单独的作用。5) chHDAC-2在其基因转录及其前mrna的替代加工过程中控制IgM h链的数量。6) chHDAC-3对DT40细胞的生存至关重要。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