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

通过基因打靶技术对 H1 和核心组蛋白变体进行功能分析

• 批准号: 09480152
• 负责人: NAKAYAMA Tatsuo
• 金额: $ 8万
• 依托单位: Miyazaki Medical College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09480152/
• 关键词: Histone variants; DT40 cells; Gene targeting technique; Chromatin structure; Transcription regulation; Histone deacetylase; IgM H-chain; Cell growth; イムノグロブリン; 2次元電気泳動; ヒストン; バリアント

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

44个鸡H1和核心组蛋白基因中有39个位于110 kb的主要组蛋白基因簇中。使用基因打靶技术，我们产生了几个DT 40突变体，分别是缺乏一个或两个特定的组蛋白基因，一个等位基因的主要基因簇的110 kb，一个约57 kb的片段的集群携带21个基因，和11的12个H1基因拷贝。此外，我们分别产生了四种缺乏chHDAC-1、2、3和4的DT 40突变体。通过对突变体的系统分析，我们得到了以下一些值得注意的结论：1）组蛋白基因家族H1、H2 A、H2 B、H3和H4具有固有的能力，可以补偿大约一半的自身组分的缺失，并基于剩余成员表达的增加，保持每个组蛋白亚型的量处于化学计量平衡，无论主要基因簇的一个等位基因的完全破坏或大约一个等位基因的破坏， ...更多信息 两个等位基因的半片段。因此，主要组蛋白基因簇的一个等位基因足以用于细胞增殖。2）12个H1基因拷贝中的11个的缺失对细胞功能，即生长速率和整体染色质结构的影响不明显，表明H1基因的一个拷贝就足以进行细胞增殖。PAGE在缺失簇的一个等位基因的情况下不改变，因为任何组蛋白变体的组成没有变化，但是在缺失簇的大约一半片段的情况下变化，并且后者加上更多的H1基因，由于H1和核心变体的质量变化。4）蛋白质模式在突变体中分别改变，缺乏特定的H1和H2 B变体。可变蛋白质对相应的突变体是特异性的，这表明H1和核心变体应该在特定基因的表达调控中发挥各自的作用，可能是通过改变位于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的交替加工步骤中控制IgM H链的量。6）chHDAC-2在其基因转录和前mRNA的交替加工步骤中控制IgM H链的量。3对DT 40细胞的存活力是必需的。少

项目成果

Takami, Y. and Nakayama, T.: "A single copy of linker H1 genes is enough for proliferation of the DT40 chicken B cell line, and linker H1 variants participate in regulation of gene expression."Genes to Cells. 2. 711-723 (1997)

Takami, Y. 和 Nakayama, T.：“接头 H1 基因的单个拷贝足以使 DT40 鸡 B 细胞系增殖，并且接头 H1 变体参与基因表达的调节。”《基因到细胞》。

Nakayama, T., Takami, Y. and Ahmad, A.: "Interaction of the p48 subunit of chicken chromatin assembly factor-1 with histone deacetylases"Current Topics in Biochemical Research. 1. 173-178 (1999)

Nakayama, T.、Takami, Y. 和 Ahmad, A.：“鸡染色质组装因子 1 的 p48 亚基与组蛋白脱乙酰酶的相互作用”生化研究当前主题。

Takami,Y.et al.: "Chicken Histone deacetylase-2 controls the amount of the IgM H-chain at the steps of both transcription"J.Biol.Chem.. 274. 23977-23990 (1999)

Takami,Y.et al.：“鸡组蛋白脱乙酰酶 2 在两个转录步骤中控制 IgM H 链的量”J.Biol.Chem.. 274. 23977-23990 (1999)

Nakayama, T. et al.: "Interaction of the p48 subunit of chicken chromatin assembly factor-1 with histone deacetylases"Current Topics in Biochemical Research. 1. 173-178 (1999)

Nakayama, T. 等人：“鸡染色质组装因子 1 的 p48 亚基与组蛋白脱乙酰酶的相互作用”生化研究当前主题。

Ahmad,A.et al.: "Wdrepeats of the p48 subunit of chicken chromatin assembly factor-1 required for in vitro interaction"J.Biol.Chem.. 274. 16646-16653 (1999)

Ahmad, A. 等人：“体外相互作用所需的鸡染色质组装因子 1 的 p48 亚基的 Wdrepeats”J.Biol.Chem.. 274. 16646-16653 (1999)