Joint study on histone modifying enzymes by gene disruption

在过去的几年里，许多HDAC家族成员，如HAT家族成员，已经在真核生物中被鉴定出来。我们已经克隆了鸡三种hdac， chhdac - 1,2,3的cdna和基因组dna。chHDAC-3定位于细胞核和细胞质中，而chHDAC-1和chhdac - 2优先定位于细胞核中。利用鸡DT40 B细胞系的基因靶向技术，我们分别获得了多种缺乏chHDAC-1、2和3的DT40突变体，以及6种H1突变体和1种H2B突变体。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)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。这些变异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来调节特定基因的表达。2) chHDAC-1参与蛋白模式变化较少，而chHDAC-2则主要参与IgM h链的积累。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。3) chHDAC-3对DT40细胞的生存至关重要。4) chHDAC-3的1-23个n端氨基酸和389-417个c端氨基酸都是细胞生存所必需的。核出口和生存能力功能都需要chHDAC-3的适当NES。chHDAC-3的去乙酰化活性也是其生存功能所必需的。此外，我们还研究了CAF-1的p46和p48亚基与HAT-1或组蛋白H2B和H4的相互作用，得到了一些值得注意的结论，如下。5) p46多肽在体外与组蛋白H2B、H4和chhat -相互作用需要不同的区域。1.6)chHAT-1的亮氨酸拉链基序在体内和体外与chca -1的p48亚基相互作用是必需的。少

In the past several years a number of HDAC family members, like those of HAT family members, have been identified in eukaryotes. We have cloned cDNAs and genomic DNAs encoding the three chicken HDACs, chHDAC-1, 2 and 3. chHDAC-3 was localized in both nuclei and the cytoplasm, although chHDAC-1 and 2 were preferentially localized in nuclei. Using the gene targeting technique for the DT40 chicken B cell line, we generated various DT40 mutants, respectively, devoid of chHDAC-1,2 and 3, and six H1 variants and one H2B variant. Systematic analyses of the resultant mutants led us to some noticeable conclusions, as follows.1) 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 genom … More ic regions.2) The participation of chHDAC-1 in protein pattern changes is slight, but chHDAC-2 predominantly participates in the accumulation of the IgM H-chain. 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.3) chHDAC-3 is essential for the viability of DT40 cells.4) Both the 1-23 N-terminal amino acids and the 389-417 C-terminal amino acids of chHDAC-3 are essential for the cell viability. The proper NES of chHDAC-3 should be required for both the nuclear export and viability function. The deacetylation activity of chHDAC-3 is also necessary for its viability function. In addition, we have studied the interactions of p46 and p48 subunits of CAF-1 with HAT-1, or histones H2B and H4, and then obtained some noticeable conclusions, as follows.5) Distinct regions of the p46 polypeptide are required for its in vitro interaction with histones H2B and H4 and chHAT-1.6) Leucine zipper motif of chHAT-1 is essential for in vivo and in vitro interactions with the p48 subunit of chCAF-1. Less