Joint study on histone modifying enzymes by gene disruption

在过去的几年里，在真核生物中已经发现了一些HDAC家族成员，如HAT家族成员。我们克隆了编码鸡hHDAC-1、chHDAC-2和chHDAC-3的cDNAs和基因组DNA。chHDAC-3定位于细胞核和细胞质，但chHDAC-1和2优先定位于细胞核。利用基因打靶技术对DT40鸡B细胞系进行基因打靶，分别获得了不含chHDAC-1、2和3的各种DT40突变体，以及6个H1突变体和1个H_2B突变体。对产生的突变体进行系统分析后，我们得出了一些值得注意的结论，如下：1)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和核心变异体可能通过改变特定基因组…中的染色质结构，在特定基因的表达调控中发挥单独的作用2)chHDAC-1在蛋白质模式改变中的参与很少，但chHDAC-2主要参与IgM H链的积累。3)chHDAC-3是DT40细胞生存所必需的。4)chHDAC-3的1-23个N端氨基酸和389-417个C端氨基酸都是细胞存活所必需的。核出口和生存功能都需要chHDAC-3的适当NES。ChHDAC-3的脱乙酰基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H_2B和H_4的相互作用，得到了一些值得注意的结论：5)p46多肽与组蛋白H_2B和H_4以及Chhat-1.6)在体内和体外与chCAF-1的P_48亚基的相互作用中，亮氨酸拉链基序是必不可少的。较少

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