Joint study on histone modifying enzymes by gene disruption

在过去的几年中，已经在真核生物中鉴定了许多HDAC家族成员，如HAT家族成员。我们已经克隆了编码三种鸡HDAC（chHDAC-1、2和3）的cDNA和基因组DNA。chHDAC-3定位在细胞核和细胞质中，尽管chHDAC-1和2优先定位在细胞核中。利用针对DT 40鸡B细胞系的基因打靶技术，我们分别产生了各种DT 40突变体，缺乏chHDAC-1、2和3，以及六种H1变体和一种H2 B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）突变体中的蛋白质模式分别发生了改变，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和核心变体在调节特定基因的表达中应该发挥各自的作用，可能是通过改变特定基因组中的染色质结构。 ...更多信息 2）chHDAC-1对蛋白质模式变化的参与很小，但chHDAC-2主要参与IgM H链的积累。chHDAC-2在其基因的转录和其前体mRNA的交替加工步骤中控制IgM H链的量。3）chHDAC-3对于DT 40细胞的生存力是必需的。4）chHDAC-3的1-23个N-末端氨基酸和389-417个C-末端氨基酸对于细胞生存力是必需的。chHDAC-3的适当内斯对于核输出和活力功能都是必需的。chHDAC-3的脱乙酰化活性对于其活力功能也是必需的。此外，我们还研究了CAF-1的p46和p48亚基与HAT-1或组蛋白H2 B和H4的相互作用，得到了一些值得注意的结论，5）p46多肽的不同区域是其与组蛋白H2 B和H4以及chHAT-1的体外相互作用所必需的。6）chHAT-1的亮氨酸拉链基序1对于与chCAF-1的p48亚基的体内和体外相互作用是必需的。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