Cdk9 is an essential kinase in Drosophila that is required for heat shock gene expression, histone methylation and elongation factor recruitment
Cdk9 is an essential kinase in Drosophila that is required for heat shock gene expression, histone methylation and elongation factor recruitment
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DOI：
10.1007/s00438-006-0164-2
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发表时间：
2007-02-01
影响因子：
3.1
通讯作者：
Michener, Dorian E.
中科院分区：
文献类型：
--
作者：
Eissenberg, Joel C.;Shilatifard, Ali;Michener, Dorian E.
大RNA聚合酶II亚基C末端结构域（CTD）的磷酸化被认为在启动子清除和募集在信使RNA合成和加工中起作用的蛋白质因子中是重要的。P-TEFb是一种靶向CTD的蛋白激酶。本研究的目标是鉴定需要体内P-TEFb活性的染色质修饰和关联。我们敲低P-TEFb的催化亚基，Cdk 9，在果蝇使用RNA干扰。cdk 9基因敲除果蝇在变态过程中死亡。CTD七肽重复序列的丝氨酸2和丝氨酸5处的磷酸化在敲低幼虫中均显著降低。Cdk 9基因敲除的幼虫热休克诱导Hsp 70 mRNA的表达减弱。组蛋白H3在赖氨酸4处的单甲基化和三甲基化均显著降低，表明CTD磷酸化与体内转录染色质中的组蛋白甲基化之间存在联系。染色体解旋酶蛋白质CHD 1的水平在Cdk 9敲低的染色体中降低，表明CHD 1通过P-TEFb依赖性组蛋白甲基化靶向染色体。组蛋白H3在赖氨酸36位的二甲基化在敲低幼虫中显著降低，暗示CTD磷酸化在这种染色质修饰的调节中。RNA聚合酶II延伸因子ELL的结合在敲低染色体中减少，表明ELL通过CTD磷酸化被募集到活性聚合酶中。
Phosphorylation of the large RNA Polymerase II subunit C-terminal domain (CTD) is believed to be important in promoter clearance and for recruiting protein factors that function in messenger RNA synthesis and processing. P-TEFb is a protein kinase that targets the (CTD). The goal of this study was to identify chromatin modifications and associations that require P-TEFb activity in vivo. We knocked down the catalytic subunit of P-TEFb, Cdk9, in Drosophila melanogaster using RNA interference. Cdk9 knockdown flies die during metamorphosis. Phosphorylation at serine 2 and serine 5 of the CTD heptad repeat were both dramatically reduced in knockdown larvae. Hsp 70 mRNA induction by heat shock was attenuated in Cdk9 knockdown larvae. Both mono- and trimethylation of histone H3 at lysine 4 were dramatically reduced, suggesting a link between CTD phosphorylation and histone methylation in transcribed chromatin in vivo. Levels of the chromo helicase protein CHD1 were reduced in Cdk9 knockdown chromosomes, suggesting that CHD1 is targeted to chromosomes through P-TEFb-dependent histone methylation. Dimethylation of histone H3 at lysine 36 was significantly reduced in knockdown larvae, implicating CTD phosphorylation in the regulation of this chromatin modification. Binding of the RNA Polymerase II elongation factor ELL was reduced in knockdown chromosomes, suggesting that ELL is recruited to active polymerase via CTD phosphorylation.