P-TEFb, the super elongation complex and mediator regulate a subset of non-paused genes during early Drosophila embryo development.
P-TEFb, the super elongation complex and mediator regulate a subset of non-paused genes during early Drosophila embryo development.
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DOI：
10.1371/journal.pgen.1004971
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发表时间：
2015-02
期刊：
影响因子：
4.5
通讯作者：
Mannervik M
中科院分区：
文献类型：
--
作者：
Dahlberg O;Shilkova O;Tang M;Holmqvist PH;Mannervik M
正转录延伸因子B（P-TEF B）是由Cdk 9和细胞周期蛋白T组成的激酶，其释放RNA聚合酶II（Pol II）进入主动延伸。它可以组装成一个更大的超伸长复合物（SEC），由额外的延伸因子组成。在这里，我们使用基于miRNA的方法来敲低早期果蝇胚胎中P-TEFb和SEC组分的母体贡献。P-TEFb或SEC耗竭导致胚胎后部细胞损失和细胞化缺陷。有趣的是，在P-TEFb胚胎中，许多含有启动子近端暂停的Pol II的模式化基因的表达相对正常。相反，P-TEFb和SEC是前细胞胚胎中一些非暂停的快速转录基因表达所必需的，包括细胞化基因Serendipity-α。我们还证明了另一个P-TEFb调控基因，末端，在胚胎发育中具有重要的功能。在敲低介体亚基后观察到相似的形态学和基因表达表型，提供了P-TEFb、SEC和介体在转录控制中协作的体内证据。令人惊讶的是，P-TEFb缺失不影响启动子处Pol II相对于3'末端的比率，尽管影响总体Pol II Ser 2磷酸化水平。相反，在P-TEFb下调的基因处Pol II占据减少。我们的结论是，一个子集的非暂停，前细胞基因是最容易减少P-TEFb，SEC和调解人在果蝇胚胎中的水平。胚胎发育涉及通过基因转录的调节形成各种细胞类型，导致细胞类型特异性RNA和蛋白质的表达。动物基因转录的关键调控步骤涉及RNA聚合酶II（Pol II）转变为主动延伸。在许多基因中，Pol II在转录起始位点下游约50个碱基对处瞬时暂停。这种启动子近端暂停的释放涉及激酶P-TEFb，其磷酸化负延伸因子，使Pol II进入有效延伸。在这项工作中，我们已经耗尽了相当数量的P-TEFb从早期果蝇胚胎。我们发现，几个暂停Pol II基因可以在P-TEFb耗竭胚胎中相对正常地表达，而一些非暂停基因的表达则大幅减少。该结果表明，非暂停基因在进入主动延伸之前也通过P-TEFb依赖性检查点。出乎意料的是，我们在P-TEFb胚胎中发现与这些非暂停基因相关的Pol II较少。我们证明，参与招募Pol II启动子的蛋白质复合物，介体复合物，表现出与P-TEFb相同的形态和基因表达表型。我们建议，调解人和P-TEFb功能在招聘Pol II的一个子集的发育基因。
Positive Transcription Elongation Factor b (P-TEFb) is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II) into active elongation. It can assemble into a larger Super Elongation Complex (SEC) consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-α. We also demonstrate that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3’ end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos. Embryo development involves formation of various cell types through the regulation of gene transcription, resulting in expression of cell type specific RNAs and proteins. A key regulatory step in transcription of animal genes involves the transition of RNA polymerase II (Pol II) into active elongation. At many genes, Pol II is transiently paused approximately 50 basepairs downstream of the transcription start site. Release from this promoter-proximal pausing involves the kinase P-TEFb, which phosphorylates negative elongation factors, allowing Pol II to enter into productive elongation. In this work, we have depleted a considerable amount of P-TEFb from early Drosophila embryos. We find that several genes with paused Pol II can be expressed relatively normally in P-TEFb depleted embryos, whereas expression of some non-paused genes is substantially reduced. This result suggests that also non-paused genes transit through a P-TEFb-dependent checkpoint before entering active elongation. Unexpectedly, we find less Pol II associated with these non-paused genes in P-TEFb embryos. We demonstrate that a protein complex involved in recruitment of Pol II to promoters, the Mediator complex, show the same morphological and gene expression phenotypes as P-TEFb. We propose that Mediator and P-TEFb function together in recruiting Pol II to a subset of developmental genes.
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DOI：
10.1016/j.cub.2009.02.055
发表时间：
2009-04-28
期刊：
Current biology : CB
影响因子：
--
作者：
Chopra VS;Hong JW;Levine M
通讯作者：
Levine M
影响因子：
64.5
作者：
Guenther, Matthew G.;Levine, Stuart S.;Young, Richard A.
通讯作者：
Young, Richard A.
影响因子：
11.4
作者：
Gerber, M;Ma, JY;Shilatifard, A
通讯作者：
Shilatifard, A
影响因子：
14.9
作者：
Nguyen D;Krueger BJ;Sedore SC;Brogie JE;Rogers JT;Rajendra TK;Saunders A;Matera AG;Lis JT;Uguen P;Price DH
通讯作者：
Price DH
影响因子：
10.5
作者：
JIANG, J;HOEY, T;LEVINE, M
通讯作者：
LEVINE, M