Analysis of regulatory mechanisms of chromatin phosphorylation by kinases

激酶对染色质磷酸化的调控机制分析

基本信息

批准号：
18570164
负责人：
HISATAKE Koji
金额：
$ 2.49万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18570164/
关键词：
chromatin phosphorylation transcription expression regulation 遺伝子キナーゼ

项目摘要

We reconstituted chromatin in vitro that were composed entirely of recombinant histories via use of chromatin assembly factors, NAP-1, ACF and Topoisomerase I. The reconstituted chromatin was found to contain about 19 nucleosomes in a regular array by micrococcal digestion and supercoiling assays. Using this chromatin, we found that Aurora kinase phosphorylated histone H3 at serine 10 and 28 efficiently; while MSK1 failed to do so, indicating that the phosphorylation of histone H3 by MSK1 is inhibited dramatically by incorporation into the chromatin. Upon further addition of CREB, ART1, SRF and Elk-1, MSK1 phosphorylated histone H3 within the chromatin. In contrast, such augmentation of phosphorylation was not seen for Aurora kinase even when the same set of transcription factors were added to the chromatin. GST pull-down assays revealed that the all the four histones did interact with Aurora Abut failed to interact with MSK1, and MSK1 interacted with all the four activators, consistent with the requirement for the activators to phosphorylate chromatin-embedded histone H3.We also found that HMGN1 facilitates the MSK1-mediated phosphorylation of histone H3 and during this process HMGN1 became phosphorylated at multiple sites by MSK1. Interestingly, this phosphorylation was not dependent upon the presence of activators. Analysis of the deletion mutants of MSK1 showed that the MSK1 possessed two interacting domains for CREB, both of which is presumably repressed by the intramolecular interactions. Indeed, removal of N-terminal 50 amino acids restored the CREB-mediated histone H3 phosphorylation, indicating that this particular region is the predominant repression domain within MSK1. The results suggested that the intra-molecular repression is de-repressed by the phosphorylation by p38 and by the interaction with CREB, revealing a complex regulatory network for this meted histone H3 phosphorylation.

我们通过使用染色体质组装因子，NAP-1，ACF和拓扑异构酶。在体外重新组成的染色质。发现重组的染色质因通过微球菌消化和超级涂料和超级涂料测定。使用该染色质，我们发现Aurora激酶在丝氨酸10和28处有效地磷酸化组蛋白H3。尽管MSK1未能做到这一点，但表明MSK1对组蛋白H3的磷酸化被掺入染色质而受到巨大抑制。在进一步添加CREB，ART1，SRF和ELK-1后，MSK1在染色质中磷酸化的组蛋白H3。相比之下，即使将相同的转录因子添加到染色质时，对于Aurora激酶，也没有看到这种磷酸化的增强。 GST pull-down assays revealed that the all the four histones did interact with Aurora Abut failed to interact with MSK1, and MSK1 interacted with all the four activators, consistent with the requirement for the activators to phosphorylate chromatin-embedded histone H3.We also found that HMGN1 facilitates the MSK1-mediated phosphorylation of histone H3 and during this process HMGN1 became phosphorylated在MSK1的多个站点上。有趣的是，这种磷酸化不取决于激活剂的存在。对MSK1的缺失突变体的分析表明，MSK1具有CREB的两个相互作用结构域，这两者都受到分子内相互作用的抑制。实际上，去除N末端50氨基酸可以恢复CREB介导的组蛋白H3磷酸化，表明该特定区域是MSK1中主要的抑制域。结果表明，通过p38的磷酸化和与CREB的相互作用，分子内的抑制作用被抑制，从而揭示了该METED组蛋白H3磷酸化的复杂调节网络。