Analysis of regulatory mechanisms of chromatin phosphorylation by kinases

我们利用染色质组装因子、NAP-1、ACF和拓扑异构酶I在体外重组了完全由重组历史组成的染色质。经微球菌消化和超螺旋实验发现，重组染色质含有大约19个核小体，排列规则。利用该染色质，我们发现Aurora激酶有效地在丝氨酸10和28处磷酸化组蛋白H3，而MSK1未能做到这一点，这表明MSK1对组蛋白H3的磷酸化被掺入染色质显著抑制。在进一步加入CREB、Art1、SRF和ELK-1后，MSK1使染色质中的组蛋白H3磷酸化。相反，即使将相同的转录因子添加到染色质中，极光激酶的磷酸化水平也没有这样的增强。GST下拉实验表明，这四个组蛋白都与Aurora结合，但不能与MSK1相互作用，而MSK1与所有四个激活剂相互作用，这与激活剂磷酸化染色质包埋组蛋白H3的要求一致。我们还发现，HMGN1促进了MSK1介导的组蛋白H3的磷酸化，在这个过程中，HMGN1被MSK1在多个位置磷酸化。有趣的是，这种磷酸化并不依赖于激活剂的存在。对MSK1缺失突变体的分析表明，MSK1有两个与CREB相互作用的结构域，这两个结构域可能都被分子内相互作用所抑制。事实上，去除N-末端50个氨基酸可以恢复CREB介导组蛋白H3的磷酸化，表明这个特定的区域是MSK1中的主要抑制域。结果表明，p38的磷酸化和与CREB的相互作用抑制了分子内的抑制，揭示了组蛋白H3磷酸化的复杂调控网络。

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.