Analysis of regulatory mechanisms of chromatin phosphorylation by kinases

我们通过使用染色质组装因子NAP-1、ACF和拓扑异构酶I在体外重建完全由重组历史组成的染色质。通过微球菌消化和超螺旋分析，发现重组染色质含有约19个规则排列的核小体。使用这种染色质，我们发现极光激酶磷酸化组蛋白H3的丝氨酸10和28有效，而MSK 1未能做到这一点，表明磷酸化组蛋白H3的MSK 1被显着抑制掺入染色质。在进一步添加CREB、ART 1、SRF和Elk-1后，MSK 1磷酸化染色质内的组蛋白H3。相比之下，即使在染色质中加入相同的转录因子，极光激酶的磷酸化也没有增加。GST pull-down分析表明，这4种组蛋白均能与Aurora A相互作用，但不能与MSK 1相互作用，而MSK 1能与4种激活剂相互作用，这与激活剂磷酸化染色质包埋的组蛋白H3的要求一致。有趣的是，这种磷酸化不依赖于激活剂的存在。对MSK 1缺失突变体的分析表明，MSK 1具有两个CREB相互作用结构域，这两个结构域可能都受到分子内相互作用的抑制。事实上，去除N-末端50个氨基酸恢复了CREB介导的组蛋白H3磷酸化，表明该特定区域是MSK 1内的主要抑制结构域。结果表明，分子内抑制通过p38的磷酸化以及与CREB的相互作用而被解除，揭示了这种代谢的组蛋白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.