Mkk6解离异染色质对小鼠体细胞重编程的调控

Somatic cell reprogramming is an ideal model to study chromatin remodeling. Previously, we described a fluorescence recovery after photobleaching method to assess the dynamics of heterochromatin/euchromatin and showed significant heterochromatin relaxation at the initial stage of reprogramming. We identified Gadd45a as a chromatin relaxer which also enhances reprogramming efficiency (EMBO R 2016). Except for Gadd45a, we have screened out a kinase, Mkk6, which not only relaxes heterochromatin, but also improves reprogramming. However, how Mkk6 mediates heterochromatin relaxation, then promotes reprogramming is unknown. With further study, we found Mkk6 enhancing reprogramming depends on its kinase activity, but does not depend on its classic downstream target P38. We propose new target proteins that phosphorylated by Mkk6 in reprogramming, and play important roles in the regulation of chromatin remodeling and cell fate transition. In this project, we intend to use Phosphoproteomics analysis, RNA-Seq, co-immunoprecipitation and ChIP methods to identify the phosphorylated target proteins of Mkk6 in reprogramming, research and verify their function in heterochromatin relaxation, and in regulation of reprogramming process. Our study will reveal the new phosphorylated targets of Mkk6 and the new regulatory approaches to chromatin remodeling, and elucidate the mechanism of Mkk6 promoting somatic reprogramming.

体细胞重编程是研究染色质重塑的理想模型。我们在前期实验中建立了染色质解离动态研究技术，发现了重编程早期异染色质的解离规律，并筛选出一个新的染色质解离蛋白Gadd45a，其能促进重编程（EMBO R 2016）。此外我们还筛选到激酶Mkk6，能解离异染色质，也能显著提高重编程效率。但是Mkk6究竟是如何介导异染色质解离，进而促进重编程的尚不清楚。进一步研究发现Mkk6促进重编程依赖于其激酶活性，但不依赖于其经典靶标P38，我们认为Mkk6有新的磷酸化靶蛋白，并在调控染色质重塑和细胞命运转换中起重要作用。本项目拟采用磷酸化蛋白组学分析、RNA-Seq、免疫共沉淀、ChIP等方法，鉴定出重编程过程中Mkk6新的磷酸化靶蛋白，研究和验证其受Mkk6磷酸化后参与解离异染色质，而对重编程过程的调控。本项目有望揭示Mkk6新的磷酸化靶标及对染色质重塑新的调控途径，阐明Mkk6促进体细胞重编程的作用机理。

体细胞重编程是研究染色质重塑和表观遗传的理想模型。我们在早期实验中描述了重编程早期的异染色质解离规律，并筛选鉴定出一些染色质解离蛋白如Gadd45a、Mkk6等，它们也能显著提高重编程效率。在本项目中，我们研究了Mkk6解离异染色质和促进重编程的机制。我们发现Mkk6促进重编程依赖于其激酶活性，但不依赖于其经典靶标P38。我们用磷酸化蛋白组学分析鉴定出Mkk6新的磷酸化靶蛋白Gatad2b。Mkk6通过磷酸化Gatad2b可以上调组蛋白乙酰化和解离异染色质，从而促进Sox2和Klf4粉背对其下游基因的结合和多能性基因的表达。我们的研究揭示了Mkk6通过磷酸化调控染色质状态进而决定细胞命运的新功能。