组蛋白去乙酰化酶亚家族class IIa HDAC在调控iPS重编程和维持胚胎干细胞多能性的机制研究

The reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) is a groundbreaking discovery with deep implications for regenerative medicine, disease modeling and drug development. Yet, to fulfill these promises it is necessary an improved understanding of the mechanisms of this technology, because it still has many caveats. We have previously demonstrated that a process of epithelialization or mesenchymal-to-epithelial transition (MET) is a central stage of reprogramming and can thus be used to divide the process into two phases. More recently, we have also shown that a subfamily of histone deacetylases (HDACs), the class IIa HDACs, regulate the MET and enhance reprogramming through transcription factor MEF2 by controlling the secretion of Tgf-beta cytokines. Importantly, our unpublished data show as well that nuclear accumulation of class IIa HDACs helps MET significantly but blocks subsequent reprogramming completely. In this grant proposal we want to pursue two main objectives. First, we want to dissect in detail how class IIa HDACs induce the MET phase of reprogramming, and second we wish to understand how they block it in the second phase. For these purposes we will use a wide range of technologies well-set up in our laboratory. Our specific hypotheses are that class IIa HDACs regulate the MET at multiple levels and not just through Tgf-beta cytokines, and also that class IIa HDACs modify the pluripotent transcription factor Klf4 to prevent the final jump to a pluripotent state, for which we have very promising preliminary data. In addition, revealing the function of class IIa HDACs in pluripotent stem cells is also necessary because this will help us understand better their role in regulating pluripotent genes. Therefore, our work is highly relevant because it will provide a deeper understanding of the mechanisms of reprogramming, and may also have implications at other levels, for example in the epithelial-to-mesenchymal transition of cancer or fibrosis. Moreover, we would be the first laboratory to show that a dynamic modification of pluripotent transcription factors (specifically Klf4 but potentially others as well) during reprogramming is an instrumental event driving the epigenetic transformation. In fact, we expect that manipulation of the posttranslational modifications in Klf4 will allow the generation of iPSCs more quickly, more efficiently and more safely.

掌握iPS细胞的重编程机制能帮助摆脱目前iPS应用的局限性。之前我们发现成纤维细胞发生间质向上皮样转化（MET）是iPS重编程进行的必然过程。最近，我们在研究组蛋白去乙酰化酶（HDAC）时发现其IIa型亚家族可通过抑制MEF2转录因子来促进MET的发生，进而提高重编程效率。我们还发现持续核内表达的IIa型HDAC突变体虽能诱导高水平的MET，但随后却彻底抑制了iPS细胞的生成。这说明IIa型HDAC在重编程前后阶段起了不同的功能。根据这些初步结果，我们拟进行以下研究：Tgfβ信号通路作为抑制重编程中MET的主要因素受到IIa型HDAC调控的具体机制是什么；IIa型HDAC在MET后抑制重编程的作用是否通过抑制外源重编程因子Klf4的转录活性而起作用的。理解这里面的调控关系不仅能帮助我们理解重编程机制，同时也揭示了一种通过翻译后修饰来调控重编程的新方式。

体细胞重编程是一个多步骤，低概率的细胞命运转换事件。在这个过程中，细胞内的染色质状态和基因表达要经历巨大的变化。而这些变化，是由过表达的四个Yamanaka因子，即SOX2， KLF4， OCT4和c-MYC来共同启动的。这些转录因子可以招募表观调节因子，将其靶定到基因组，从而对基因组的修饰和基因的表达实施调控。目前关于多能性基因难以有效激活的表观调控机制尚未完全阐明。本项目主要研究体细胞重编程过程中组蛋白去乙酰化酶调控组蛋白去乙酰化在重编程中的作用，我们发现衰减内源性的class IIa HDAC因影响了重编程细胞早期的MET过程而降低了重编程效率。相反，若过表达核内的的class IIa HDAC虽促进了重编程早期的MET过程，但是抑制了重编程后期多能性基因的激活，且这种抑制作用是依赖于NCoR/SMRT-HDAC3抑制复合物的作用。另外，我们也发现在重编程过程中，SOX2， KLF4，OCT4和c-MYC，尤其是c-MYC可以将共抑制复合物NCoR/SMRT-HDAC3招募到基因组。该复合物的酶活性中心，HDAC3则对靶位点的组蛋白乙酰化修饰进行调节，降低其乙酰化水平，抑制基因的表达。在重编程前期，NCoR/SMRT-HDAC3复合物被招募到基因组，促进高表达的体细胞基因沉默，有利于重编程；而在重编程后期，NCoR/SMRT-HDAC3复合物被招募到多能性基因位点，抑制多能性基因的有效激活，阻碍重编程。干扰NCoR/SMRT-HDAC3复合物的功能会显著地提高重编程效率，说明该复合物对多能性基因的抑制作用是重编程的主要障碍之一。通过该研究，我们揭示了NCoR/SMRT共抑制蛋白家族和HDAC3在重编程过程中对基因表达的抑制作用,也揭示了c-MYC在重编程前期和后期的不同功能，至少在很大程度上解释了为什么c-MYC的存在会导致更多的不完全重编程细胞即pre-iPSCs、组蛋白去乙酰化酶抑制剂如VPA和Butyrate在四因子的条件下能发挥更为明显的功能。同时，该研究对于c-MYC和NCoR/SMRT-HDAC3在其他体系如癌症和发育中的作用机制提出了新的可能性。