PUMA在血细胞重编程中的表观遗传调控研究

Reprogramming technology such as induced pluripotent stem cells (iPSC) holds a great promise in regenerative medicine. However, further improvements in reprogramming efficiency and quality measures are the key to its successful applications in the clinic. Epigenetic regulation is a driving force for cellular reprogramming. Our previous studies demonstrated that different epigenetic states represented in different types of blood cells determine the efficiency of reprogramming as shown by nuclear transfer. On other hand, disruption of epigenetic makeup may also cause cancer. We have recently demonstrated an essential role of biallelic mutations of SETD2 (encoding a histone H3K36 methyltransferase) in both initiation and progression of human leukemia. In addition, reprogramming and tumor development are shown to share the p53 pathway- the most representative and broadly operated suppressive mechanism in human cancers. We have recently identified PUMA as a unique target in the p53 pathway, as PUMA deficiency led to a better survival rate associated surprisingly with reduced DNA damage and fewer chromosomal aberrations in iPSC whereas loss of p21 or p53 resulted in an opposite outcome. Based on these previous results, in our current proposal, we plan to firstly identify new epigenetic factors which regulate PUMA expression during blood cell reprogramming toward iPSC. We will then study the functional role of those new epigenetic factors, Finally, we will optimize the current reprogramming protocols for human blood cells toward iPSC with high quality involving the use of new combinations of reprogramming factors.

重编程技术在再生医学中有巨大潜力，提高体细胞重编程效率及编程后细胞质量是其临床应用需要突破的关键技术瓶颈。我们前期的工作表明，不同分化阶段血细胞的表观遗传状态影响了体细胞重编程效率；单一表观遗传基因SETD2突变后，导致全基因组H3K36me3甲基化缺失，促进血细胞恶性转化、导致白血病发生和发展；下调抑癌基因p53信号通路的下游基因Puma，可以提高诱导多能干细胞（iPSC）的生成效率，但不影响基因组的稳定性。在本课题中，我们将以p53- PUMA信号通路中PUMA基因作为靶点，首先，通过高通量测序筛选测序重编程过程中对PUMA有调控作用的新的表观遗传因子；其次，验证新的表观遗传因子对血细胞重编程效率及基因组稳定性的影响；最后，通过新的重编程因子组合，优化重编程体系，确立高效、安全的iPSC诱导方法，为iPSC的临床应用提供可靠的依据。

多能干细胞在再生医学中有巨大潜力，提高多能干细胞的质量和安全性是临床应用亟待解决的问题。我们从细胞恶性转化和基因组稳定性密切相关的P53-PUMA这一独特且重要的信号通路入手，阐明PUMA在放射损伤条件下不产生肿瘤的作用机制及对人多能干细胞质量和安全性的影响。我们发现，在小鼠多能干细胞中抑制PUMA表达可以显著减低放射条件下γH2AX的表达，PUMA缺失抑制Fbh1进入细胞核进而抑制RAD51泛素化，提高同源重组修复；进一步，抑制PUMA的表达可以提高人多能干细胞生存和体外分化造血干祖细胞和血细胞的形成；同时，PUMA的小分子抑制剂可以使人重编程效率提高2.3倍；为多能干细胞的临床应用奠定了基础。我们也发现， Dnmt3b的缺失加速MLL-AF9白血病进展，并能够与Dnmt3a缺失协同促进白血病发展，为DNA甲基化转移酶在白血病发生中的作用提供了新的见解。