Prdm16在脊椎动物断肢再生与肢体发育中的功能及转录表达机制研究

两栖动物爪蛙可以再生断肢，但局限于肢体发育早期，晚期肢体则不再生。因此对爪蛙断肢再生调控机制的深入了解有助于诱导脊椎动物断肢再生。在前期两个独立研究中，我们发现Prdm16是断肢再生中起重要作用的Evi5的下游因子，Prdm16亦能诱导多能干细胞分化为肢芽前体细胞。功能分析表明Prdm16是爪蛙肢体发育及断肢再生所必需的，而其过表达则可以刺激晚期爪蛙的断肢再生。Prdm16是锌指转录因子，其PR结构域可能有甲基转移酶活性。我们初步实验结果亦显示其可以调控组蛋白修饰。但Prdm16在肢体发育与再生的作用及机制尚不清楚。本项目拟以转录组学、组蛋白修饰、ATAC-seq及CRISPR/dCas9-ChIP-MS多种手段，阐释Prdm16在肢体发育与断肢再生中的作用机制及其转录表达调控机理，探讨表观遗传调控对断肢再生的影响，为进一步诱导断肢再生提供理论基础。

The anuran amphibian Xenopus can regenerate the limb, but only in the young tadpole stages. Understanding of the molecular mechanisms underlying this ontogenetic decline of limb regeneration may provide critical insights for stimulating limb regeneration in mammals. .We have investigated the function and molecular targets of Xenopus evi5, a gene that is highly upregulated in the regenerating salamander limb but not changed in the non-regenerating Xenopus frog limb stumps. One candidate gene we found was prdm16. This gene was also identified in our separate screening of transcription factors that could induce limb progenitor-like cells from pluripotent stem cells. These findings suggested the importance of Prdm16 in limb regeneration..Our ongoing experiments confirmed that Prdm16 is required for Xenopus tadpole limb regeneration, as knocking-down of prdm16 expression by morpholinos inhibits limb regeneration, while over-expression of prdm16 DNA induce limb regeneration in the regeneration-deficient late-stage tadpoles. Interestingly, we found that prdm16 down expression also interferes with limb development. Thus, Prdm16 regulates limb regeneration and limb development in the Xenopus tadpoles. However, the molecular mechanism is not clear..Prdm16 contains multiple zinc finger domains, and a PR/SET domain at the N-terminus. This suggests that Prdm16 has the potential to function as a methyl transferase, in addition of being a transcription factor. However, whether and how Xenopus Prdm16 modify the epigenetic status of the limb blastema cells remains to be explored. .Thus in this study, we will analyze how Prdm16 regulates limb regeneration by transcriptome analysis, Histone modification profiling, and ATAT-seq analysis. We will also utilize the recently developed CRISPR/dCas9-ChIP-MS system to understand the transcriptional regulation of prdm16 gene in the developing and regenerating limb. The finding of this study will facilitate the development of measurements for further stimulating limb regeneration.