KRAB型锌指蛋白ZNF599对细胞核形态的影响及其机制研究

As the control center of the eukaryotic cells, the cell nucleus is precisely regulated to keep the cell’s normal morphology and function. Nuclear distortion is related to different kinds of diseases such as progeria, Parkinson’s disease and tumor. Our recent work uncovered a previously uncharacterized protein-KRAB zinc finger protein ZNF599 whose disruption leads to an irregular nuclear shape with blebbings, dents and ridges. However, the underlying mechanism is largely unknown. SUN4 belongs to SUN family of proteins and acts as an important component of nuclear membrane. Ectopic expression of SUN4 in wild type cells shows a similar effect on nuclear morphology as observed in ZNF599 knock out cells. Besides, we observed that the expression of SUN4 significantly increased in ZNF599 knock out cells, which can be rescued to that of wild type cells in ZNF599 knock out cells stably expressing GFP-ZNF599. Based on the domain structure analysis and its intracellular localization, we hypothesize that ZNF599 plays an important role in nuclear envelope organization by exerting epigenetic regulation of transcriptional repression of SUN4. To test this hypothesis, we will investigate transcription repression activity of ZNF599, the binding of ZNF599 on SUN4 gene, the level of histone modifications of SUN4 as well as protein expression level in ZNF599 knock out cells compared to wild type cells. These experiments will help us determine the mechanism by which ZNF599 regulates nuclear shape by controlling the expression of nuclear shape related factor SUN4. To achieve the objective of our project, we will use genomics sequencing techniques including ChIP-seq and RNA-seq to determine the target genes of ZNF599 and nuclear shape related pathways. The outcome of this study will help us determine the detailed mechanism of nuclear shape regulation, and also provide new insights into the pathology of related human diseases.

细胞核是细胞的遗传控制中心，其形态受到严格调控以保证其功能正常发挥。核形畸变与早老症、帕金森和肿瘤等多种疾病相关。我们前期工作发现一个全新蛋白-KRAB型锌指蛋白ZNF599的缺失导致细胞核形畸变，但其影响核形的分子机制完全未知。SUN4作为LINC复合物成员，是构成核膜的重要组分；我们发现SUN4过表达可使核形畸变，而且SUN4表达水平在ZNF599敲除后显著增加，回转ZNF599可使其恢复至正常。结合分析ZNF599的核内定位及结构特征，我们推测ZNF599通过表观遗传学机制抑制性调节SUN4，从而参与核形调控。为验证该假说，本项目拟通过检测ZNF599对SUN4的转录抑制活性，其在SUN4基因区域的结合以及其敲除对该区域组蛋白修饰水平的影响来阐明ZNF599的核形调控机制。同时利用ChIP和RNA测序技术筛选其靶标基因和参与通路，系统阐明其核形调控途径，为相关疾病的机制研究提供基础。