晶状体纤维细胞脱核是晶状体正常发育的关键环节之一，对形成具有聚焦功能的透明晶状体至关重要。纤维细胞脱核障碍又是导致多种先天性白内障的直接原因。 然而，晶状体纤维细胞脱核的机制目前尚不清楚。我们的前期研究结果显示多种原因引起泛素介导的p27降解阻滞，导致Cdk1 抑制和纤维细胞脱核受阻、 晶状体发育异常和先天性白内障。 根据前期实验结果及相关文献，我们提出如下的科学假说：泛素介导的p27降解、Cdk1激活、Brg1 表达和染色质重塑是晶状体纤维细胞脱核的核心机制。本项目拟通过利用自建的K6W泛素转基因小鼠和p27敲除小鼠为模型，研究在晶状体发育过程中上述分子之间的相互作用及信号传导途径。 通过研究泛素介导的p27降解、Cdk1激活、 核膜纤层蛋白磷酸、核膜裂解、Brg1表达、染色质修饰、DNA酶II 表达及入核等环节， 阐明晶状体纤维脱核的调控机制，为防治先天性白内障提供理论和实验依据。

Ocular lens is avascular transparent organ comprised of only two types of cells called lens epithelial cells and lens fiber cells. Lens fiber cells are differentiated from lens epithelial cells. Coordinated denucleation and removal of other organelles are critical for lens transparency. Failure to execute the denucleation process in the lens fibers results in abnormal lens fiber cell differentiation and cataract formation. Many congenital cataracts are associated with retention of fiber nuclei in center of the lenses. However, the molecular mechanisms for the controlled denucleation process during lens development remains largely unknown. Our recent work demonstrated that ubiquitin-dependent degradation of p27 and activation of Cdk1 are required for lens fiber denuleation. Impairment of the ubiquitin-dependent p27 degradation is causally associated with retention of the nuclei and congenital cataracts in several animal models. It was reported that Brg1 was also essential in lens fiber denucleation process via modulating chromatin remodeling. Although previous studies demonstrate that ubiquitin plays a role in chromatin remodeling and regulation gene expression, but the role of ubiquitin-dependent p27 degradation and Cdk1 activation in regulating Brg1 expression and chromatin remodeling has not been reported. In this proposed project, we will utilize the novel K6W- mutant ubiquitin transgenic mice and p27 knockout/knockin mice as model systems to elucidate function of ubiquitin-dependent p27 degradation in regulating Cdk1 activation, Brg1 expression and chromatin remodeling as well as their upstream signals in lens fiber denucleation process during lens development and fiber differentiation. The specific aims of this proposal are: 1) systematically compare wt lens and K6W-mutant-ubiquitin-expressing lenses by histological, biochemical, proteomic and epigenical approaches in order to identify key molecules that are controlled by the ubiquitin during lens fiber differentiation and essential for denucleation process; 2) to determine the role of ubiquitin-dependent p27 degradation in controlling Brg1 expression and chromatin modifications during lens fiber differentiation and denucleation; 3) to investigate the role of ubiquitin-dependent p27 degradation in regulating Cdk1 activation, phosphorylation and disassemble of nuclear envelope, as well as degradation of nuclear DNA during lens fiber differentiation and maturation. All together, successful completion of the proposed experiments will advance our understanding of the critical molecular controls for removal of nuclei and other organelles in order to establish lens transparency. This information will provide guidance for prevention and treatment of congenital cataracts that are associated with lens fiber nuclei retention in the center of the lenses.