Functional analysis of Polycomb-group gene, Pcgf5 in cardiac morphogenesis

为了明确多梳组蛋白（Polycomb group，PcG）Pcgf 5在心脏形态发生中的作用，以及了解PcG基因突变是否与人类先天性心脏病相关，对Pcgf 5在小鼠胚胎中的表达进行了详细分析，并利用Cre-loxP系统建立了条件性Pcgf 5基因敲除小鼠。在整装原位杂交分析中，在发育的早期阶段的心脏中观察到强的Pcgf 5表达，这意味着Pcgf 5可能参与心脏发育。Western blotting和免疫组化结果显示，PcG蛋白、Bmi 1、Ring 1b和Rae 28/Phc 1在胚胎心肌细胞和内皮细胞核中均有分布。这些蛋白的表达水平在早期阶段较高，并在围产期下降。免疫共沉淀分析（Co-IP）和原位近端连接试验（PLA）证实了Ring 1b与Bmi 1、Ring 1b与Rae 28/Phc 1在胚胎心脏发育过程中的物理相互作用，表明Bmi 1、Ring 1b和Rae 28/Phc 1在胚胎心脏中形成了与其他组织一样的Polycomb复合物。另一方面，Pcgf 5与Ring 1b相互作用，但不与Rae 28/Phc 1相互作用。这些发现表明，某些类型的多梳复合体在发育中的心脏中起作用。

To define the functions of one of the Polycomb group(PcG) protein, Pcgf5 for cardiac morphogenesis and know whether mutations of PcG genes are associated with human congenital heart disease, detail analysis of Pcgf5 expression in mouse embryos was performed, and conditional Pcgf5 knockout mice were generated using Cre-loxP system. In the whole-mount in situ hybridization analysis, strong Pcgf5 expression was observed in the developing heart at early stages, meaning Pcgf5 could be involved in cardiac development. The results of western blotting analysis and immunohistochemical analysis showed that some PcG protein, Bmi1, Ring1b and Rae28/Phc1 distributed in the nuclei of embryonic cardiomyocytes and endocardial cells. Expression levels of these proteins were higher at early stages and decreased at the perinatal period. Physical interaction between Ring1b and Bmi1, Ring1b and Rae28/Phc1 in the developing hearts was confirmed with co-immunoprecipitation analysis(Co-IP) and in situ proximal ligation assay(PLA), indicating that Bmi1, Ring1b and Rae28/Phc1 make a Polycomb complex in the embryonic hearts as shown in other tissues. On the other hand, Pcgf5 interacted with Ring1b but not with Rae28/Phc1. These findings suggest that some kinds of Polyocomb complex work in the developing hearts.