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

为了明确Polycomb group（PcG）蛋白之一Pcgf5在心脏形态发生中的功能，了解PcG基因突变是否与人类先天性心脏病相关，我们详细分析了Pcgf5在小鼠胚胎中的表达，并利用Cre-loxP系统生成了条件Pcgf5基因敲除小鼠。在全载原位杂交分析中，在早期发育的心脏中观察到强Pcgf5表达，这意味着Pcgf5可能参与心脏发育。western blotting和免疫组化分析结果显示，胚胎心肌细胞和心内膜细胞的细胞核内分布有部分PcG蛋白、Bmi1、Ring1b和Rae28/Phc1。这些蛋白的表达水平在早期阶段较高，在围产期降低。通过共免疫沉淀分析（Co-IP）和原位近端结联实验（PLA）证实了Ring1b与Bmi1、Ring1b与Rae28/Phc1在发育中的物理相互作用，表明Bmi1、Ring1b和Rae28/Phc1在胚胎心脏中形成Polycomb复合物，这在其他组织中也有体现。另一方面，Pcgf5与Ring1b相互作用，但与Rae28/Phc1不相互作用。这些发现表明，某些种类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.