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

Polycomb组基因Pcgf5在心脏形态发生中的功能分析

基本信息

批准号：
21591405
负责人：
SHIRAI Manabu
金额：
$ 2.91万
依托单位：
National Cardiovascular Center Research Institute
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2009
资助国家：
日本
起止时间：
2009 至 2011
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-21591405/
关键词：
ポリコーム遺伝子形態形成発生医学

项目摘要

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.

为了明确Pcg蛋白Pcgf5在心脏形态发生中的作用，了解PcG基因突变是否与人类先天性心脏病相关，我们对Pcgf5在小鼠胚胎中的表达进行了详细的分析，并利用Cre-loxP系统建立了条件性Pcgf5基因敲除小鼠。在整体原位杂交分析中，Pcgf5在发育早期心脏中有较强的表达，这意味着Pcgf5可能参与心脏的发育。Western blotting分析和免疫组织化学分析结果表明，PcG蛋白、Bmi1、Ring1b和Rae28/Phc1分布于胚胎心肌细胞和心内膜细胞的细胞核中。这些蛋白在早期的表达水平较高，在围产期表达水平下降。用免疫共沉淀法(Co-IP)和原位近端连接试验(LA)证实了Ring1b与Bmi1、Ring1b与RAE28/Phc1在胚胎心脏中的物理相互作用，表明Bmi1、Ring1b和RAE28/Phc1在胚胎心脏中形成了与其他组织一样的多梳复合体。另一方面，Pcgf5与Ring1b相互作用，但不与Rae28/Phc1相互作用。这些发现表明，某些类型的多元梳状复合体在发育中的心脏中起作用。