Requirement for sex comb on midleg protein interactions in drosophila polycomb group repression
Requirement for sex comb on midleg protein interactions in drosophila polycomb group repression
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DOI：
10.1534/genetics.104.027474
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发表时间：
2004-07-01
期刊：
影响因子：
3.3
通讯作者：
Simon, JA
中科院分区：
文献类型：
--
作者：
Peterson, AJ;Mallin, DR;Simon, JA
果蝇足中段性梳（SCM）蛋白是Polycomb组（PcG）的转录抑制因子。虽然遗传学研究证实SCM是一个重要的PcG成员，但其分子作用尚不清楚。为了研究SCM如何与PcG复合物连接，我们分析了一个保守的蛋白质相互作用模块SPM结构域在体内的作用。该结构域存在于SCM和另一种PcG蛋白Polyhomeotic（PH）中，其是Polycomb抑制复合物1（PRC1）的核心组分。体外SCM-PH相互作用由其各自的SPM结构域介导。使用酵母双杂交和体外结合测定来分离和表征SCM的SPM结构域中的> 30个错义突变。遗传拯救试验表明，SCM阻遏物在体内的功能被破坏的突变，损害SPM结构域的相互作用在体外。此外，一个孤立的，野生型SPM结构域的过表达产生的PcG功能丧失的表型在苍蝇。SCM与重构的PRC1核心复合物的共组装表明SCM可以与PRC1合作。然而，凝胶过滤色谱法表明，在胚胎核提取物中，SCM的大部分与PH是生物化学可分离的。这些结果表明，SCM，虽然不是PRC1的核心组成部分，相互作用和PRC1基因沉默的功能。
The Drosophila Sex Comb on Midleg (SCM) protein is a transcriptional repressor of the Polycomb group (PcG). Although genetic studies establish SCM as a crucial PcG member, its molecular role is not known. To investigate how SCM might link to PcG complexes, we analyzed the in vivo role of a conserved protein interaction module, the SPM domain. This domain is found in SCM and in another PcG protein, Polyhomeotic (PH), which is a core component of Polycomb repressive complex 1 (PRC1). SCM-PH interactions in vitro are mediated by their respective SPM domains. Yeast two-hybrid and in vitro binding assays were used to isolate and characterize >30 missense mutations in the SPM domain of SCM. Genetic rescue assays showed that SCM repressor function in vivo is disrupted by mutations that impair SPM domain interactions in vitro. Furthermore, overexpression of an isolated, wild-type SPM domain produced PcG loss-of-function phenotypes in flies. Coassembly of SCM with a reconstituted PRC1 core complex shows that SCM can partner with PRC1 However, gel filtration chromatography showed that the bulk of SCM is biochemically separable from PH in embryo nuclear extracts. These results suggest that SCM, although not a core component of PRC1, interacts and functions with PRC1 in gene silencing.