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Peterson, AJ, Mallin, DR, Francis, NJ, Ketel, CS, Stamm, J, Voeller, RK, Kingston, RE and Simon, JA (2004) Requirement for sex comb on midleg protein interactions in Drosophila polycomb group repression. Genetics 167:1225-39
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.
Amino Acid Sequence; Animals; Blotting, Western; Chromatography, Gel; DNA Primers; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Drosophila/genetics; Drosophila Proteins/genetics; Drosophila Proteins/metabolism; Gene Components; Gene Silencing; Immunoprecipitation; Molecular Sequence Data; Mutation, Missense/genetics; Nucleoproteins/genetics; Nucleoproteins/metabolism; Phenotype; Protein Binding; Protein Structure, Tertiary; Repressor Proteins/genetics; Repressor Proteins/metabolism; Two-Hybrid System Techniques