ABSTRACT
Reconstituting posttranslational modification with SUMO in vitro is an essential tool in the analysis of sumoylation. In this article, we provide detailed protocols that allow to set up and perform sumoylation reactions using a purified recombinant sumoylation machinery. The protocols include purification of the SUMO E1 enzyme His-Aos1/Uba2, untagged E2 enzyme Ubc9, untagged SUMO, and the RanBP2 E3 ligase fragment IR1 + M. Using these components, we provide step-by-step instructions to set up sumoylation reactions. Two established SUMO model substrates, His-RanGAPtail and HisYFP-Sp100, complement the described tool box; these proteins serve as positive controls in E3 ligase-independent and -dependent sumoylation reactions and are valuable instruments to adjust the reaction conditions if necessary.
Subject(s)
Small Ubiquitin-Related Modifier Proteins/metabolism , Sumoylation , GTPase-Activating Proteins/metabolism , Humans , Ubiquitin-Activating Enzymes/metabolism , Ubiquitin-Conjugating Enzymes/metabolismABSTRACT
The Polycomb group of proteins (PcG) maintains stable epigenetic silencing of over 100 genes via PcG response elements (PREs). Here we investigate the relationship between Polycomb binding, transcriptional status, and histone H3 methylation at lysine 9 (H3K9Me) and 27 (H3K27Me) for over 30 PcG targets in Drosophila. We show that H3K9Me and H3K27Me have distinct distributions at different loci. Our data show that Polycomb binding and histone methylation at the promoter do not prevent strong transcriptional activity, and indicate instead that silencing requires methylation of both PRE and promoter. In addition, we show that trimethylated H3K9 and H3K27 peptides can compete Polycomb from polytene chromosomes, with different effects at different loci, which correlate with differences in methylation status and transcriptional activity. We use mathematical modeling to examine these data, and propose that weak Polycomb-histone tail interactions enable PcG complexes to bind dynamically to chromatin, offering opportunities for regulation.
Subject(s)
Histones/metabolism , Homeodomain Proteins/metabolism , Lysine/metabolism , Animals , Binding Sites , Chromatin Immunoprecipitation , Chromosome Mapping , Chromosomes/metabolism , Drosophila , Drosophila Proteins/metabolism , Epigenesis, Genetic , Gene Expression Regulation , Gene Silencing , Genes, Reporter , Histones/genetics , Models, Biological , Polycomb Repressive Complex 1 , Promoter Regions, Genetic , Protein Binding , Repressor Proteins/metabolism , Salivary Glands/cytology , Transcription, Genetic , TransgenesABSTRACT
Alternative sigma factors have been detected in the myxobacterium Stigmatella aurantiaca during indole-induced sporulation, fruiting body formation and heat shock using an antiserum raised against sigma factor SigB. The time course of sigB gene expression was analysed by RT-PCR and by determining beta-galactosidase activity during development in a merodiploid strain that harboured a sigB-lacZ fusion gene. Inactivation of the sigB gene by insertion of the neo gene resulted in the loss of one sigma factor as shown by Western analysis. Neither fruiting body formation nor sporulation, nor the production of possible SigB targets, such as DnaK, GroEL or HspA, were affected.
