title: Isolation of In Vivo SUMOylated Chromatin-Bound Proteins.
abstract: SUMO posttranslational modification directs gene transcription and epigenetic programming to support normal cell function. The dynamic nature of SUMO-modification makes it difficult to identify endogenous protein substrates. Isolation of chromatin-bound SUMO targets is exceptionally challenging, as conventional immunoprecipitation assays are inefficient at concentrating this protein population. This chapter describes a protocol that effectively precipitates chromatin-associated fractions of SUMOylated heterochromatin protein 1α in cultured cells. Techniques to enrich endogenous SUMO substrates at the chromatin are also demonstrated and discussed. This approach could be adapted to evaluate chromatin-bound SUMO targets in additional in vivo systems.
authors.affiliation: Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, 3517 Cullen Blvd, SERC Bldg, Rm 3010, Houston, TX, 77204-5056, USA. tbawa-khalfe@uh.edu.
authors.forename: Tasneem
authors.initials: T
authors.lastname: Bawa-Khalfe
mesh_terms: D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D061986:MCF-7 Cells; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D058207:Sumoylation; D014452:Ubiquitins / Q000235:genetics / Q000378:metabolism*
chemical_list: C000715594:CBX5 protein, human; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C492189:SUMO3 protein, human; D014452:Ubiquitins; D000090266:Chromobox Protein Homolog 5; D010450:Endopeptidases; C533959:SENP7 protein, human
keywords: Chromatin-bound; HP1α; SENP7; SUMO