1. Locus-Specific Proteomics by TChP: Targeted Chromatin Purification
- Author
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Eric Soler, Ernie de Boer, Sjaak Philipsen, Supat Thongjuea, Frank Grosveld, Jeroen Demmers, Sara ten Have, Farzin Pourfarzad, Thamar B. van Dijk, Sima Kheradmandkia, Nynke Gillemans, Marieke von Lindern, Ali Aghajanirefah, Ralph Stadhouders, Landsteiner Laboratory, Cell biology, Neurosciences, Hematology, and Biochemistry
- Subjects
Proteomics ,Transcription, Genetic ,Cell ,Locus (genetics) ,Mice, Transgenic ,beta-Globins ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Mass Spectrometry ,03 medical and health sciences ,Mice ,0302 clinical medicine ,SDG 3 - Good Health and Well-being ,hemic and lymphatic diseases ,Gene expression ,medicine ,Animals ,Humans ,Promoter Regions, Genetic ,Transcription factor ,lcsh:QH301-705.5 ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,Gene knockdown ,Molecular biology ,Chromatin ,3. Good health ,Cell biology ,medicine.anatomical_structure ,lcsh:Biology (General) ,Regulatory sequence ,030220 oncology & carcinogenesis ,Gene Knockdown Techniques - Abstract
Here, we show that transcription factors bound to regulatory sequences can be identified by purifying these unique sequences directly from mammalian cells in vivo. Using targeted chromatin purification (TChP), a double-pull-down strategy with a tetracycline-sensitive "hook" bound to a specific promoter, we identify transcription factors bound to the repressed gamma-globin gene-associated regulatory regions. After validation of the binding, we show that, in human primary erythroid cells, knockdown of a number of these transcription factors induces gamma-globin gene expression. Reactivation of gamma-globin gene expression ameliorates the symptoms of beta-thalassemia and sickle cell disease, and these factors provide potential targets for the development of therapeutics for treating these patients.
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