Locus-specific proteomics by TChP: targeted chromatin purification

Farzin Pourfarzad, Ali Aghajanirefah, Ernie de Boer, Sara Ten Have, Thamar Bryn van Dijk, Sima Kheradmandkia, Ralph Stadhouders, Supat Thongjuea, Eric Soler, Nynke Gillemans, Marieke von Lindern, Jeroen Demmers, Sjaak Philipsen, Frank Grosveld

    Research output: Contribution to journalArticlepeer-review

    22 Citations (Scopus)

    Abstract

    Here, we show that transcription factors bound to regulatory sequences can be identified by purifying these unique sequences directly from mammalian cells invivo. 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 ?-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 ?-globin gene expression. Reactivation of ?-globin gene expression ameliorates the symptoms of ß-thalassemia and sickle cell disease, and these factors provide potential targets for the development of therapeutics for treating these patients. •Purification of single-copy locus chromatin•Proteomic identification of protein factors bound to this chromatin•Recruitment of the identified factors to the ß-globin chromatin hub•Functional analysis of identified factors on ?-globin gene transcriptional regulation. Grosveld and colleagues show that proteins bound to unique sequences in the genome can be purified at levels sufficient for identification by mass spectrometry. Applying this technique, the authors identified a number of known factors as well as additional proteins that are potentially involved in the suppression of the human ?-globin genes.
    Original languageEnglish
    Pages (from-to)589-600
    Number of pages12
    JournalCell Reports
    Volume4
    Issue number3
    DOIs
    Publication statusPublished - 15 Aug 2013

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