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 journalArticle

    17 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

    Fingerprint

    Globins
    Proteomics
    Chromatin
    Purification
    Gene expression
    Transcription Factors
    Genes
    Gene Expression
    Erythroid Cells
    Proteins
    Thalassemia
    Hooks
    Nucleic Acid Regulatory Sequences
    Sickle Cell Anemia
    Tetracycline
    Statistical Factor Analysis
    Mass spectrometry
    Mass Spectrometry
    Cells
    Genome

    Cite this

    Pourfarzad, F., Aghajanirefah, A., de Boer, E., Ten Have, S., Bryn van Dijk, T., Kheradmandkia, S., ... Grosveld, F. (2013). Locus-specific proteomics by TChP: targeted chromatin purification. Cell Reports, 4(3), 589-600. https://doi.org/10.1016/j.celrep.2013.07.004
    Pourfarzad, Farzin ; Aghajanirefah, Ali ; de Boer, Ernie ; Ten Have, Sara ; Bryn van Dijk, Thamar ; Kheradmandkia, Sima ; Stadhouders, Ralph ; Thongjuea, Supat ; Soler, Eric ; Gillemans, Nynke ; von Lindern, Marieke ; Demmers, Jeroen ; Philipsen, Sjaak ; Grosveld, Frank. / Locus-specific proteomics by TChP : targeted chromatin purification. In: Cell Reports. 2013 ; Vol. 4, No. 3. pp. 589-600.
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    author = "Farzin Pourfarzad and Ali Aghajanirefah and {de Boer}, Ernie and {Ten Have}, Sara and {Bryn van Dijk}, Thamar and Sima Kheradmandkia and Ralph Stadhouders and Supat Thongjuea and Eric Soler and Nynke Gillemans and {von Lindern}, Marieke and Jeroen Demmers and Sjaak Philipsen and Frank Grosveld",
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    Pourfarzad, F, Aghajanirefah, A, de Boer, E, Ten Have, S, Bryn van Dijk, T, Kheradmandkia, S, Stadhouders, R, Thongjuea, S, Soler, E, Gillemans, N, von Lindern, M, Demmers, J, Philipsen, S & Grosveld, F 2013, 'Locus-specific proteomics by TChP: targeted chromatin purification', Cell Reports, vol. 4, no. 3, pp. 589-600. https://doi.org/10.1016/j.celrep.2013.07.004

    Locus-specific proteomics by TChP : targeted chromatin purification. / Pourfarzad, Farzin; Aghajanirefah, Ali; de Boer, Ernie; Ten Have, Sara; Bryn van Dijk, Thamar; Kheradmandkia, Sima; Stadhouders, Ralph; Thongjuea, Supat; Soler, Eric; Gillemans, Nynke; von Lindern, Marieke; Demmers, Jeroen; Philipsen, Sjaak; Grosveld, Frank.

    In: Cell Reports, Vol. 4, No. 3, 15.08.2013, p. 589-600.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Locus-specific proteomics by TChP

    T2 - targeted chromatin purification

    AU - Pourfarzad, Farzin

    AU - Aghajanirefah, Ali

    AU - de Boer, Ernie

    AU - Ten Have, Sara

    AU - Bryn van Dijk, Thamar

    AU - Kheradmandkia, Sima

    AU - Stadhouders, Ralph

    AU - Thongjuea, Supat

    AU - Soler, Eric

    AU - Gillemans, Nynke

    AU - von Lindern, Marieke

    AU - Demmers, Jeroen

    AU - Philipsen, Sjaak

    AU - Grosveld, Frank

    PY - 2013/8/15

    Y1 - 2013/8/15

    N2 - 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.

    AB - 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.

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    Pourfarzad F, Aghajanirefah A, de Boer E, Ten Have S, Bryn van Dijk T, Kheradmandkia S et al. Locus-specific proteomics by TChP: targeted chromatin purification. Cell Reports. 2013 Aug 15;4(3):589-600. https://doi.org/10.1016/j.celrep.2013.07.004