title: Expanding the Circuitry of Pluripotency by Selective Isolation of Chromatin-Associated Proteins.
abstract: Maintenance of pluripotency is regulated by a network of transcription factors coordinated by Oct4, Sox2, and Nanog (OSN), yet a systematic investigation of the composition and dynamics of the OSN protein network specifically on chromatin is still missing. Here we have developed a method combining ChIP with selective isolation of chromatin-associated proteins (SICAP) followed by mass spectrometry to identify chromatin-bound partners of a protein of interest. ChIP-SICAP in mouse embryonic stem cells (ESCs) identified over 400 proteins associating with OSN, including several whose interaction depends on the pluripotent state. Trim24, a previously unrecognized protein in the network, converges with OSN on multiple enhancers and suppresses the expression of developmental genes while activating cell cycle genes. Consistently, Trim24 significantly improved efficiency of cellular reprogramming, demonstrating its direct functionality in establishing pluripotency. Collectively, ChIP-SICAP provides a powerful tool to decode chromatin protein composition, further enhanced by its integrative capacity to perform ChIP-seq.
authors: German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120 Heidelberg, Germany; Excellence Cluster CellNetworks, Heidelberg University, 69120 Heidelberg, Germany; European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors: Mahmoud-Reza
authors: MR
authors: Rafiee
authors: European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors: Charles
authors: C
authors: Girardot
authors: German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120 Heidelberg, Germany; Excellence Cluster CellNetworks, Heidelberg University, 69120 Heidelberg, Germany.
authors: Gianluca
authors: G
authors: Sigismondo
authors: German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, 69120 Heidelberg, Germany; Excellence Cluster CellNetworks, Heidelberg University, 69120 Heidelberg, Germany; European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Meyerhofstrasse 1, 69117 Heidelberg, Germany. Electronic address: j.krijgsveld@dkfz.de.
authors: Jeroen
authors: J
authors: Krijgsveld
mesh_terms: D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation; D065150:Cellular Reprogramming; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism; D047369:Chromatin Immunoprecipitation / Q000379:methods; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D000071317:Nanog Homeobox Protein / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D050814:Octamer Transcription Factor-3 / Q000235:genetics* / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D011485:Protein Binding; D055748:SOXB1 Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D002843:Chromatin; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D009687:Nuclear Proteins; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D014157:Transcription Factors; C093517:transcriptional intermediary factor 1
keywords: biotinylation; chromatin; embryonic stem cells; pluripotency; protein interactions; proteomics; reprogramming