Abstract
SummaryMaintenance 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.
Highlights
In embryonic stem cells (ESCs), the three master transcription factors Oct4, Sox2, and Nanog constitute the core transcriptional circuitry (Boyer et al, 2005; Loh et al, 2006), which on the one hand promotes the expression of pluripotency genes, while on the other hand suppresses lineage commitment and differentiation (Boyer et al, 2006; Laugesen and Helin, 2014; Lee et al, 2006)
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 on chromatin is still missing
ChIP-selective isolation of chromatin-associated proteins (SICAP) in mouse embryonic stem cells (ESCs) identified over 400 proteins associating with OSN, including several whose interaction depends on the pluripotent state
Summary
In ESCs, the three master transcription factors Oct, Sox, and Nanog constitute the core transcriptional circuitry (Boyer et al, 2005; Loh et al, 2006), which on the one hand promotes the expression of pluripotency genes, while on the other hand suppresses lineage commitment and differentiation (Boyer et al, 2006; Laugesen and Helin, 2014; Lee et al, 2006). Since transcription factors, including pluripotency TFs, execute their function in chromatin, we aimed to identify proteins that associate with OSN in their DNA-bound state as opposed to interactions that may occur in soluble form. Despite the large diversity of available methods to identify protein interactions (reviewed by Dunham et al, 2012), very few of them differentiate between interactions that depend on the subcellular location. This is a critical shortcoming, especially for proteins that dynamically change location, either between or within organelles (e.g., nucleosol or chromatin bound). To identify proteins in their DNA-bound state, we developed a method for the selective isolation of chromatin-associated proteins (SICAP). We demonstrate the power of ChIPSICAP by the discovery of Trim as a component of the pluripotency network
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