Proteomics

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Locus-specific proteomics identification of OCT4 regulators


ABSTRACT: We set out to develop an unbiased, genome-wide method to screen for proteins that locate to the OCT4 proximal enhancer (PE) in hESCs; for this purpose, we developed an optimized locus-specific proteomics approach in hESCs. First, we designed TALEN (Transcription Activator-Like Effector Nuclease) constructs to target the sequences that are near the OCT4 PE, located in a region of DNaseI hypersensitivity. TALEN constructs with the highest cutting efficiency were chosen for locus-specific proteomics. We then made modifications to the original TALEN protein to transform it into a catalytically-dead TALE (dTALE) protein that is optimized for locus-specific proteomics in hESCs via three steps: 1) The nuclease-domain FokI at the C-terminus was replaced by a GFP (green fluorescence protein); 2) a 3X FLAG tag at the N-terminus was included for following pull-down analysis; 3) the existing CMV promoter was replaced with an EF1alpha promoter that has robust expression in hESCs. This dTALE protein could then be chemically crosslinked to the OCT4 locus together with all the other proteins that bind to the locus. We verified that dTALE protein binds to the targeted locus by chromatin immunoprecipitation (ChIP)-qPCR. Following crosslinking, chromatin was sheared, and all the associated proteins were immunoprecipitated using an anti-FLAG antibody. Immunoprecipitation pulled down the dTALE protein as well as other proteins and complexes that are also attached to that region. Crosslinking was then reversed, and the samples were subjected to mass spectrometry to enable generation of a list of proteins that potentially bind to OCT4 PE locus.

OTHER RELATED OMICS DATASETS IN: GSE118638

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Embryonic Stem Cell

SUBMITTER: Daniel Chelsky  

LAB HEAD: Daniel Chelsky

PROVIDER: PXD010963 | Pride | 2019-01-07

REPOSITORIES: Pride

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20131027_AB.raw Raw
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Publications

A distinct isoform of ZNF207 controls self-renewal and pluripotency of human embryonic stem cells.

Fang Fang F   Xia Ninuo N   Angulo Benjamin B   Carey Joseph J   Cady Zackery Z   Durruthy-Durruthy Jens J   Bennett Theo T   Sebastiano Vittorio V   Reijo Pera Renee A RA  

Nature communications 20181022 1


Self-renewal and pluripotency in human embryonic stem cells (hESCs) depends upon the function of a remarkably small number of master transcription factors (TFs) that include OCT4, SOX2, and NANOG. Endogenous factors that regulate and maintain the expression of master TFs in hESCs remain largely unknown and/or uncharacterized. Here, we use a genome-wide, proteomics approach to identify proteins associated with the OCT4 enhancer. We identify known OCT4 regulators, plus a subset of potential regula  ...[more]

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