PXD010963

PXD010963 is an original dataset announced via ProteomeXchange.

Title	Locus-specific proteomics identification of OCT4 regulators
Description	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.
HostingRepository	PRIDE
AnnounceDate	2019-01-07
AnnouncementXML	Submission_2019-01-07_06:41:01.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Daniel Chelsky
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	monohydroxylated residue; deamidated residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2018-08-31 02:20:49	ID requested
⏵ 1	2019-01-07 06:41:02	announced

Fang F, Xia N, Angulo B, Carey J, Cady Z, Durruthy-Durruthy J, Bennett T, Sebastiano V, Reijo Pera RA, A distinct isoform of ZNF207 controls self-renewal and pluripotency of human embryonic stem cells. Nat Commun, 9(1):4384(2018) [pubmed]

curator keyword: Biological

submitter keyword: OCT4, TALEN, cross-linking, LC-MSMS

Daniel Chelsky
contact affiliation	Caprion Biosciences Inc.
contact email	dchelsky@caprion.com
lab head
Daniel Chelsky
contact affiliation	Caprion Biosciences
contact email	dchelsky@caprion.com
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2019/01/PXD010963

PRIDE project URI

• PRIDE
  1. PXD010963
     1. Label: PRIDE project
     2. Name: Locus-specific proteomics identification of OCT4 regulators