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Tytuł:
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Discovery of Klf2 interactors in mouse embryonic stem cells by immunoprecipitation-mass spectrometry utilizing exogenously expressed bait.
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Autorzy:
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Seo L; Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, South Korea.
Kim YI; Center for Bioanalysis, Korea Research Institute of Standards and Science, Daejeon 34113, South Korea.
Kim H; Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, South Korea.
Hyun K; Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, South Korea.
Kim J; Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 34141, South Korea. Electronic address: .
Lee JE; Division of Policy and Strategy, Korea Research Institute of Standards and Science, Daejeon 34113, South Korea. Electronic address: .
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Źródło:
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Biochimica et biophysica acta. Proteins and proteomics [Biochim Biophys Acta Proteins Proteom] 2021 Sep; Vol. 1869 (9), pp. 140672. Date of Electronic Publication: 2021 May 14.
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Typ publikacji:
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Journal Article; Research Support, Non-U.S. Gov't
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Język:
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English
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Imprint Name(s):
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Original Publication: Amsterdam : Elsevier
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MeSH Terms:
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Immunoprecipitation/*methods
Kruppel-Like Transcription Factors/*metabolism
Mass Spectrometry/*methods
Animals ; Cell Line ; DNA-Binding Proteins ; Gene Expression/genetics ; Gene Expression Regulation, Developmental/genetics ; Kruppel-Like Transcription Factors/genetics ; Kruppel-Like Transcription Factors/physiology ; Mice ; Mouse Embryonic Stem Cells/metabolism ; Protein Binding ; Transcription Factors
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Contributed Indexing:
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Keywords: Exogenous bait; Immunoprecipitation-mass spectrometry; Klf2; Mouse embryonic stem cells; Snd1
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Substance Nomenclature:
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0 (DNA-Binding Proteins)
0 (Klf2 protein, mouse)
0 (Kruppel-Like Transcription Factors)
0 (Transcription Factors)
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Entry Date(s):
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Date Created: 20210517 Date Completed: 20210922 Latest Revision: 20210922
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Update Code:
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20240104
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DOI:
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10.1016/j.bbapap.2021.140672
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PMID:
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34000451
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Krüppel-like factor 2 (Klf2) is a DNA-binding transcription factor that regulates embryonic stem cell-specific gene expression. Transcription cofactors such as p300 acetyltransferase and Erk kinases interact with Klf2, providing an additional layer of transcription regulation in embryonic stem cells. To carry out a thorough survey of the Klf2 interactome in embryonic stem cells and identify novel transcription cofactors, we designed a modified immunoprecipitation-mass spectrometry (IP-MS) method. In this method, recombinant Klf2, expressed and purified from Sf9 insect cells instead of ectopically expressed in cells, was used as bait. Using this modified IP-MS method, we discovered nine Klf2-interacting proteins, including the previously reported Crebbp and p300. These proteins showed at least an 8-fold increase in signal intensity in Klf2 pull-downs compared with controls, with P-values <0.010. Among the identified Klf2-binding proteins confirmed using our IP-MS workflow was Snd1, which we found to interact directly with Klf2 and function as a transcriptional coactivator of Klf2 to drive the Oct4 gene expression. Collectively, our IP-MS protocol may offer a useful tool for identifying novel transcription cofactors in stem cells.
(Copyright © 2021. Published by Elsevier B.V.)
