Discovery of Klf2 interactors in mouse embryonic stem cells by immunoprecipitation-mass spectrometry utilizing exogenously expressed bait

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Kruppel-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 Pvalues <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.
Publisher
ELSEVIER
Issue Date
2021-09
Language
English
Article Type
Article
Citation

BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, v.1869, no.9, pp.140672

ISSN
1570-9639
DOI
10.1016/j.bbapap.2021.140672
URI
http://hdl.handle.net/10203/287071
Appears in Collection
BS-Journal Papers(저널논문)
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