Discovery of Klf2 Interactors in Mouse Embryonic Stem Cells by Immunoprecipitation-Mass Spectrometry Utilizing Exogenously Expressed Bait
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ABSTRACT: Krüppel-like factor 2 (Klf2) is a DNA-binding transcription factor that regulates embryonic stem cell-specific gene expression. Transcription cofactors such as p300 acetyltransferases 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 Oct4 gene expression. Collectively, our IP-MS protocol may offer a useful tool for identifying novel transcription cofactors in stem cells.
ORGANISM(S): Mus Musculus (mouse)
SUBMITTER: J. Eugene Lee
PROVIDER: PXD022123 | JPOST Repository | Mon Sep 20 00:00:00 BST 2021
REPOSITORIES: jPOST
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