Proteomics

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Discovery of Klf2 Interactors in Mouse Embryonic Stem Cells by Immunoprecipitation-Mass Spectrometry Utilizing Exogenously Expressed Bait


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

Dataset's files

Source:
Action DRS
190506_IP_mESC_NE_1_control_03.raw Raw
190506_IP_mESC_NE_2_control_01.raw Raw
190506_IP_mESC_NE_2_control_02.raw Raw
190506_IP_mESC_NE_2_control_03.raw Raw
190506_IP_mESC_NE_4_Klf2_01.raw Raw
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Publications

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

Seo Lin L   Kim Yong-In YI   Kim Hyoungmin H   Hyun Kwangbeom K   Kim Jaehoon J   Lee J Eugene JE  

Biochimica et biophysica acta. Proteins and proteomics 20210514 9


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  ...[more]

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