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A Lentiviral Functional Proteomics Approach Identifies Chromatin Remodeling Complexes Important for the Induction of Pluripotency*

By Anthony B. Mak, Zuyao Ni, Johannes A. Hewel, Ginny I. Chen, Guoqing Zhong, Konstantina Karamboulas, Kim Blakely, Sandra Smiley, Edyta Marcon, Denitza Roudeva, Joyce Li, Jonathan B. Olsen, Cuihong Wan, Thanuja Punna, Ruth Isserlin, Sergei Chetyrkin, Anne-Claude Gingras, Andrew Emili, Jack Greenblatt and Jason Moffat

Abstract

Protein complexes and protein-protein interactions are essential for almost all cellular processes. Here, we establish a mammalian affinity purification and lentiviral expression (MAPLE) system for characterizing the subunit compositions of protein complexes. The system is flexible (i.e. multiple N- and C-terminal tags and multiple promoters), is compatible with GatewayTM cloning, and incorporates a reference peptide. Its major advantage is that it permits efficient and stable delivery of affinity-tagged open reading frames into most mammalian cell types. We benchmarked MAPLE with a number of human protein complexes involved in transcription, including the RNA polymerase II-associated factor, negative elongation factor, positive transcription elongation factor b, SWI/SNF, and mixed lineage leukemia complexes. In addition, MAPLE was used to identify an interaction between the reprogramming factor Klf4 and the Swi/Snf chromatin remodeling complex in mouse embryonic stem cells. We show that the SWI/SNF catalytic subunit Smarca2/Brm is up-regulated during the process of induced pluripotency and demonstrate a role for the catalytic subunits of the SWI/SNF complex during somatic cell reprogramming. Our data suggest that the transcription factor Klf4 facilitates chromatin remodeling during reprogramming

Topics: Stem Cell Biology
Publisher: The American Society for Biochemistry and Molecular Biology
OAI identifier: oai:pubmedcentral.nih.gov:2871416
Provided by: PubMed Central
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