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Mol Cell Proteomics. 2010 May;9(5):811-23. doi: 10.1074/mcp.M000002-MCP201. Epub 2010 Mar 19.

A lentiviral functional proteomics approach identifies chromatin remodeling complexes important for the induction of pluripotency.

Author information

  • 1Banting and Best Department of Medical Research, Donnelly Centre, University of Toronto, Toronto M5S3E1, Canada.

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 Gateway 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.

PMID:
20305087
[PubMed - indexed for MEDLINE]
PMCID:
PMC2871416
Free PMC Article

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