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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Line
  • Cellular Reprogramming / genetics
  • Chromatography, Affinity
  • Chromosomal Proteins, Non-Histone / metabolism*
  • Humans
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors / metabolism
  • Lentivirus / metabolism*
  • Mice
  • Molecular Sequence Data
  • Multiprotein Complexes / metabolism
  • Pluripotent Stem Cells / cytology
  • Pluripotent Stem Cells / metabolism*
  • Protein Binding
  • Proteomics / methods*
  • Transcription Factors / metabolism*
  • Transcription, Genetic

Substances

  • Chromosomal Proteins, Non-Histone
  • KLF4 protein, human
  • Klf4 protein, mouse
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors
  • Multiprotein Complexes
  • SWI-SNF-B chromatin-remodeling complex
  • Transcription Factors