title: Determination of protein interactome of transcription factor Sox2 in embryonic stem cells engineered for inducible expression of four reprogramming factors.
abstract: Unbiased proteomic screens provide a powerful tool for defining protein-protein interaction networks. Previous studies employed multidimensional protein identification technology to identify the Sox2-interactome in embryonic stem cells (ESC) undergoing differentiation in response to a small increase in the expression of epitope-tagged Sox2. Thus far the Sox2-interactome in ESC has not been determined. To identify the Sox2-interactome in ESC, we engineered ESC for inducible expression of different combinations of epitope-tagged Sox2 along with Oct4, Klf4, and c-Myc. Epitope-tagged Sox2 was used to circumvent the lack of suitable Sox2 antibodies needed to perform an unbiased proteomic screen of Sox2-associated proteins. Although i-OS-ESC differentiate when both Oct4 and Sox2 are elevated, i-OSKM-ESC do not differentiate even when the levels of the four transcription factors are coordinately elevated ∼2-3-fold. Our findings with i-OS-ESC and i-OSKM-ESC provide new insights into the reasons why ESC undergo differentiation when Sox2 and Oct4 are elevated in ESC. Importantly, the use of i-OSKM-ESC enabled us to identify the Sox2-interactome in undifferentiated ESC. Using multidimensional protein identification technology, we identified >70 proteins that associate with Sox2 in ESC. We extended these findings by testing the function of the Sox2-assoicated protein Smarcd1 and demonstrate that knockdown of Smarcd1 disrupts the self-renewal of ESC and induces their differentiation. Together, our work provides the first description of the Sox2-interactome in ESC and indicates that Sox2 along with other master regulators is part of a highly integrated protein-protein interaction landscape in ESC.
authors.affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198-5950, USA.
authors.forename: Zhiguang
authors.initials: Z
authors.lastname: Gao
authors.forename: Jesse L
authors.initials: JL
authors.lastname: Cox
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Gilmore
authors.forename: Briana D
authors.initials: BD
authors.lastname: Ormsbee
authors.forename: Sunil K
authors.initials: SK
authors.lastname: Mallanna
authors.forename: Michael P
authors.initials: MP
authors.lastname: Washburn
authors.forename: Angie
authors.initials: A
authors.lastname: Rizzino
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D060846:Cell Engineering; D002868:Chromosomal Proteins, Non-Histone / Q000172:deficiency / Q000235:genetics; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D018604:Epitope Mapping; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D006801:Humans; D016547:Kinesins / Q000235:genetics / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping; D040901:Proteomics; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics* / Q000378:metabolism*
chemical_list: D002868:Chromosomal Proteins, Non-Histone; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D016271:Proto-Oncogene Proteins c-myc; D055748:SOXB1 Transcription Factors; C529549:Smarcd1 protein, mouse; C527783:Sox2 protein, mouse; C089655:Kif4 protein, mouse; D016547:Kinesins