Controlling for Gene Expression Changes in Transcription Factor Protein Networks

The development of affinity purification technologies combined with mass spectrometric analysis of purified protein mixtures has been used both to identify new protein–protein interactions and to define the subunit composition of protein complexes. Transcription factor protein interactions, however, have not been systematically analyzed using these approaches. Here, we investigated whether ectopic expression of an affinity tagged transcription factor as bait in affinity purification mass spectrometry experiments perturbs gene expression in cells, resulting in the false positive identification of bait-associated proteins when typical experimental controls are used. Using quantitative proteomics and RNA sequencing, we determined that the increase in the abundance of a set of proteins caused by overexpression of the transcription factor RelA is not sufficient for these proteins to then co-purify non-specifically and be misidentified as bait-associated proteins. Therefore, typical controls should be sufficient, and a number of different baits can be compared with a common set of controls. This is of practical interest when identifying bait interactors from a large number of different baits. As expected, we found several known RelA interactors enriched in our RelA purifications (NFκB1, NFκB2, Rel, RelB, IκBα, IκBβ, and IκBε). We also found several proteins not previously described in association with RelA, including the small mitochondrial chaperone Tim13. Using a variety of biochemical approaches, we further investigated the nature of the association between Tim13 and NFκB family transcription factors. This work therefore provides a conceptual and experimental framework for analyzing transcription factor protein interactions. The development of affinity purification technologies combined with mass spectrometric analysis of purified protein mixtures has been used both to identify new protein–protein interactions and to define the subunit composition of protein complexes. Transcription factor protein interactions, however, have not been systematically analyzed using these approaches. Here, we investigated whether ectopic expression of an affinity tagged transcription factor as bait in affinity purification mass spectrometry experiments perturbs gene expression in cells, resulting in the false positive identification of bait-associated proteins when typical experimental controls are used. Using quantitative proteomics and RNA sequencing, we determined that the increase in the abundance of a set of proteins caused by overexpression of the transcription factor RelA is not sufficient for these proteins to then co-purify non-specifically and be misidentified as bait-associated proteins. Therefore, typical controls should be sufficient, and a number of different baits can be compared with a common set of controls. This is of practical interest when identifying bait interactors from a large number of different baits. As expected, we found several known RelA interactors enriched in our RelA purifications (NFκB1, NFκB2, Rel, RelB, IκBα, IκBβ, and IκBε). We also found several proteins not previously described in association with RelA, including the small mitochondrial chaperone Tim13. Using a variety of biochemical approaches, we further investigated the nature of the association between Tim13 and NFκB family transcription factors. This work therefore provides a conceptual and experimental framework for analyzing transcription factor protein interactions. Mapping the complex network of interactions between proteins provides insight into how a cell's protein machinery functions (1.Sowa M.E. Bennett E.J. Gygi S.P. Harper J.W. 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Gingras A.-C. Nesvizhskii A.I. Tyers M. A global protein kinase and phosphatase interaction network in yeast.Science. 2010; 328: 1043-1046Crossref PubMed Scopus (524) Google Scholar). Much progress has been made in uncovering these networks, broadening our understanding of many proteins' biological functions and elucidating the architecture of many multi-subunit protein complexes (5.Sardiu M.E. Cai Y. Jin J. Swanson S.K. Conaway R.C. Conaway J.W. Florens L. Washburn M.P. Probabilistic assembly of human protein interaction networks from label-free quantitative proteomics.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1454-1459Crossref PubMed Scopus (195) Google Scholar). Despite this, there have been few studies focused on investigating proteins associated with transcription factors (3.Bouwmeester T. Bauch A. Ruffner H. Angrand P.O. Bergamini G. Croughton K. Cruciat C. Eberhard D. Gagneur J. Ghidelli S. Hopf C. Huhse B. Mangano R. Michon A.M. Schirle M. Schlegl J. Schwab M. Stein M.A. Bauer A. Casari G. Drewes G. Gavin A.C. Jackson D.B. Joberty G. Neubauer G. Rick J. Kuster B. Superti-Furga G. A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway.Nat. Cell Biol. 2004; 6: 97-105Crossref PubMed Scopus (865) Google Scholar, 6.Giambruno R. Grebien F. Stukalov A. Knoll C. Planyavsky M. Rudashevskaya E.L. Colinge J. Superti-Furga G. Bennett K.L. Affinity purification strategies for proteomic analysis of transcription factor complexes.J. Proteome Res. 2013; 12: 4018-4027Crossref PubMed Scopus (17) Google Scholar). There are technical challenges to address when trying to identify transcription-factor-associated proteins. The endogenous transcription factors might be expressed at relatively low levels, and their interactions might vary according to the state of the cell or in response to signals. If affinity-tagged transcription factors are expressed ectopically at higher levels, it might be possible to uncover bona fide interactions, but this can result in gene expression changes in transcription-factor-expressing cells. Such changes need to be considered when analyzing experimental data. Here we focused on identifying transcription-factor-associated proteins using affinity-tagged, constitutively expressed transcription factors as bait in affinity purification mass spectrometry experiments. Multidimensional protein identification technology (MudPIT) 1The abbreviations used are: MudPIT, multidimensional protein identification technology; dNSAF, distributed normalized spectral abundance factor; FDR, false discovery rate; FKPM, fragments per kilobase per million fragments measured; HEK293, human embryonic kidney 293 cell line; NFκB, nuclear factor κ-light-chain-enhancer of activated B cells; PLGEM, Power Law Global Error Model. 1The abbreviations used are: MudPIT, multidimensional protein identification technology; dNSAF, distributed normalized spectral abundance factor; FDR, false discovery rate; FKPM, fragments per kilobase per million fragments measured; HEK293, human embryonic kidney 293 cell line; NFκB, nuclear factor κ-light-chain-enhancer of activated B cells; PLGEM, Power Law Global Error Model. has been widely used to identify “prey” proteins that co-purify with an affinity-tagged “bait” protein (7.Banks C. Kong S. Washburn M. Affinity purification of protein complexes for analysis by multidimensional protein identification technology.Protein Expr. Purif. PubMed Scopus Google Scholar). as affinity of the endogenous protein the of this is not as with higher and a constitutively bait is used S. T. J. J. purification for the and identification of protein complexes from PubMed Scopus Google Scholar). There are and to this of bait might be for the identification of bona fide bait interactors that are in low in cells. tagged baits are it is to a number of bait proteins for new interactors in studies using these this not the typical expression of the endogenous of the bait in and interactions might be strategies for affinity purification mass spectrometry using that expression but baits at or that that expression of the bait can be as a of is it is to identify bait-associated proteins that with the bait and not that not result from bait experiments are used to the of proteins that are enriched by the of the bait protein purification and that not on the bait proteins that with the affinity used for D. T. S. M.E. A. A. D. D. M. Lin Pawson T. D. B. R. Superti-Furga G. Colinge J. Choi H. M. S. Bennett K.L. Washburn M.P. B. Gingras A.C. Nesvizhskii A. The a for affinity purification mass spectrometry 2013; Scholar). the set of proteins with the bait has been compared to the set of proteins purified from the affinity J. H. M. Washburn M.P. the of human RNA complexes using technology to quantitative Proteome Res. PubMed Scopus Google or to proteins purified from an cell S. C. Florens L. B. Swanson S.K. Jin J. Cai Y. Washburn M.P. Conaway J.W. Conaway R.C. A set of by multidimensional protein identification 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). A is that are not to the bait protein and therefore not have the to to the of the bait as the experimental If the bait is a protein that might have on the abundance of proteins as a transcription it is possible that these the of proteins in the used for As a the of proteins with the bait might be This is of when using as for identifying protein–protein interactions and when using as affinity-tagged transcription that might to an increase in the expression of many proteins. to address this we used the transcription factor RelA as a bait for identifying proteins S. D. and B of the subunit of a Full Text PDF PubMed Scopus Google Scholar). we a label-free quantitative proteomics to of RelA interactors using typical experimental controls. we that the in the controls the proteins. by of RNA that overexpression of a tagged transcription factor bait caused changes in global of gene we used an set of experiments to whether these of gene expression sufficient to false positive of proteins. we focused on of the proteins that we Tim13 H. The human family of mitochondrial PubMed Scopus Google and investigated the nature of association with RelA and the NFκB family R. M. C. E.J. C. of the subunit of human nuclear factor the of is by or factor Natl. Acad. Sci. PubMed Scopus Google Scholar). and from from affinity from The human used for from the and The and by a a or a between the and of the fragments in using the of and or and the of the bait proteins by and then between the and in the and with of the proteins in the and in Cell at and and the in of and We then the by the a and the then at for at to of cell with of and at for at bait complexes purified The with of for at The with and and proteins from the with of to of affinity bait complexes purified using affinity for at with from of according to the The with and proteins from the with of in of and for at then from and further analyzed by of using an protein in A and to a in A The with a from to in and for or into and using the expression at in with the and in of and at for at RNA from or the using with to RNA used to for according to the for with using of an The resulting to the human using the C. L. with 2009; PubMed Scopus Google Scholar). expression and RNA from or then compared with RNA from to identify expressed using C. G. A. G. L. assembly and by and cell 2010; PubMed Scopus Google Scholar, A. H. C. L. of in using PubMed Scopus Google Scholar). Affinity purified proteins with with and in and S. Florens L. Washburn M.P. and analysis of multidimensional protein identification technology Biol. 2009; PubMed Scopus Google Scholar). with and the then with to then with and as described The resulting with of from the into the mass for analysis using (7.Banks C. Kong S. Washburn M. Affinity purification of protein complexes for analysis by multidimensional protein identification technology.Protein Expr. Purif. PubMed Scopus Google Scholar). The resulting the by the to from to human protein the of using the J.R. to mass spectral of with in a protein PubMed Scopus Google Scholar). not The mass set at for and for A of to to for that not using to the assembly of a protein for J.R. and for and protein from Proteome Res. PubMed Scopus Google Scholar). for a of of and a of and a of Using these we an spectral false discovery for the of and the protein A of proteins in the of experimental using and J.R. and for and protein from Proteome Res. PubMed Scopus Google Scholar). The in used to proteins that of If proteins by the set of at to the set to between number used to the the abundance of these proteins in experimental and using spectral to Y. Washburn M.P. Florens L. to how to with by 2010; PubMed Scopus Google proteins with a of enriched in experimental but not determined using the Swanson S.K. M. Florens L. Washburn M.P. between and quantitative proteomics 2008; Full Text Full Text PDF PubMed Scopus Google Scholar, C. Liu Y. H. Y. H. proteomics analysis of 2010; Full Text Full Text PDF PubMed Scopus Google Scholar). then according to and from determined C. Liu Y. H. Y. H. proteomics analysis of 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Y. the false discovery a practical and to R. B. Scholar). proteins then according to their associated as to or to identify proteins associated with the transcription factor RelA, we expressed the or in the resulting cell to affinity we proteins in purification using M.P. D. J.R. analysis of the by multidimensional protein identification PubMed Scopus Google in C. Kong S. Washburn M. Affinity purification of protein complexes for analysis by multidimensional protein identification technology.Protein Expr. Purif. PubMed Scopus Google we used the J.R. to mass spectral of with in a protein PubMed Scopus Google and J.R. and for and protein from Proteome Res. PubMed Scopus Google to a of proteins in L. Washburn M.P. analysis by multidimensional protein identification Biol. 328: Google Scholar). We determined proteins enriched in the but not in the proteins might co-purify as a result of physical interactions between the bait and the or interactions by that be in we proteins that in of the experimental the number of false positive at the of a of bait interactors this of possible we of these enriched in experimental to this quantitative we the of protein in by for protein Y. Washburn M.P. Florens L. to how to with by 2010; PubMed Scopus Google and we then used the as for the Swanson S.K. M. Florens L. Washburn M.P. between and quantitative proteomics 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). we used the by to C. Liu Y. H. Y. H. proteomics analysis of 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Y. the false discovery a practical and to R. 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S. A. are in Res. PubMed Scopus Google Scholar). interactions are in their and J. of protein–protein J. PubMed Scopus Google Scholar, J.R. C. and network 2010; Full Text Full Text PDF PubMed Scopus Google Scholar). whether we the association between Tim13 and we our purified to on a The protein the and at the the This be with a interaction between Tim13 and that not the of has been used widely in affinity purification mass spectrometry experiments to define of proteins that co-purify with affinity-tagged bait proteins (5.Sardiu M.E. Cai Y. Jin J. Swanson S.K. Conaway R.C. Conaway J.W. Florens L. Washburn M.P. Probabilistic assembly of human protein interaction networks from label-free quantitative proteomics.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1454-1459Crossref PubMed Scopus (195) Google Scholar). of the there is an of small of protein that co-purify and be the of but of proteins might be as a result of their abundance or to an affinity for the used for the are also purifications using the affinity-tagged bait and as bait interactors analysis studies have compared from the bait protein with a tagged bait or with with the J. H. M. Washburn M.P. the of human RNA complexes using technology to quantitative Proteome Res. PubMed Scopus Google Scholar, S. C. Florens L. B. Swanson S.K. Jin J. Cai Y. Washburn M.P. Conaway J.W. Conaway R.C. A set of by multidimensional protein identification 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). 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Tim13 of and and endogenous Tim13 also with these are with a physical interaction between Tim13 and for a an we to define a of for association with Tim13 We found that Tim13 associated with NFκB that to to This the between and of The has previously been to be in the of into the transcription factor S. S. A. are in Res. PubMed Scopus Google Scholar). we to the of the interactions in are and many interactions with have affinity are PubMed Scopus Google Scholar). of a a interaction between Tim13 and we found that these proteins when to the of the in to we the proteins enriched in our RelA purifications in to the previously NFκB proteins. The of interactions between transcription factors and their associated proteins has the to the complex transcription factor studies have focused on transcription factor interaction using a variety of and used a to identify proteins associated with the transcription factor B. J. S. M. R. interactions of the transcription factor Biol. 12: PubMed Scopus Google these proteins in including cell cell and studies have to identify transcription factor interactors by affinity-tagged factors the of and analyzing purified complexes affinity purification mass these into associated proteins K. of 2010; PubMed Scopus Google Scholar, R. B. A. J.R. A. H. J.R. identification of proteins using a combined 6: PubMed Scopus Google and NFκB associated proteins (3.Bouwmeester T. Bauch A. Ruffner H. Angrand P.O. Bergamini G. Croughton K. Cruciat C. Eberhard D. Gagneur J. Ghidelli S. Hopf C. Huhse B. Mangano R. Michon A.M. Schirle M. Schlegl J. Schwab M. Stein M.A. Bauer A. Casari G. Drewes G. Gavin A.C. Jackson D.B. Joberty G. Neubauer G. Rick J. Kuster B. Superti-Furga G. A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway.Nat. Cell Biol. 2004; 6: 97-105Crossref PubMed Scopus (865) Google Scholar). Such have used a variety of controls to the set of proteins with the affinity-tagged these proteins with an tagged bait (3.Bouwmeester T. Bauch A. Ruffner H. Angrand P.O. Bergamini G. Croughton K. Cruciat C. Eberhard D. Gagneur J. Ghidelli S. Hopf C. Huhse B. Mangano R. Michon A.M. Schirle M. Schlegl J. Schwab M. Stein M.A. Bauer A. Casari G. Drewes G. Gavin A.C. Jackson D.B. Joberty G. Neubauer G. Rick J. Kuster B. Superti-Furga G. A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway.Nat. Cell Biol. 2004; 6: 97-105Crossref PubMed Scopus (865) Google proteins with bait the affinity K. of 2010; PubMed Scopus Google and proteins with the affinity R. B. A. J.R. A. H. J.R. identification of proteins using a combined 6: PubMed Scopus Google Scholar). The expression of an affinity-tagged transcription factor as bait can to a in gene expression in the bait to controls not with the transcription We have whether purifications the are sufficient for identifying a set of bait-associated proteins that are enriched in affinity-tagged bait or whether using controls in false positive of bait-associated proteins. the we have we have that controls are sufficient and not result in false positive This is for studies using as in mass is and using a common set of controls when a variety of different tagged baits be We and Kong for their of the and of for technical the of this with