title: Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics.
abstract: The cellular abundance of transcription factors (TFs) is an important determinant of their regulatory activities. Deriving TF copy numbers is therefore crucial to understanding how these proteins control gene expression. We describe a sensitive selected reaction monitoring-based mass spectrometry assay that allowed us to determine the copy numbers of up to ten proteins simultaneously. We applied this approach to profile the absolute levels of key TFs, including PPARγ and RXRα, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from ∼250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DNA binding model for PPARγ based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPARγ binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPARγ copy number.
authors.affiliation: Laboratory of Systems Biology and Genetics, Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
authors.forename: Jovan
authors.initials: J
authors.lastname: Simicevic
authors.forename: Adrien W
authors.initials: AW
authors.lastname: Schmid
authors.forename: Paola A
authors.initials: PA
authors.lastname: Gilardoni
authors.forename: Benjamin
authors.initials: B
authors.lastname: Zoller
authors.forename: Sunil K
authors.initials: SK
authors.lastname: Raghav
authors.forename: Irina
authors.initials: I
authors.lastname: Krier
authors.forename: Carine
authors.initials: C
authors.lastname: Gubelmann
authors.forename: Frédérique
authors.initials: F
authors.lastname: Lisacek
authors.forename: Felix
authors.initials: F
authors.lastname: Naef
authors.forename: Marc
authors.initials: M
authors.lastname: Moniatte
authors.forename: Bart
authors.initials: B
authors.lastname: Deplancke
mesh_terms: D041721:3T3-L1 Cells; D000818:Animals; D002454:Cell Differentiation; D004247:DNA / Q000378:metabolism; D051379:Mice; D047495:PPAR gamma / Q000032:analysis / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D047490:Retinoid X Receptor alpha / Q000032:analysis; D014157:Transcription Factors / Q000032:analysis*
chemical_list: D047495:PPAR gamma; D047490:Retinoid X Receptor alpha; D014157:Transcription Factors; D004247:DNA