title: NLS-tagging: an alternative strategy to tag nuclear proteins.
abstract: The characterization of transcription factor complexes and their binding sites in the genome by affinity purification has yielded tremendous new insights into how genes are regulated. The affinity purification requires either the use of antibodies raised against the factor of interest itself or by high-affinity binding of a C- or N-terminally added tag sequence to the factor. Unfortunately, fusing extra amino acids to the termini of a factor can interfere with its biological function or the tag may be inaccessible inside the protein. Here, we describe an effective solution to that problem by integrating the 'tag' close to the nuclear localization sequence domain of the factor. We demonstrate the effectiveness of this approach with the transcription factors Fli-1 and Irf2bp2, which cannot be tagged at their extremities without loss of function. This resulted in the identification of novel proteins partners and a new hypothesis on the contribution of Fli-1 to hematopoiesis.
authors.affiliation: Department of Cell Biology, Erasmus Medical Center, Faculty building, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Giraud
authors.affiliation: Department of Cell Biology, Erasmus Medical Center, Faculty building, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
authors.forename: Ralph
authors.initials: R
authors.lastname: Stadhouders
authors.affiliation: Department of Cell Biology, Erasmus Medical Center, Faculty building, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
authors.forename: Andrea
authors.initials: A
authors.lastname: Conidi
authors.affiliation: Proteomics Center, Erasmus University Medical Center, Faculty building, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
authors.forename: Dick H W
authors.initials: DH
authors.lastname: Dekkers
authors.affiliation: Department of Cell Biology, Erasmus Medical Center, Faculty building, PO Box 2040, 3000 CA Rotterdam, The Netherlands Laboratory of Molecular Biology (Celgen), Department of Development and Regeneration, KU Leuven, Herestraat 49, B-3000 Leuven, Belgium.
authors.forename: Danny
authors.initials: D
authors.lastname: Huylebroeck
authors.affiliation: Proteomics Center, Erasmus University Medical Center, Faculty building, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
authors.forename: Jeroen A A
authors.initials: JA
authors.lastname: Demmers
authors.affiliation: Department of Cell Biology, Erasmus Medical Center, Faculty building, PO Box 2040, 3000 CA Rotterdam, The Netherlands Laboratory of Hematopoiesis and Leukemic Stem Cells (LSHL), CEA/INSERM U967, Fontenay-aux-Roses, France Center for Biomedical Genetics and Medical Epigenetics Consortium, Erasmus Medical Center, Faculty building, PO Box 2040, 3000 CA Rotterdam, The Netherlands.
authors.forename: Eric
authors.initials: E
authors.lastname: Soler
authors.affiliation: Department of Cell Biology, Erasmus Medical Center, Faculty building, PO Box 2040, 3000 CA Rotterdam, The Netherlands Center for Biomedical Genetics and Medical Epigenetics Consortium, Erasmus Medical Center, Faculty building, PO Box 2040, 3000 CA Rotterdam, The Netherlands Center for Biomedical Genetics, Erasmus Medical Center, Faculty building, PO Box 2040, 3000 CA Rotterdam, The Netherlands f.grosveld@erasmusmc.nl.
authors.forename: Frank G
authors.initials: FG
authors.lastname: Grosveld
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000032:analysis* / Q000737:chemistry; D051789:Proto-Oncogene Protein c-fli-1 / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
chemical_list: D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D051789:Proto-Oncogene Protein c-fli-1; D014157:Transcription Factors