title: Probing the kinetic landscape of Hox transcription factor-DNA binding in live cells by massively parallel Fluorescence Correlation Spectroscopy.
abstract: Hox genes encode transcription factors that control the formation of body structures, segment-specifically along the anterior-posterior axis of metazoans. Hox transcription factors bind nuclear DNA pervasively and regulate a plethora of target genes, deploying various molecular mechanisms that depend on the developmental and cellular context. To analyze quantitatively the dynamics of their DNA-binding behavior we have used confocal laser scanning microscopy (CLSM), single-point fluorescence correlation spectroscopy (FCS), fluorescence cross-correlation spectroscopy (FCCS) and bimolecular fluorescence complementation (BiFC). We show that the Hox transcription factor Sex combs reduced (Scr) forms dimers that strongly associate with its specific fork head binding site (fkh250) in live salivary gland cell nuclei. In contrast, dimers of a constitutively inactive, phospho-mimicking variant of Scr show weak, non-specific DNA-binding. Our studies reveal that nuclear dynamics of Scr is complex, exhibiting a changing landscape of interactions that is difficult to characterize by probing one point at a time. Therefore, we also provide mechanistic evidence using massively parallel FCS (mpFCS). We found that Scr dimers are predominantly formed on the DNA and are equally abundant at the chromosomes and an introduced multimeric fkh250 binding-site, indicating different mobilities, presumably reflecting transient binding with different affinities on the DNA. Our proof-of-principle results emphasize the advantages of mpFCS for quantitative characterization of fast dynamic processes in live cells.
authors.affiliation: Max-Planck Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany. Electronic address: papadopo@mpi-cbg.de.
authors.forename: Dimitrios K
authors.initials: DK
authors.lastname: Papadopoulos
authors.affiliation: Department of Clinical Neuroscience (CNS), Center for Molecular Medicine (CMM), Karolinska Institutet, 17176 Stockholm, Sweden; Institute of Physics, University of Belgrade, 11080 Belgrade, Serbia.
authors.forename: Aleksandar J
authors.initials: AJ
authors.lastname: Krmpot
authors.affiliation: Department of Clinical Neuroscience (CNS), Center for Molecular Medicine (CMM), Karolinska Institutet, 17176 Stockholm, Sweden; Institute of Physics, University of Belgrade, 11080 Belgrade, Serbia.
authors.forename: Stanko N
authors.initials: SN
authors.lastname: Nikolić
authors.affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, 10691 Stockholm, Sweden.
authors.forename: Robert
authors.initials: R
authors.lastname: Krautz
authors.affiliation: Department of Clinical Neuroscience (CNS), Center for Molecular Medicine (CMM), Karolinska Institutet, 17176 Stockholm, Sweden.
authors.forename: Lars
authors.initials: L
authors.lastname: Terenius
authors.affiliation: Max-Planck Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany.
authors.forename: Pavel
authors.initials: P
authors.lastname: Tomancak
authors.affiliation: Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm, Sweden; Laboratory of Biomedical Optics, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Rigler
authors.affiliation: Department of Cell Biology, Biozentrum, University of Basel, 4056 Basel, Switzerland.
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Gehring
authors.affiliation: Department of Clinical Neuroscience (CNS), Center for Molecular Medicine (CMM), Karolinska Institutet, 17176 Stockholm, Sweden. Electronic address: vladana.vukojevic@ki.se.
authors.forename: Vladana
authors.initials: V
authors.lastname: Vukojević
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004330:Drosophila / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000378:metabolism*; D005453:Fluorescence; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D005801:Genes, Homeobox / Q000235:genetics*; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology*; D013050:Spectrometry, Fluorescence / Q000379:methods; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D014157:Transcription Factors; D004247:DNA
keywords: Bimolecular Fluorescence Complementation; Dimer formation; Hox genes; Massively parallel Fluorescence Correlation Spectroscopy; Sex combs reduced; Transcription factor–DNA binding