Probing the kinetic landscape of Hox transcription factor–DNA binding in live cells by massively parallel Fluorescence Correlation Spectroscopy

Dimitrios K. Papadopoulos, Aleksandar J. Krmpot, Stanko N. Nikolić, Robert Krautz, Lars Terenius, Pavel Tomancak, Rudolf Rigler, Walter J. Gehring, Vladana Vukojević

Research output: Contribution to journalArticlepeer-review

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.
Original languageEnglish
Pages (from-to)218-225
Number of pages8
JournalMechanisms of Development
Volume138 Part 2
Early online date30 Sep 2015
DOIs
Publication statusPublished - Nov 2015

Keywords

  • Animals
  • Binding Sites
  • Cell Nucleus
  • DNA
  • DNA-Binding Proteins
  • Drosophila
  • Drosophila Proteins
  • Fluorescence
  • Gene Expression Regulation, Developmental
  • Genes, Homeobox
  • Homeodomain Proteins
  • Protein Binding
  • Spectrometry, Fluorescence
  • Transcription Factors
  • Journal Article
  • Research Support, Non-U.S. Gov't

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