Abstract
GAGA-motif binding proteins control transcriptional activation or repression of homeotic genes. Interestingly, there are no sequence similarities between animal and plant proteins. Plant BBR/BPC-proteins can be classified into two distinct groups: Previous studies have elaborated on group I members only and so little is known about group II proteins. Here, we focused on the initial characterization of AtBPC6, a group II protein from Arabidopsis thaliana. Comparison of orthologous BBR/BPC sequences disclosed two conserved signatures besides the DNA binding domain. A first peptide signature is essential and sufficient to target AtBPC6-GFP to the nucleus and nucleolus. A second domain is predicted to form a zipper-like coiled-coil structure. This novel type of domain is similar to Leucine zippers, but contains invariant alanine residues with a heptad spacing of 7 amino acids. By yeast-2-hybrid and BiFC-assays we could show that this Alanine zipper domain is essential for homotypic dimerization of group II proteins in vivo. Interhelical salt bridges and charge-stabilized hydrogen bonds between acidic and basic residues of the two monomers are predicted to form an interaction domain, which does not follow the classical knobs-into-holes zipper model. FRET-FLIM analysis of GFP/RFP-hybrid fusion proteins validates the formation of parallel dimers in planta. Sequence comparison uncovered that this type of domain is not restricted to BBR/BPC proteins, but is found in all kingdoms.
Highlights
Eukaryotic gene expression is tightly controlled by enhancer and silencer elements
For group II proteins, such as AtBPC6 from Arabidopsis, only two domains were predictable from sequence analyses, which are a coiled-coil signature and the conserved DNA-binding domain shared with group I members
It was shown that the Barley B Recombinant (BBR)/Basic Pentacysteine (BPC) proteins are capable of DNA-bending in vitro and it has been assumed they might harbor an orthologous function to Drosophila Trl or Psq in planta [13,18]
Summary
Eukaryotic gene expression is tightly controlled by enhancer and silencer elements In between these DNA regions, insulator elements have been identified in animals, which are bound by proteins, that mediate insulator function and prevent illegitimate activation or repression of neighboring loci [1]. In Drosophila melanogaster about twenty years ago the GAGA-factor (GAF) encoded by the Trithorax-like (Trl) gene was identified to bind to GAGA DNA-motifs inside such insulator regions [2,3]. Functions of GAGA-motif binding proteins are more diverse and can be linked to epigenetic regulation of homeotic genes e.g. by recruiting silencing factors to specific sites, as well as influencing the promoter-proximal pausing of RNA Polymerase II (Pol II) [5,9,10,11]
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