γ-Tubulin interacts with E2F transcription factors to regulate proliferation and endocycling in Arabidopsis.

B M Kállai,C Papdi,J Chumová,Z Magyar,P Binarová,L Bögre,L Trögelová,T Mészáros,V Filimonenko,H Kourová,P Hozák,O Kofroňová

doi:10.1093/jxb/erz498

Abstract

γ-Tubulin is associated with microtubule nucleation, but evidence is accumulating in eukaryotes that it also functions in nuclear processes and in cell division control independently of its canonical role. We found that in Arabidopsis thaliana, γ-tubulin interacts specifically with E2FA, E2FB, and E2FC transcription factors both in vitro and in vivo. The interaction of γ-tubulin with the E2Fs is not reduced in the presence of their dimerization partners (DPs) and, in agreement, we found that γ-tubulin interaction with E2Fs does not require the dimerization domain. γ-Tubulin associates with the promoters of E2F-regulated cell cycle genes in an E2F-dependent manner, probably in complex with the E2F-DP heterodimer. The up-regulation of E2F target genes PCNA, ORC2, CDKB1;1, and CCS52A under γ-tubulin silencing suggests a repressive function for γ-tubulin at G1/S and G2/M transitions, and the endocycle, which is consistent with an excess of cell division in some cells and enhanced endoreduplication in others in the shoot and young leaves of γ-tubulin RNAi plants. Altogether, our data show ternary interaction of γ-tubulin with the E2F-DP heterodimer and suggest a repressive role for γ-tubulin with E2Fs in controlling mitotic activity and endoreduplication during plant development.

Highlights

Introduction γTubulin is a highly conserved microtubule nucleator in eukaryotic cells
Results γ-Tubulin binds to E2FA, E2FB and E2FC proteins in vitro Our previous finding on the nuclear localization of γ-tubulin and the meristem organisation phenotypes of γ-tubulin silenced plants (Binarova et al, 2000; Binarova et al, 2006) implied a cell cycle-related function for γ-tubulin
The obtained pull-down results c demonstrated that γ-tubulin and E2FA form a complex even under stringent washing s conditions, while no detectable amount of γ-tubulin is associated with the MYB66-coated u magnetic beads (Fig. 1A). n we co-translated hexaHis-labelled -tubulin and GST-tagged E2FA, E2FB and E2FC a proteins for pull-down experiments and showed that all three E2Fs have the ability to interact with -tubulin (Fig. 1B)

Summary

Introduction

Introduction γTubulin is a highly conserved microtubule nucleator in eukaryotic cells. The specificity of E2F/γ-tubulin interaction was p demonstrated by mixing and incubating 20 μl of GST-MYB66/E2FA and 40 μl of His12-γe tubulin containing in vitro translation mixtures for 1 h at room temperature and pulling down c the complexes with 2.5 μl of Pierce Glutathione Magnetic Beads.

Results

Conclusion