Regulation of AUXIN RESPONSE FACTOR condensation and nucleo-cytoplasmic partitioning

Auxin critically regulates plant growth and development. Auxin-driven transcriptional responses are mediated through the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. ARF protein condensation attenuates ARF activity, resulting in dramatic shifts in the auxin transcriptional landscape. Here, we perform a forward genetics screen for ARF hypercondensation, identifying an F-box protein, which we named AUXIN RESPONSE FACTOR F-BOX1 (AFF1). Functional characterization of SC

Inactivating UBE2M Impacts the DNA Damage Response and Genome Integrity Involving Multiple Cullin Ligases

Protein neddylation is involved in a wide variety of cellular processes. Here we show that the DNA damage response is perturbed in cells inactivated with an E2 Nedd8 conjugating enzyme UBE2M, measured by RAD51 foci formation kinetics and cell based DNA repair assays. UBE2M knockdown increases DNA breakages and cellular sensitivity to DNA damaging agents, further suggesting heightened genomic instability and defective DNA repair activity. Investigating the downstream Cullin targets of UBE2M revealed that silencing of Cullin 1, 2, and 4 ligases incurred significant DNA damage. In particular, UBE2M knockdown, or defective neddylation of Cullin 2, leads to a blockade in the G1 to S progression and is associated with delayed S-phase dependent DNA damage response. Cullin 4 inactivation leads to an aberrantly high DNA damage response that is associated with increased DNA breakages and sensitivity of cells to DNA damaging agents, suggesting a DNA repair defect is associated. siRNA interrogation of key Cullin substrates show that CDT1, p21, and Claspin are involved in elevated DNA damage in the UBE2M knockdown cells. Therefore, UBE2M is required to maintain genome integrity by activating multiple Cullin ligases throughout the cell cycle

Inhibiting CUL2 neddylation leads to impaired G1-S transition.

<p><b>A</b>. Double thymidine block experiments were performed in HEY cells treated with DMSO control or MLN4924 and UBE2M siRNA. See Experimental procedure for detailed protocol. The red line was established by selecting the peak value of the cells in G1 (2N) for the control siRNA sample at the zero hour time point. The red line was then kept constant between samples to provide a means of comparison. <b>B</b>. Double thymidine block experiments were performed in HEY cells individually knockdown with indicated cullins. <b>C</b>. Expression of siRNA-resistant CUL2 wild type (WT), but not the empty vector (EV) nor the CUL2 mutant (C689R; ΔNedd8 in the figure), partially rescues the G1-S arrest phenotype. CUL2 siRNA #3 targets the 3′UTR of the CUL2 mRNA. Western blot confirms the knockdown efficiency and ectopic expression of CUL2 proteins. <b>D</b>. Double thymidine block experiments were performed using the HCT116 wild type or p21-/- cells that are treated with either control or CUL2 siRNAs. <b>E</b>. Induction rate of RAD51 foci was measured in HEY cells treated with control or CUL2 siRNAs. The counting was <i>normalized</i> to the 0 time point to indicate the fold increase.</p

Model.

<p>UBE2M inhibition impacts DNA damage response and genome integrity involving multiple Cullin ligases.</p

Disruption of genomic integrity upon UBE2M silencing.

<p><b>A</b>. γ-H2AX foci was measured upon expression of shRNA targeting UBE2M 3′UTR, then rescued by expressing siRNA-resistant UBE2M WT or C111S mutant. The western blot analysis shows the knockdown efficiency and the expression of FLAG-HA tagged UBE2M WT and C11S mutant. (∼3 kDa shift is predicted). <b>B</b>. Neutral comet assay. HEY cells were transfected with control or two independent UBE2M siRNAs for ∼72 hours before harvest for the analysis. The tail moment is the length of the tail times the density of the tail. % tail DNA is the density of the tail divided by the density of the tail plus the density of the head.</p

Effects of individual Cullin silencing in genome integrity.

<p><b>A</b>. γ-H2AX foci induction was measured in HEY cells treated siRNAs against indicated cullins. Knockdown efficiency is shown in right. <b>B</b>. Formation of double strand breaks were measured using neutral comet assay, in HEY cells treated with siRNAs against indicated cullins.</p

Silencing of CUL4 leads to G2-M checkpoint activation that is associated with DNA repair defects.

<p><b>A</b>. Resolution of RAD51 foci was measured upon knockdown of individual Cullins. Schematic of the experiment shown in left. <b>B</b>. RAD51 foci kinetics was performed in cells in which CDT2 was stably knockdown. <b>C</b>. Prior depletion of CDT1 or p21 by siRNAs partially rescues the hype-RAD51 foci formation in MLN4924 treated cells. <b>D</b>. Clonogenic assays were performed for the HEY cells knockdown with CUL4A or CDT2. <b>E</b>. HR repair assays <b>F</b>. NHEJ assay.</p

DNA damage response is perturbed by UBE2M silencing.

<p>A. Growth suppression by UBE2M silencing is enhanced by DNA damaging agents. Growth sensitivity of HEY cells in the presence of CPT and PARP inhibitor ABT888 (Figure S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101844#pone.0101844.s001" target="_blank">File S1</a>) was monitored using clonogenic assay. <b>B</b>. Treatment of HeLa cells with MLN4924 (0.3uM) leads to elevated BRCA1 and RAD51 foci formation. *indicates neddylated form. <b>C</b>. Time course study of RAD51 foci recruitment and resolution upon MLN4924 treatment. D. Time course study of RAD51 foci in UBE2M knockdown cells. <b>E</b>. Cells depleted of UBE2M were analyzed for HR (<b>E</b>) and NHEJ repair (F) assays.</p