Figure 2. A reduction of H3 and H4 genes copy number suppresses the growth defect caused by Abo1 depletion.

(A) Scheme of the conditional knock-down strategy of abo1 using the auxin-inducible degron system. TIR1 F-box proteins from Arabidopsis thaliana and Oryza sativa (rice) are expressed fused to S. pombe Skp1 (green rectangle TIR1), whereas endogenous Abo1 is fused to the 2xHA-IAA degron (blue triangle, IAA17) double tag. Addition of synthetic auxin 1-naphthaleneacetic acid (NAA) to the medium promotes association between Skp1–Cullin–F-box (SCF) E3 ubiquitin ligases complex and the degron tag, leading to subsequent ubiquitination of Abo1 and its degradation by the proteasome. (B) Western blot showing the level of Abo1-HA₂-IAA17 fusion protein, 6 h after addition of DMSO (−) or NAA (+) in cells expressing TIR1-Skp1 proteins (right panel, Abo1-HA₂-IAA17, SPV 4530). Protein extracts from untagged cells (left panel, untagged, SPV 4451) show the absence of cross-reaction with the α-HA antibody. Molecular weight markers (kD) are shown on the left. Kinetics of the effect of the conditional knock-down of Abo1 on cell growth are shown in Fig S4. (C) Growth curves of wt (SPV 4,451), abo1-aid (SPV 4,530, 4,531, and 4,532), abo1-aid, hht1∆, hhf1∆ (SPV 4,817, 4,818 and 4,819) and abo1-aid, hht2∆, hhf2∆ (SPV 4,822, 4,823 and 4,824) cells obtained from three biological replicates issued from two different clones. Cells were grown in liquid medium containing DMSO (upper graph, − NAA) or 0.5 mM NAA (lower graph, + NAA) for the indicated times before measuring the OD at 600 nm. In the presence of NAA, t test indicates a significant difference in abo1-aid cells’ growth, compared to all three other cell cultures, ***P < 0.001.