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. 2019 Apr 23;38(11):e100368. doi: 10.15252/embj.2018100368

Figure 7. The ub‐hotspot pathway is required to regulate aberrant Euc1 function.

Figure 7

  1. Deletion of EUC1 does not lead to ub‐HS adjacent gene deregulation, but rather widespread transcriptome changes. Total RNA isolated from WT and euc1∆ cells was polyA‐enriched and sequenced (RNAseq, triplicates). Significance testing was based on the Wald test, see Materials and Methods for details. See also Appendix Fig S8A for quantification of selected transcripts by qPCR. baseMean: mean expression levels across all samples.
  2. RNAseq transcriptome analysis of EUC1 overexpression as in (A). ∆euc1 cells with pGAL‐EUC1 or pEUC1‐EUC1 (control) integrated at the URA3 locus (YIplac211) were grown to mid‐log phase, and 2% galactose was added for 3 h. See also Appendix Fig S8E.
  3. EUC1 overexpression is toxic at elevated temperatures and upon exposure to the membrane‐fluidizing drug benzyl alcohol (BenzAlc). For (C, D, F), serial dilutions of the indicated strains were spotted and grown on YPD control (or selective media) plates or under conditions as indicated. See also Fig EV4A and B.
  4. EUC1 overexpression toxicity depends on DNA binding and transactivation. The indicated EUC1 alleles with endogenous or galactose‐inducible promoters were integrated at the URA3 locus (YIplac211, euc1∆ background) and spotted on glucose control or galactose plates to induce EUC1 overexpression. See also Appendix Fig S9A–C.
  5. Aberrant binding of overexpressed EUC1 partially depends on Euc1 DNA binding. ChIP‐qPCR of Euc1 after 3‐h galactose induction. Note that IP/input ratios of Euc1 signals are shown, also for the control locus (contr., TOS1 promoter) to highlight Euc1 binding at non‐ub‐HS sites. STI1‐CIN5: intergenic region. TEC1‐us: upstream (promoter) region. Data represent means ± SD (n = 3). A.U.: arbitrary units. See also Fig EV4C and D.
  6. Simultaneous overexpression of SLX5 and SLX8 rescue EUC1 toxicity. Experiment as in (D), but with concomitant plasmid‐borne overexpression (ADH promoter) of SLX5 and SLX8 (bottom panels) and on media selecting for SLX5/SLX8 plasmids. See also Fig EV4E and Appendix Fig S9D.