Supplemental Material

This article contains the following supporting material:

• Results -

• Figure 1 - Sir2p levels are normal in esa1 mutants. Whole cell extracts were prepared for the indicated strains and two dilutions examined for each as indicated by protein immunoblotting. Immunoreactivity was detected for Sir2p (upper panel) and a non-specific loading control (lower panel). At both 30°C and 34°C, Sir2p protein levels were comparable in the wild-type and esa1 strains.

• Figure 2 - esa1p HAT activity is required for rDNA and telomeric silencing. (A) Expression of the dominant negative HAT catalytic mutant ESA1-E338Q disrupted wild-type rDNA silencing. The ESA1 wild-type strain transformed with an esa1-E338Q TRP1 2-micron plasmid (LPY6220-LPY6223) was partially defective for rDNA silencing on plates containing canavanine (right, -trp rDNA silencing), compared to the ESA1 (LPY6219) and vector control (LPY6217) transformants. However, no growth defect was observed in the ESA1-E338Q transformants (center, -trp). (B) Moreover, the esa1-414 mutant strain transformed with the ESA1-E338Q TRP1 2-micron plasmid (LPY6228-LPY6231) was completely defective for rDNA silencing at 28°C, compared to ESA1 (LPY6227) and vector control (LPY6225) transformants. In the esa1-414 mutant, some growth defects were observed in the ESA1-E338Q transformants (center, -trp). (C) Similarly, expression of the ESA1-E338Q dominant negative mutant disrupted telomeric silencing in wild-type and (D) esa1-414 mutant strains. (C) A wild-type strain transformed with esa1-E338Q (LPY6184-LPY6187) displayed no growth defects (center, -trp) but had strong telomeric silencing defects on 5-FOA plates (right, -trp 5-FOA) compared to vector (LPY6181) and ESA1 (LPY6182) transformants. (D) In an esa1-414 mutant strain transformed with ESA1-E338Q (LPY6192-LPY6195), growth was somewhat compromised (center, -trp) and telomeric silencing was completely disrupted on 5-FOA plates (right, -trp 5-FOA) compared to vector (LPY6189) and ESA1 (LPY6190) transformants. All assays were performed at 28°C and the -trp rDNA silencing plates in (A) and (B) contained 32 μg/ml canavanine, an intermediate concentration (see Figure 2 legend) to compare directly rDNA silencing effects in wild-type and esa1-414 mutant strains.