Sir2 association with the HIS3-marked repeat is variable

It is possible that rDNA-PE is the result of different levels of Sir2 associating with different HIS3-marked rDNA repeat units. Chromatin immunoprecipitation (ChIP) was performed to measure the association of Sir2 protein with the HIS3-marked rDNA repeat using yeast strains from the NTS2::HIS3 and NTS1::HIS3 collections. The

expectation is that the level of Sir2 associated with the HIS3-marked repeat will correlate inversely with the strength of silencing; Sir2 association will be lower in less silent rDNA repeats compared to the more silent rDNA repeats.

DNA from chromatin immunoprecipitated with Sir2 antisera was purified and analyzed by quantitative PCR (qPCR). Two primer sets were used, one to assess the association of Sir2 with the HIS3-marked rDNA repeat unit, and a second set to measure the association of Sir2 with unmarked rDNA repeat units. The %IP of Sir2 at the HIS3- marked rDNA repeat was normalized to the %IP of Sir2 in all unmarked rDNA repeats (Figure 3-9A). ChIP experiments revealed that yeast strain MBY2617, which exhibited strong silencing of the HIS3 gene in the rDNA (Figure 3-3A, HIS3/ACT1 mRNA = 0.07), had significantly more Sir2 associated with the HIS3-marked rDNA repeat than two of the other NTS2::HIS3 strains, MBY2603 and MBY2608 (Figure 3-9A). MBY2617 also had higher average Sir2 association with the HIS3-marked repeat than MBY2508, but the numbers were not statistically significant. Similar levels of transcript from the HIS3 gene in the rDNA were detected in yeast strains MBY2508 and

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Figure 3-9. Chromatin immunoprecipitation (ChIP) with Sir2 antibody shows association of Sir2 with different HIS3-marked or unmarked rDNA repeats. (A) %IP of Sir2 with the HIS3 gene in yeast strains from the NTS2::HIS3 collection. (B) %IP of Sir2 with the HIS3 gene in yeast strains from the NTS1::HIS3 collection. (C) %IP of Sir2 with NTS2 in yeast strains from the NTS2::HIS3 collection. (D) %IP of Sir2 with NTS1 in yeast strains from the NTS1::HIS3 collection. Yeast strain numbers are shown on the X axis. %IP values are shown on the Y axis. The %IP Sir2 with the

HIS3 reporter in NTS2 (A) or NTS1 (B) was normalized to %IP Sir2 with NTS2 (C) or

NTS1 (D) of unmarked rDNA repeats. *p<0.05; **p<0.01.

A. B.

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MBY2603 (Figure 3-3A, HIS3/ACT1 mRNA ratios of 0.22 and 0.15, respectively), and Sir2 association with the HIS3-marked repeat in these strains was similar (Figure 3-9A). Consistent with this trend, yeast strain MBY2608, that had the highest level of transcript from the HIS3 gene in the rDNA (Figure 3-3A, HIS3/ACT1 mRNA ratio = 0.44) had significantly less Sir2 associated with the HIS3-marked rDNA repeat than the three other NTS2::HIS3 strains tested (Figure 3-9A). These data support the hypothesis that the level of Sir2 associated with the HIS3-marked rDNA repeat correlates with the strength of Pol II gene silencing in NTS2; more Sir2 was associated with the most silent HIS3- marked rDNA repeat unit and less Sir2 was associated with the least silent HIS3-marked rDNA repeat unit.

The association of Sir2 with HIS3-marked rDNA repeats was analyzed in four NTS1::HIS3 strains using ChIP and qPCR as described above. MBY2625, one of the least silent strains in the NTS1::HIS3 strain collection (Figure 3-3B, HIS3/ACT1 mRNA ratio = 0.48) had significantly less Sir2 associated with the HIS3-marked repeat than two strains in which silencing of HIS3 was stronger, MBY2636 and MBY2624 (Figure 3-3B,

HIS3/ACT1 mRNA ratio = 0.06 and 0.22, respectively) (Figure 3-9B). These results

corroborate the observations from the NTS2::HIS3 strains (Figure 3-9A) and suggest that less Sir2 is associated with the HIS3-marked rDNA repeats that are more highly

expressed.

While these data support the hypothesis that Sir2 association is lower with less silent HIS3-marked rDNA repeats, some strains in the NTS1::HIS3 collection had results that were inconsistent with this simple hypothesis. For example, Sir2 association with

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the HIS3-marked rDNA repeat in yeast strain MBY2636 was not significantly different from that in MBY2624, despite the difference in normalized HIS3 transcript levels (Figure 3-3B, 0.06 and 0.22, respectively). Further, in MBY2622, the steady state level of HIS3 mRNA was identical to that in yeast strain MBY2625 (Figure 3-3B, HIS3/ACT1 mRNA, 0.48); based on the simple hypothesis, MBY2622 was expected to have a lower level of Sir2 associated with the HIS3-marked repeat, similar to the level of association of Sir2 with HIS3 in yeast strain MBY2625. However, MBY2622 had significantly more Sir2 associated with the HIS3-marked repeat than MBY2625, with a level of Sir2 association similar to strains MBY2636 and MBY2624 that have more silent HIS3- marked rDNA repeats. These contrary data suggest that Sir2 association with HIS3- marked repeats in NTS1 is not the sole determinant of the strength of silent chromatin. It is possible that other proteins or differences in chromatin structure specific to NTS1 may be involved in determining the strength of silent chromatin.

The association of Sir2 with the NTS2 and NTS1 regions of the unmarked rDNA repeats was also analyzed by qPCR (Figure 3-9 C&D). No differences were found in the level of association of Sir2 with unmarked rDNA repeats, consistent with our results showing that the level of Sir2 protein in whole cell extracts from these strains was similar (Figure 3-8). These data indicate that variation in the level of Sir2 detected by ChIP is specific to the HIS3-marked repeat and not a reflection of variation in the average association of Sir2 across the entire rDNA array.

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3.3.8 The level of acetylated histone H3 at the HIS3-marked repeat is lower