FIG 4.

The Yng2 PHD and CHD-containing Eaf3 genetically and functionally interact in NuA4 function/activity. (A) Spot assays showing the effect of the W247A mutation of yng2 alone or in combination with deletion of eaf3 or eaf7 on cell growth. Tenfold serial dilutions of WT (strain QY3207), yng2-W247A (strain QY3208), Δeaf3 (strain QY3209), yng2-W247A Δeaf3 (strain QY3210), Δeaf7 (strain QY3219), and yng2-W247A Δeaf7 (strain QY3220) cells were grown for 2 to 10 days on YPD at the indicated temperatures (top) or at 30°C on the indicated drug-containing YPD (bottom). (B) Growth assays using the cells mentioned in panel A, which were spotted on the DNA-damaging agents methyl methanesulfonate (MMS; 0.03%) and hydroxyurea (HU; 130 mM). The cells were grown for 2 to 3 days at 30°C. (C) Spot assays with cells producing Eaf3 but lacking its chromodomain. Tenfold serial dilutions of Δeaf3 and Δeaf3 yng2-W247A cells containing plasmid pFL36 expressing Flag-Eaf3 WT or ΔCHD (amino acids 77 to 120) or the empty plasmid were grown on minimum medium (synthetic complete medium without Leu) at 30°C and 37°C and at 30°C on minimum medium containing the indicated drugs. (Note that the different levels of sensitivity to drugs compared to those in panel A are due to the use of minimal versus rich medium.) (D) Western blot analysis of wild-type and mutant Eaf3 expression in whole-cell extracts from cells used in the assay whose results are presented in panel C. Anti-Flag signals are compared to the anti-histone H3 signal as a loading control. (E) Western blot analysis of NuA4 complexes purified from WT, yng2-W247A, Δeaf3, and yng2-W247A Δeaf3 cells, using antibodies against the indicated subunits. no-TAP, a fraction purified from untagged cells by the method used for tagged cells. (F) Silver-stained gel of the NuA4 complexes analyzed in panel E and used in the HAT assays. Molecular size markers (in kilodaltons) are indicated on the left, and subunits are identified on the right. (G and H) Kinetics of HAT assays using NuA4 complexes purified from WT and yng2-W247A (Yng2W-A) Δeaf3 cells on native chromatin purified from WT (G) and Δset1 mutant (H) cells. After preincubation at room temperature for 15 min, acetyl-CoA was added to the reaction mixture, and the reactions were stopped at the indicated time points. Purified complexes were normalized on the basis of their activity on free histones. Values are based on those from two independent assays, and standard deviations are shown.