FACS analysis of DNA content for bar1Δ and bar1Δyta7Δ or wild-type and yta7Δ cells. (A) bar1Δ and bar1Δyta7Δ cells were blocked with α-factor, released, and samples were taken at the indicated times for FACS analysis. (B) Histograms of FACS data from A showing 1N and 2N DNA contents as a percentage of gated cells. (C) Wild-type and yta7Δ cells were blocked with nocodazole, released, and samples were taken at the indicated times for FACS analysis. The asterisks highlight the key differences between the WT and yta7Δ cells. (D) Histograms of FACS data from C showing 1N and 2N DNA contents as a percentage of gated cells.

Yta7 activity overlaps with the activities of other described histone regulators. (A) Temperature sensitivity. Indicated cells were 10-fold serially diluted and incubated at 25° or 34° for the indicated days (d). (B) Hydroxyurea (HU) sensitivity. Cells were 10-fold serially diluted and incubated at 25° for the indicated days. (C) Methyl methanesulfonate (MMS) sensitivity. Cells were 10-fold serially diluted and incubated at 25° for the indicated days.

Timing of CLN1 gene transcription and new bud formation in α-factor synchronized bar1Δ and yta7Δ bar1Δ cells and in nocodazole synchronized wild-type and yta7Δ cells. Cells were blocked with α-factor (A and B) or nocodazole (C and D), released, and samples were taken at the indicated times. For each time point, the amount of late G₁-cyclin CLN1 transcript relative to ACT1 transcript was determined by real-time PCR (A and C). Error bars are the standard deviation. The formation of new buds was also monitored (B and D).

Yta7 regulates histone gene transcription. bar1Δ (solid circles) and yta7Δ bar1Δ (open squares) cells were blocked with α-factor, released, and samples were taken at the indicated times. For each time point, cDNA was prepared and the amount of indicated transcript (relative to ACT1 transcript) was determined by real-time PCR. Darkly shaded regions emphasize the increased histone gene transcription upon gene deletion of YTA7. Error bars are the standard deviation. Asterisks show significant differences determined by one-tailed t-testing (P < 0.05).

Nocodazole blocking alters the observed yta7Δ transcriptional defect in histone gene regulation. (A) Wild-type (solid circles) and yta7Δ (open squares) cells were blocked with nocodazole and released. Samples were taken at the indicated times and analyzed as in Figure 3. Darkly shaded regions emphasize the increased histone gene transcription upon gene deletion of YTA7. Error bars are the standard deviation. (B) HTB1 gene transcription is upregulated upon deletion of HIR2. Wild-type (solid circles) and hir2Δ (open squares) cells were blocked with nocodazole and released. Samples were taken at the indicated times and analyzed as in Figure 3. Error bars are the standard deviation. Asterisks show significant differences determined by one-tailed t-testing (P < 0.05).

Yta7 does not direct or correlate with general H3K56 acetylation. (A) H3K56ac is not dependent on Yta7. ChIP to histone H3K56ac was performed in wild-type, yta7Δ, and H3K56R cells. Real-time PCR was used to measure enrichment at the intergenic region between HTA1 and HTB1 relative to ACT1. ChIP to unmodified H3 was used to control for nucleosome occupancy. Error bars are the standard deviation of the mean. The asterisk shows the significant difference determined by t-testing (P < 0.01). (B) Genomewide Yta7 binding does not correlate with H3K56ac. Over the 177 sites identified from ChIP-chip analysis of Yta7, only 6.8% were overlapping with previously reported H3K56ac-binding sites (Xu et al. 2005).

The Yta7 protein localizes to histone gene loci. ChIP-chip analysis of Yta7-Myc from asynchronous cells using 50-bp resolution tiled arrays (NimbleGen Systems). (A) Yta7 associated DNA occupancy is plotted as a function of relative coordinates on an average gene flanked with intergenic regions. (B) Shown is Yta7-Myc binding at the four histone loci: HTB1 and HTA1, HHF1 and HHT1, HHT2 and HHF2, and HTA2 and HTB2. The log₂ intensity ratio of immunoprecipitated to control DNA is plotted as a function of genomic position. Error bars are the standard deviation of triplicate array analyses.

Yta7bd interacts with histone H3. (A) Yta7 protein preferentially interacts with histone H3. Yta7bd-GST fusion, or GST alone, was incubated with purified histones and isolated on glutathione resin. Histones associated with Yta7 were visualized by immunoblotting. Input (I) and bound (B) lanes are shown. (B) Yta7bd interacts with the N-terminal tail of histone H3. Recombinant Yta7bd, or GST alone, was incubated with streptavidin Dynabeads coated with biotinylated peptides representing different regions and modification states of histone H3 (methylation and acetylation). Bound Yta7bd was resolved by SDS–PAGE and visualized by Coomassie staining. (C) Quantification of Yta7bd binding from B. Yta7bd binding is shown as a percentage relative to H3 1-20 binding. (D) Full-length Yta7 protein interacts with the N-terminal tail of histone H3. Purified Yta7-PrA was incubated with streptavidin Dynabeads coated with biotinylated peptides representing different regions and modification states of histone H3. Bound Yta7-PrA was resolved by SDS–PAGE and visualized by immunoblotting for the PrA tag. (E) Yta7 associated histone H3 was acetylated at Lys56. Yta7-PrA was immunopurified and coenriching proteins were resolved by SDS–PAGE/Coomassie staining. Copurifying histone H3 was treated in-gel with d6-acetic anhydride to isotopically mark unmodified lysines and then digested with trypsin. Shown is a section of a mass spectrum containing the H3K56 peptide as either acetylated (K56ac) or unacetylated (K56). In accordance to the monoisotopic peak areas, H3K56 was 33% acetylated.