The functional overlap of Yta7 and certain histone regulators is bromodomain dependent. (A) To assay temperature sensitivity, indicated cells were 10-fold serially diluted and incubated at 25°C or 34°C for the indicated number of days (d). To assay HU sensitivity, cells were 10-fold serially diluted and incubated at 25°C for the indicated number of days. (B) Deletion/mutation of the Yta7BD, Asf1, or Spt16 does not affect protein levels of Yta7 or Yta7 with a bromodomain deletion. Western blotting was used to detect the PrA tag on Yta7 or the internal PrA tag used to delete the Yta7BD in the indicated strains. Western blotting for histone H3 served as a loading control.

The Yta7BD binds to histones in a PTM-independent manner. (A) Recombinant Yta7BD-GST or GST alone was incubated with purified histones and isolated on glutathione resin. Associated histones were resolved on Tris-glycine gels (4 to 20%) and visualized by Coomassie blue staining. (B) Yng1 PHD finger and the CBP bromodomain show specific binding under the Yta7BD binding assay conditions. Recombinant GST, GST-Yng1 PHD finger, or GST-CBP bromodomain was incubated with the indicated biotinylated histone peptides immobilized on streptavidin-coated Dynabeads. Bound protein was resolved by denaturing gel electrophoresis and visualized by Coomassie blue staining. (C) Quantitative analysis of histone acetylation copurifying with Yta7BD-GST. Copurifying and input histones shown in panel A were treated with d6-acetic anhydride and digested with trypsin. Shown are overlaid MALDI spectra of the N-terminal tail of histone H4 corresponding to peptide 4-17 containing K5, K8, K12, and K16 that have been chemically (45 Da) or in vivo (42 Da) acetylated. No significant difference in histone acetylation was observed. The complete set of results is in Table 2.

The bromodomain of Yta7 is required for barrier activity. The YTA7 gene or the bromodomain region of the gene was deleted in strains harboring a URA3 reporter gene at the transcriptionally active barrier (ROY508) or silent region (ROY648) of HMR. Cells were 10-fold serially diluted on medium with or without 5-FOA. WT, wild type.

Deletion of the bromodomain genomically redistributes Yta7. (A) ChIP-chip analysis of Yta7-Myc and Yta7BDΔ-Myc. A Venn diagram shows a redistribution of Yta7 binding with deletion of the bromodomain. Binding sites from the microarray are reported at the 95% confidence level. (B) Yta7 binding at GLN4 was lost with bromodomain deletion. Binding is reported as the log₂ of the immunopurified signal to whole-cell lysate array signals. The asterisk indicates significant binding (95% confidence level). (C) Bromodomain deletion resulted in detectable Yta7 binding at YCR095C. The asterisk indicates significant binding (95% confidence level). (D) Deletion of the bromodomain did not affect Yta7 association at the HTB1-HTA1 histone locus. The asterisks indicate significant binding (95% confidence level). (E) Conventional ChIP for the Myc tag on Yta7 and Yta7BDΔ confirms the redistribution shown in panels B, C, and D. Error bars are the standard errors from triplicate analyses. (F) Bromodomain deletion does not affect HTB1 transcription. Transcript levels of HTB1 relative to ACT1 in asynchronous cells harboring full-length Yta7 or the bromodomain deletion. Error bars are the standard deviations. Asterisks show significant differences determined by one-tailed testing (P < 0.001). WT, wild type.

Yta7 contains histone binding regions distinct from the bromodomain. (A) Schematic of Yta7 constructs used to determine histone binding. The shapes represent: PrA tag (circles), AAA ATPase domain (filled squares), putative AAA ATPase domain (striped squares), and bromodomain (open squares). Constructs were made genomically. (B) Affinity purification of the constructs in panel A. Yta7 constructs were purified on immunoglobulin G-coated Dynabeads under conditions that preserve in vivo protein associations. PrA-tagged Yta7 and associated proteins were resolved by denaturing gel electrophoresis and visualized by Coomassie blue staining. Yta7 constructs and histones were identified by MS.