Figure 5.

HDA2 and HDA3 are required for deacetylation of histones H3 and H2B by HDA1 at the ENA1 promoter. Chr-IP demonstrating the effects of hda1Δ, hda2Δ, and hda3Δ on the acetylation of (A) H4 sites K5, K8, K12, and K16; (B) H3 sites K9, K14, K18, K23, and K27; and (C) H2A site K7 and H2B sites K11 and K16 at the ENA1, GAL10, and HO promoters. Amplification of a 138-bp fragment 0.5 kb from the telomere (Tel) of chromosome VI-R was used as reference to ensure equal loading of samples. Yeast strains used for Chr-IP are WT (YDS2), hda1Δ (WJY127), hda2Δ, (WJY128), and hda3Δ (WJY129). HO was found to be relatively unaffected by HDA1 disruption and was used as a negative control. (D) Quantification of increases in H3 and H2B acetylation in hda1Δ, hda2Δ, and hda3Δ cells relative to WT cells. ³²P-α-dATP was added to the PCR mixture, and the PhosphorImager was used to quantitate the intensity of ENA1 PCR bands in the hda1Δ, hda2Δ, and hda3Δ mutants relative to WT after normalizing to the Tel bands.