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. 2006 May;26(9):3339–3352. doi: 10.1128/MCB.26.9.3339-3352.2006

FIG. 4.

FIG. 4.

Analysis of Ubp8p recruitment and H2B-K123 ubiquitination at the PHO84, ADH1, and CUP1 genes, using a modified ChIP assay (see Materials and Methods). (A) Ubp8p is recruited to the UASs of the SAGA-dependent (PHO84, ADH1, and CUP1) genes. For analysis of the recruitment of Ubp8p to the PHO84 and ADH1 UASs, the yeast strains were grown in YPD to an OD600 of 1.0 at 30°C prior to formaldehyde cross-linking. The CUP1 gene was induced with 1 mM CuSO4 for 25 min in synthetic complete medium at 30°C before treatment with formaldehyde. Immunoprecipitations were performed using anti-Myc antibody. An anti-HA antibody (F-7; Santa Cruz Biotechnology, Inc.) was used to monitor the background signal. Primer pairs targeting the UASs of PHO84, ADH1, and CUP1 (see Materials and Methods) were used for PCR analysis of the immunoprecipitated DNA samples. (B) Analysis of H2B-K123 ubiquitination at PHO84, ADH1, and CUP1. Yeast cells (YKH045 [H2B wild type] and YKH046 [H2B-K123R point mutant]) were grown and cross-linked as described above. Immunoprecipitation was performed against HA-tagged ubiquitin using an anti-HA antibody. The anti-Myc antibody was used to monitor the background signal. Primer pairs targeting the UASs, core promoters, and ORFs of PHO84, ADH1, and CUP1 were used for PCR analysis of the immunoprecipitated DNA samples. ORF1 and ORF2 are located towards the 5′ and 3′ ends of the ORF, respectively. (C) The level of H2B-K123 ubiquitination is significantly elevated at the promoters but not the ORFs of PHO84, ADH1, and CUP1 in the Δubp8 mutant. The yeast strains YKH045, expressing HA-tagged ubiquitin, and ASY19 (derived by deleting UBP8 from YKH045) were grown as described above. Immunoprecipitation and PCR analysis were performed as described above. (D) Analysis of Ubp8p recruitment and H2B-K123 ubiquitination at the RPS5 UAS. (Top panel) Yeast strains expressing multiple-Myc-epitope-tagged Ubp8p were grown in YPD to an OD600 of 1.0 at 30°C, followed by formaldehyde-based cross-linking. Immunoprecipitation was carried out as described above. The primer pair targeting the RPS5 UAS was used in the PCR analysis. (Bottom panel) The yeast strains YKH045, YKH046, and ASY19 were grown and immunoprecipitated as described above.