Journal: Molecular Biology of the Cell

Article Title: Interaction of Gcn4 with target gene chromatin is modulated by proteasome function

doi: 10.1091/mbc.E16-03-0192

Figure Lengend Snippet: Stimulation of Gcn4 target genes by SCF Cdc4 and the proteasome. (A) CDC34 (W303-1a) and cdc34-2 (MT670) yeast were grown to log phase at 30°C in minimal medium and then shifted to 37°C or maintained at 30°C for 1 h as indicated. Strains were then treated with SM or DMSO for 1.5 h, at which time RNA was collected and ARG1 mRNA levels quantified by RT-qPCR. Relative mRNA levels for ARG1 were normalized to the CDC34 strain treated with SM at 30°C. n = 3 . (B) As in A, except using CDC4 (W303-1a) and cdc4-1 (MT668) strains. n = 3 . (C) Anchor-away strains expressing HA-tagged Gcn4 (GHY139), FRB-tagged Cdc34 (GHY149), or FRB-tagged Gcn4 (GHY145) were grown to log phase in minimal medium, treated with either DMSO or rapamycin for 1 h, and then further treated with either DMSO or SM for 1.5 h. RNA was collected, and ARG1 mRNA levels were measured by RT-qPCR, as in A. Relative mRNA levels for ARG1 were normalized to the GCN4-HA strain treated with SM at 30°C. n = 3 . (D–F) Yeast bearing the pup1–T30A pre3–T20A mutations (GHY010) were grown to log phase in minimal medium and treated with either DMSO or MG132 for 1 h. Strains were then treated with SM or DMSO for 1.5 h, at which time RNA was collected and ARG1 (D), ARG4 (E), and HIS4 (F) mRNA levels quantified by RT-qPCR. Relative mRNA levels were then normalized to the SM-induced, DMSO-treated sample for each gene. n = 4 . Error bars represent SEM. (G) Yeast expressing either native Gcn4 (GHY010) or HA-tagged Gcn4 (GHY025) were grown to log phase in minimal medium and treated with DMSO or MG132 for 1 h. Strains were then treated with SM or DMSO for 1.5 h, at which time protein was extracted, resolved by SDS–PAGE, and subject to Western blotting with antibodies against the HA epitope or β-actin (Act).

Article Snippet: A previous study ( Lipford et al ., 2005 ) reported that activation of Gcn4 target genes is reduced by mutations in the Cdc34 and Cdc4 components of the SCF Cdc4 Ub ligase ( Chi et al ., 2001 ), as well as by inhibition of proteasomal proteolysis.

Techniques: Quantitative RT-PCR, Expressing, SDS Page, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Interaction of Gcn4 with target gene chromatin is modulated by proteasome function

doi: 10.1091/mbc.E16-03-0192

Figure Lengend Snippet: Inhibiting the proteasome impedes the ability of Gcn4 to bind target gene chromatin. (A) CDC4 (W303-1a), CDC4 GCN4-HA (GHY107), cdc4-1 (MT668), and cdc4-1 GCN4-HA (GHY107) strains were grown to log phase at 30°C in minimal medium, shifted to the restrictive temperature of 37°C for 1 h, and then induced with SM for an additional 1.5 h. At this time, ChIP was performed with an antibody against the HA-epitope tag. Coprecipitating ARG1 promoter DNA was quantified by qPCR, expressed relative to the percentage of input DNA. n = 3 . (B) GCN4-HA (GHY025) yeast were grown to log phase at 30°C in minimal medium, treated with either DMSO or MG132 for 1 h, and induced with SM for 1.5 h. ChIP was performed using antibodies against the HA-epitope tag or TBP. Coprecipitating ARG1 promoter DNA was quantified by qPCR. n = 3 . (C) As in B, except that coprecipitating DNAs from the anti-HA ChIP were quantified by qPCR, using primer pairs that amplify Gcn4-binding sites in the indicated genes. n = 3 . Error bars represent SEM. (D) GCN4-GFP HTB2-mCherry (GHY339) yeast were grown to log phase at 30°C in minimal medium, treated with either DMSO or MG132 for 1 h, and induced with SM for 1.5 h. Samples were imaged using either fluorescence (top) or differential interference contrast microscopy (bottom). Scale bars, 5 μm.

Article Snippet: A previous study ( Lipford et al ., 2005 ) reported that activation of Gcn4 target genes is reduced by mutations in the Cdc34 and Cdc4 components of the SCF Cdc4 Ub ligase ( Chi et al ., 2001 ), as well as by inhibition of proteasomal proteolysis.

Techniques: Binding Assay, Fluorescence, Microscopy