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Results: 6

1.
Figure 2

Figure 2. From: Zinc-responsive coactivator recruitment by the yeast Zap1 transcription factor.

Zap1 protein accumulation is not affected in most coactivator mutant strains. Coactivator deletion mutants that affected YOR387C-lacZ expression were grown to mid-log phase in LZM supplemented with 1 μM ZnCl2. Protein extracts were prepared and analyzed by immunoblotting using an antibody raised against the DNA binding domain of Zap1. Pgk1 phosphoglycerate kinase was used as a loading control.

Avery G Frey, et al. Microbiologyopen. 2012 June;1(2):105-114.
2.
Figure 3

Figure 3. From: Zinc-responsive coactivator recruitment by the yeast Zap1 transcription factor.

Coactivators required for activation of YOR387C-lacZ are not activation domain specific. The indicated coactivator mutant strains lacking chromosomal ZAP1 (zap1Δ) and expressing either Zap1WT (filled columns), Zap1AD1 (hatched columns), or Zap1AD2 (open columns) were transformed with DPP1-lacZ (A) or ZRT1-lacZ (B) reporters. These cells were grown to mid-log phase in LZM supplemented with 1 μM ZnCl2 and then assayed for β-galactosidase activity. The shown values are the means of three independent cultures and are expressed as a percentage of the isogenic zap1Δ strain expressing the corresponding Zap1 allele but lacking any coactivator mutation. The values shown are the means of three independent cultures and the error bars represent ±1 SD. The dashed line indicates 100% activity measured in the control strains.

Avery G Frey, et al. Microbiologyopen. 2012 June;1(2):105-114.
3.
Figure 5

Figure 5. From: Zinc-responsive coactivator recruitment by the yeast Zap1 transcription factor.

Coactivator recruitment by Zap1 is strongly interdependent. Results from chromatin immunoprecipitation of Ada2-TAP (A), Spt3-TAP (B), Swi3-TAP (C), and Med15-TAP (D) are shown. Isogenic ada2Δ and swi3Δ mutants were used to determine the effects of disrupting one complex on the recruitment of others. The indicated strains were grown to mid-log phase in LZM supplemented with 1 μM ZnCl2. Cells were then cross-linked with formaldehyde, harvested, and chromatin immunoprecipitation analysis was performed using IgG-Sepharose to immunoprecipitate TAP-tagged proteins. Coprecipitation of specific DNA fragments was then assessed by real-time PCR using primers flanking the ZREs of the ZRT1 promoter. The values shown are the means of three independent cultures and the error bars represent 1 SD.

Avery G Frey, et al. Microbiologyopen. 2012 June;1(2):105-114.
4.
Figure 4

Figure 4. From: Zinc-responsive coactivator recruitment by the yeast Zap1 transcription factor.

Recruitment of Swi3, Med15, and Spt3 is zinc responsive and Zap1 dependent. Untagged wild-type (BY4741), and isogenic Swi3-TAP, Med15-TAP, and Spt3-TAP tagged strains were grown in LZM supplemented with either 1 μM (−, lanes 1–4) or 1000 μM (+, lanes 5–8) ZnCl2. Isogenic zap1Δ cells grown in low zinc were used in lanes 9–12. The cells were cross-linked with formaldehyde, harvested, and chromatin immunoprecipitation analysis was performed using IgG-Sepharose to immunoprecipitate the TAP-tagged proteins. Coprecipitation of specific DNA fragments was then assessed by PCR using primers flanking the ZREs of the ZRT1 and ZPS1 promoters. Primers specific for the CMD1 promoter were used as a negative control. PCR amplification of 10-fold serial dilutions of input samples was used to confirm the quantitative nature of the assay.

Avery G Frey, et al. Microbiologyopen. 2012 June;1(2):105-114.
5.
Figure 1

Figure 1. From: Zinc-responsive coactivator recruitment by the yeast Zap1 transcription factor.

YOR387C induction by Zap1 is AD1 specific. (A) Zap1 alleles used in this study. (B) zap1Δ cells expressing either Zap1WT, Zap1AD1, Zap1AD2, or the empty vector were grown to mid-log phase in LZM supplemented with either 1 μM (−) or 1000 μM (+) ZnCl2. Total RNA was then isolated and YOR387C mRNA levels were measured by S1 nuclease protection assays. Expression of CMD1, which encodes calmodulin, was used as a loading control. (C) Cells as described in panel B bearing the YOR387C-lacZ reporter were grown to mid-log phase in LZM supplemented with either 1 μM (filled columns) or 1000 μM (open columns) ZnCl2. Cells were then harvested, and β-galactosidase assays were performed. The values shown are the means of three independent cultures and the error bars represent ±1 SD.

Avery G Frey, et al. Microbiologyopen. 2012 June;1(2):105-114.
6.
Figure 6

Figure 6. From: Zinc-responsive coactivator recruitment by the yeast Zap1 transcription factor.

Coactivator recruitment by AD2 is weaker than AD1-mediated recruitment. Chromosomal ZAP1 was deleted from strains expressing Med15-TAP, Spt3-TAP, or Swi3-TAP. The resulting strains were transformed with Zap1AD1, Zap1AD2, or the empty vector and grown to mid-log phase in LZM supplemented with 1 μM ZnCl2. These cells were then cross-linked with formaldehyde, harvested, and chromatin immunoprecipitation analysis was performed using IgG-Sepharose to immunoprecipitate TAP-tagged proteins. Coprecipitation of the ZRT1 promoter was then assessed by real-time PCR using primers flanking the ZRT1 ZREs. The values shown are the means of three independent cultures and the error bars represent 1 SD. The letters denote values that are significantly different from each other (P < 0.05) as determined using ANOVA. The dashed lines mark the background level of recruitment observed in the vector-only cells.

Avery G Frey, et al. Microbiologyopen. 2012 June;1(2):105-114.

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