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Antagonistic Gcn5-Hda1 interactions revealed by mutations to the Anaphase Promoting Complex in yeast.

Islam A, Turner EL, Menzel J, Malo ME, Harkness TA - Cell Div (2011)

Bottom Line: Occlusion of Gcn5 recruitment to these promoters involved Hda1 and Tup1.Our data suggests large Gcn5 and Hda1 containing complexes may compete for space on promoters that utilize the Ssn6/Tup1 repressor complex.We predict that in apc5CA cells the accumulation of an APC target may compensate for the loss of both GCN5 and HDA1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Cell Biology, University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada. troy.harkness@usask.ca.

ABSTRACT

Background: Histone post-translational modifications are critical for gene expression and cell viability. A broad spectrum of histone lysine residues have been identified in yeast that are targeted by a variety of modifying enzymes. However, the regulation and interaction of these enzymes remains relatively uncharacterized. Previously we demonstrated that deletion of either the histone acetyltransferase (HAT) GCN5 or the histone deacetylase (HDAC) HDA1 exacerbated the temperature sensitive (ts) mutant phenotype of the Anaphase Promoting Complex (APC) apc5CA allele. Here, the apc5CA mutant background is used to study a previously uncharacterized functional antagonistic genetic interaction between Gcn5 and Hda1 that is not detected in APC5 cells.

Results: Using Northerns, Westerns, reverse transcriptase PCR (rtPCR), chromatin immunoprecipitation (ChIP), and mutant phenotype suppression analysis, we observed that Hda1 and Gcn5 appear to compete for recruitment to promoters. We observed that the presence of Hda1 can partially occlude the binding of Gcn5 to the same promoter. Occlusion of Gcn5 recruitment to these promoters involved Hda1 and Tup1. Using sequential ChIP we show that Hda1 and Tup1 likely form complexes at these promoters, and that complex formation can be increased by deleting GCN5.

Conclusions: Our data suggests large Gcn5 and Hda1 containing complexes may compete for space on promoters that utilize the Ssn6/Tup1 repressor complex. We predict that in apc5CA cells the accumulation of an APC target may compensate for the loss of both GCN5 and HDA1.

No MeSH data available.


Related in: MedlinePlus

Histone H3 acetylation at promoter regions is elevated specifically in apc5CA hda1Δ cells at 37°C. (A) ChIP was performed using lysates derived from the mutants shown and antibodies against total H3 or H3 acetylated at both K9 and K14. A mock treatment lacking antibody was used as a control. Once the crosslinks were reversed and DNA recovered, "end point" PCR was performed using primers against the genes shown that amplified 200 bp regions of the promoter. 10% of the reaction was used as input. (B) Two independent experiments were quantified, with the means and standard errors represented graphically, as previously described [66,70].
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Figure 3: Histone H3 acetylation at promoter regions is elevated specifically in apc5CA hda1Δ cells at 37°C. (A) ChIP was performed using lysates derived from the mutants shown and antibodies against total H3 or H3 acetylated at both K9 and K14. A mock treatment lacking antibody was used as a control. Once the crosslinks were reversed and DNA recovered, "end point" PCR was performed using primers against the genes shown that amplified 200 bp regions of the promoter. 10% of the reaction was used as input. (B) Two independent experiments were quantified, with the means and standard errors represented graphically, as previously described [66,70].

Mentions: Our data suggests the apc5CA allele enhances the transcript levels of some of the tested genes in hda1Δ cells. We next tested whether promoter acetylation of these genes was similarly impacted using chromatin immunoprecipitation (ChIP) with antibodies that recognized acetylated lysines 9 and 14 on histone H3 (H3K9/14Ac), and primers that amplified 200 basepair fragments immediately upstream of the transcriptional start site of the genes studied above. We used H3K9/K14Ac antibodies to capture acetylation of both H3K9 and H3K14 as our studies show these residues are targeted by Gcn5 and Hda1. We assessed promoter acetylation in gcn5Δ, hda1Δ and gcn5Δ hdaΔ1 mutants in the apc5CA background (Figure 3A). Antibodies against total H3 and a no antibody mock treatment were used as controls. The bands in all experiments were quantified and analyzed (Figure 3B). Once background densities were subtracted from all bands, the H3K9/14Ac/total H3 ratio was determined. The values represent two independent experiments, as described previously [66,70]. H3 promoter acetylation was reduced in both apc5CA gcn5Δ and apc5CA gcn5 hda1Δ cells, similar to the transcript patterns at 37°C (Figure 2C), strengthening the notion that Gcn5 HAT activity is tightly correlated with transcription. However, it is interesting to note that while promoter acetylation is equally low in apc5CA gcn5Δ cells at 30 and 37°C, transcript defects are only obvious at 37°C. Notably, a previous study observed that Gcn5-dependent transcription and promoter histone acetylation activities could be uncoupled [71].


Antagonistic Gcn5-Hda1 interactions revealed by mutations to the Anaphase Promoting Complex in yeast.

Islam A, Turner EL, Menzel J, Malo ME, Harkness TA - Cell Div (2011)

Histone H3 acetylation at promoter regions is elevated specifically in apc5CA hda1Δ cells at 37°C. (A) ChIP was performed using lysates derived from the mutants shown and antibodies against total H3 or H3 acetylated at both K9 and K14. A mock treatment lacking antibody was used as a control. Once the crosslinks were reversed and DNA recovered, "end point" PCR was performed using primers against the genes shown that amplified 200 bp regions of the promoter. 10% of the reaction was used as input. (B) Two independent experiments were quantified, with the means and standard errors represented graphically, as previously described [66,70].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3141613&req=5

Figure 3: Histone H3 acetylation at promoter regions is elevated specifically in apc5CA hda1Δ cells at 37°C. (A) ChIP was performed using lysates derived from the mutants shown and antibodies against total H3 or H3 acetylated at both K9 and K14. A mock treatment lacking antibody was used as a control. Once the crosslinks were reversed and DNA recovered, "end point" PCR was performed using primers against the genes shown that amplified 200 bp regions of the promoter. 10% of the reaction was used as input. (B) Two independent experiments were quantified, with the means and standard errors represented graphically, as previously described [66,70].
Mentions: Our data suggests the apc5CA allele enhances the transcript levels of some of the tested genes in hda1Δ cells. We next tested whether promoter acetylation of these genes was similarly impacted using chromatin immunoprecipitation (ChIP) with antibodies that recognized acetylated lysines 9 and 14 on histone H3 (H3K9/14Ac), and primers that amplified 200 basepair fragments immediately upstream of the transcriptional start site of the genes studied above. We used H3K9/K14Ac antibodies to capture acetylation of both H3K9 and H3K14 as our studies show these residues are targeted by Gcn5 and Hda1. We assessed promoter acetylation in gcn5Δ, hda1Δ and gcn5Δ hdaΔ1 mutants in the apc5CA background (Figure 3A). Antibodies against total H3 and a no antibody mock treatment were used as controls. The bands in all experiments were quantified and analyzed (Figure 3B). Once background densities were subtracted from all bands, the H3K9/14Ac/total H3 ratio was determined. The values represent two independent experiments, as described previously [66,70]. H3 promoter acetylation was reduced in both apc5CA gcn5Δ and apc5CA gcn5 hda1Δ cells, similar to the transcript patterns at 37°C (Figure 2C), strengthening the notion that Gcn5 HAT activity is tightly correlated with transcription. However, it is interesting to note that while promoter acetylation is equally low in apc5CA gcn5Δ cells at 30 and 37°C, transcript defects are only obvious at 37°C. Notably, a previous study observed that Gcn5-dependent transcription and promoter histone acetylation activities could be uncoupled [71].

Bottom Line: Occlusion of Gcn5 recruitment to these promoters involved Hda1 and Tup1.Our data suggests large Gcn5 and Hda1 containing complexes may compete for space on promoters that utilize the Ssn6/Tup1 repressor complex.We predict that in apc5CA cells the accumulation of an APC target may compensate for the loss of both GCN5 and HDA1.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Cell Biology, University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada. troy.harkness@usask.ca.

ABSTRACT

Background: Histone post-translational modifications are critical for gene expression and cell viability. A broad spectrum of histone lysine residues have been identified in yeast that are targeted by a variety of modifying enzymes. However, the regulation and interaction of these enzymes remains relatively uncharacterized. Previously we demonstrated that deletion of either the histone acetyltransferase (HAT) GCN5 or the histone deacetylase (HDAC) HDA1 exacerbated the temperature sensitive (ts) mutant phenotype of the Anaphase Promoting Complex (APC) apc5CA allele. Here, the apc5CA mutant background is used to study a previously uncharacterized functional antagonistic genetic interaction between Gcn5 and Hda1 that is not detected in APC5 cells.

Results: Using Northerns, Westerns, reverse transcriptase PCR (rtPCR), chromatin immunoprecipitation (ChIP), and mutant phenotype suppression analysis, we observed that Hda1 and Gcn5 appear to compete for recruitment to promoters. We observed that the presence of Hda1 can partially occlude the binding of Gcn5 to the same promoter. Occlusion of Gcn5 recruitment to these promoters involved Hda1 and Tup1. Using sequential ChIP we show that Hda1 and Tup1 likely form complexes at these promoters, and that complex formation can be increased by deleting GCN5.

Conclusions: Our data suggests large Gcn5 and Hda1 containing complexes may compete for space on promoters that utilize the Ssn6/Tup1 repressor complex. We predict that in apc5CA cells the accumulation of an APC target may compensate for the loss of both GCN5 and HDA1.

No MeSH data available.


Related in: MedlinePlus