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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

Tup1 occludes Gcn5 recruitment. A) ChIP was performed using the cells shown expressing GALpro-GCN5-HA following a 5 hour galactose induction, as described above. (B) Two independent experiments were scanned and processed using ImageJ, with the means and standard errors shown. (C) Strains lacking TUP1 were constructed in WT and apc5CA backgrounds. Growth phenotypes were assessed by spot-dilutions, followed by incubation at 34°C and 37°C.
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Figure 6: Tup1 occludes Gcn5 recruitment. A) ChIP was performed using the cells shown expressing GALpro-GCN5-HA following a 5 hour galactose induction, as described above. (B) Two independent experiments were scanned and processed using ImageJ, with the means and standard errors shown. (C) Strains lacking TUP1 were constructed in WT and apc5CA backgrounds. Growth phenotypes were assessed by spot-dilutions, followed by incubation at 34°C and 37°C.

Mentions: We next tested whether the impact of Hda1 on Gcn5 promoter accessibility involved the corepressor complex Ssn6/Tup1. Several reports have demonstrated that the Ssn6/Tup1 corepressor utilizes Hda1 to repress transcription of target genes [36,41,68]. Furthermore, Tup1 has been shown to recruit Gcn5 to repressed promoters [73-75]. It was proposed that this may set the stage for derepression of silent genes. Thus, GALproGCN5-HA was induced in hda1Δ and tup1Δ cells as the only source of Gcn5, followed by ChIP. Gcn5-HA expression in hda1Δ cells was reduced compared to WT, but expression in tup1Δ cells was unchanged (data not shown). We found that in otherwise WT strains (gcn5Δ + GALproGCN5-HA), Gcn5-HA was weakly recruited to the tested promoters (Figures 6A and 6B). In strains lacking HDA1 or TUP1, Gcn5-HA promoter occupancy was observed to increase. We also observed that in cells lacking SSN6, promoter recruitment of Gcn5-HA increased (data not shown). These results suggest that Hda1 may work together with the Ssn6/Tup1 corepressor complex to impede access of Gcn5 to the tested promoters. However, Hda1 and Gcn5 may also compete for Tup1 interactions. It is also feasible that Tup1 utilizes different mechanisms to reduce Gcn5 promoter occupancy.


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)

Tup1 occludes Gcn5 recruitment. A) ChIP was performed using the cells shown expressing GALpro-GCN5-HA following a 5 hour galactose induction, as described above. (B) Two independent experiments were scanned and processed using ImageJ, with the means and standard errors shown. (C) Strains lacking TUP1 were constructed in WT and apc5CA backgrounds. Growth phenotypes were assessed by spot-dilutions, followed by incubation at 34°C and 37°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Tup1 occludes Gcn5 recruitment. A) ChIP was performed using the cells shown expressing GALpro-GCN5-HA following a 5 hour galactose induction, as described above. (B) Two independent experiments were scanned and processed using ImageJ, with the means and standard errors shown. (C) Strains lacking TUP1 were constructed in WT and apc5CA backgrounds. Growth phenotypes were assessed by spot-dilutions, followed by incubation at 34°C and 37°C.
Mentions: We next tested whether the impact of Hda1 on Gcn5 promoter accessibility involved the corepressor complex Ssn6/Tup1. Several reports have demonstrated that the Ssn6/Tup1 corepressor utilizes Hda1 to repress transcription of target genes [36,41,68]. Furthermore, Tup1 has been shown to recruit Gcn5 to repressed promoters [73-75]. It was proposed that this may set the stage for derepression of silent genes. Thus, GALproGCN5-HA was induced in hda1Δ and tup1Δ cells as the only source of Gcn5, followed by ChIP. Gcn5-HA expression in hda1Δ cells was reduced compared to WT, but expression in tup1Δ cells was unchanged (data not shown). We found that in otherwise WT strains (gcn5Δ + GALproGCN5-HA), Gcn5-HA was weakly recruited to the tested promoters (Figures 6A and 6B). In strains lacking HDA1 or TUP1, Gcn5-HA promoter occupancy was observed to increase. We also observed that in cells lacking SSN6, promoter recruitment of Gcn5-HA increased (data not shown). These results suggest that Hda1 may work together with the Ssn6/Tup1 corepressor complex to impede access of Gcn5 to the tested promoters. However, Hda1 and Gcn5 may also compete for Tup1 interactions. It is also feasible that Tup1 utilizes different mechanisms to reduce Gcn5 promoter occupancy.

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