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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

Gcn5 promoter occupancy is kept at an equilibrium in WT cells, but increases over time in hda1 cells. (A) Western analyses of Gcn5-HA expression in gcn5Δ and gcn5Δ hda1Δ cells following a 5 hour 4% galactose-induction. Antibodies against GAPDH were used as a load control. (B) A galactose-induction time-course was performed in gcn5Δ and gcn5Δ hda1Δ cells expressing GALpro-GCN5-HA. Protein samples were removed at the times shown for Western analyses with antibodies against HA and GAPDH. (C) From the time-course described above, samples were also removed for ChIP. Recovered DNA was used as a template in "end point" PCR reactions. S, sample with antibody; C, control without antibody; I, 10% lysate input. (D) The gel in (C) was scanned, analyzed using ImageJ and plotted.
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Figure 5: Gcn5 promoter occupancy is kept at an equilibrium in WT cells, but increases over time in hda1 cells. (A) Western analyses of Gcn5-HA expression in gcn5Δ and gcn5Δ hda1Δ cells following a 5 hour 4% galactose-induction. Antibodies against GAPDH were used as a load control. (B) A galactose-induction time-course was performed in gcn5Δ and gcn5Δ hda1Δ cells expressing GALpro-GCN5-HA. Protein samples were removed at the times shown for Western analyses with antibodies against HA and GAPDH. (C) From the time-course described above, samples were also removed for ChIP. Recovered DNA was used as a template in "end point" PCR reactions. S, sample with antibody; C, control without antibody; I, 10% lysate input. (D) The gel in (C) was scanned, analyzed using ImageJ and plotted.

Mentions: It is possible that increased Gcn5-HA recruitment is due to increased GALpro-driven Gcn5 expression in hda1Δ cells, since Hda1 represses galactose-induced gene activation [72]. We assessed expression of Gcn5-HA in the strains used above and observed that GALproGCN5-HA expression after a 5 hour induction period was reduced in hda1Δ cells (Figure 5A). Therefore, it is unlikely that the decreased levels of GAL-promoter driven GCN5 in hda1Δ cells are due to Hda1's influence on the GAL promoter. Considering that hda1Δ cells express less GCN5 than WT, yet recruit a greater amount of Gcn5 to promoters, a much greater proportion of Gcn5-HA must be available for recruitment in hda1Δ cells. To examine this possibility, we performed ChIP using lysates prepared from GALproGCN5-HA expressing cells after 1, 3 and 5 hours of induction (Figure 5B). Gcn5-HA was recruited to each tested promoter (Figures 5C and 5D). When normalized to input, Gcn5-HA recruitment in HDA1 cells was similar at each induction timepoint (Figure 5D). In hda1Δ cells however, Gcn5-HA recruitment was again increased, and recruitment increased the longer the induction. Together, our data suggests that in the absence of Hda1, Gcn5-HA continually gains access to the tested promoters.


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)

Gcn5 promoter occupancy is kept at an equilibrium in WT cells, but increases over time in hda1 cells. (A) Western analyses of Gcn5-HA expression in gcn5Δ and gcn5Δ hda1Δ cells following a 5 hour 4% galactose-induction. Antibodies against GAPDH were used as a load control. (B) A galactose-induction time-course was performed in gcn5Δ and gcn5Δ hda1Δ cells expressing GALpro-GCN5-HA. Protein samples were removed at the times shown for Western analyses with antibodies against HA and GAPDH. (C) From the time-course described above, samples were also removed for ChIP. Recovered DNA was used as a template in "end point" PCR reactions. S, sample with antibody; C, control without antibody; I, 10% lysate input. (D) The gel in (C) was scanned, analyzed using ImageJ and plotted.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Gcn5 promoter occupancy is kept at an equilibrium in WT cells, but increases over time in hda1 cells. (A) Western analyses of Gcn5-HA expression in gcn5Δ and gcn5Δ hda1Δ cells following a 5 hour 4% galactose-induction. Antibodies against GAPDH were used as a load control. (B) A galactose-induction time-course was performed in gcn5Δ and gcn5Δ hda1Δ cells expressing GALpro-GCN5-HA. Protein samples were removed at the times shown for Western analyses with antibodies against HA and GAPDH. (C) From the time-course described above, samples were also removed for ChIP. Recovered DNA was used as a template in "end point" PCR reactions. S, sample with antibody; C, control without antibody; I, 10% lysate input. (D) The gel in (C) was scanned, analyzed using ImageJ and plotted.
Mentions: It is possible that increased Gcn5-HA recruitment is due to increased GALpro-driven Gcn5 expression in hda1Δ cells, since Hda1 represses galactose-induced gene activation [72]. We assessed expression of Gcn5-HA in the strains used above and observed that GALproGCN5-HA expression after a 5 hour induction period was reduced in hda1Δ cells (Figure 5A). Therefore, it is unlikely that the decreased levels of GAL-promoter driven GCN5 in hda1Δ cells are due to Hda1's influence on the GAL promoter. Considering that hda1Δ cells express less GCN5 than WT, yet recruit a greater amount of Gcn5 to promoters, a much greater proportion of Gcn5-HA must be available for recruitment in hda1Δ cells. To examine this possibility, we performed ChIP using lysates prepared from GALproGCN5-HA expressing cells after 1, 3 and 5 hours of induction (Figure 5B). Gcn5-HA was recruited to each tested promoter (Figures 5C and 5D). When normalized to input, Gcn5-HA recruitment in HDA1 cells was similar at each induction timepoint (Figure 5D). In hda1Δ cells however, Gcn5-HA recruitment was again increased, and recruitment increased the longer the induction. Together, our data suggests that in the absence of Hda1, Gcn5-HA continually gains access to the tested promoters.

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