Limits...
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 can inhibit Hda1-Tup1 associations at some promoters. (A) Sequential ChIP was used to observe Hda1-Tup1 physical interactions at specific promoters. WT, hda1Δ and gcn5Δ hda1Δ cells expressing combinations of GALpro-HDA1-HA and CUP1pro-TUP1-GST were induced using 4% galactose for 5 hours and 0.4 mM CuSO4 for 3 hours. ChIP reactions were first performed with antibodies against HA. Bound proteins were eluted from beads using 10 mM DTT for 30 minutes at 37°C. The eluted proteins were then incubated with anti-GST antibodies. The immune complexes were isolated again, cross links were reversed, and "end point" PCR was performed using the recovered DNA as template. (B) Two independent experiments were performed and processed using ImageJ. The means and standard errors are shown. (C) Westerns showing expression of the proteins used in the sequential ChIP experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3141613&req=5

Figure 7: Gcn5 can inhibit Hda1-Tup1 associations at some promoters. (A) Sequential ChIP was used to observe Hda1-Tup1 physical interactions at specific promoters. WT, hda1Δ and gcn5Δ hda1Δ cells expressing combinations of GALpro-HDA1-HA and CUP1pro-TUP1-GST were induced using 4% galactose for 5 hours and 0.4 mM CuSO4 for 3 hours. ChIP reactions were first performed with antibodies against HA. Bound proteins were eluted from beads using 10 mM DTT for 30 minutes at 37°C. The eluted proteins were then incubated with anti-GST antibodies. The immune complexes were isolated again, cross links were reversed, and "end point" PCR was performed using the recovered DNA as template. (B) Two independent experiments were performed and processed using ImageJ. The means and standard errors are shown. (C) Westerns showing expression of the proteins used in the sequential ChIP experiment.

Mentions: Others have also shown Tup1 and Hda1 functionally interact to repress gene transcription [36,41,68], and to associate in vitro [36], but not necessarily in vivo [46]. To investigate whether Tup1 and Hda1 do function together, we asked if Tup1 and Hda1 can physically interact at promoters, and if Gcn5 can influence this. To do so we performed sequential ChIP in cells expressing a combination of Hda1-HA and/or GST-Tup1. ChIP was first performed using antibodies against HA. Bound proteins were released, recovered, and incubated with antibodies against GST. Bound protein/DNA complexes were again isolated and PCR was performed using primers against the test promoters. The results show that in cells expressing either Hda1-HA or GST-Tup1, no bound DNA was recovered (Figure 7A and 7B). However, in cells co-expressing the plasmids, PCR fragments were obtained for all promoters tested. This supports the idea that Tup1 and Hda1 can associate in vivo at specific promoters. Nonetheless, this could also reflect close, but independent Hda1 and Tup1 binding on the same promoter. In gcn5Δ cells co-expressing the plasmids, putative complex formation was again observed, and was visibly increased at CDC20, PDS1, and BCY1 promoters, suggesting Gcn5 may negatively impact this interaction. Figure 7C shows that the proteins were expressed similarly in the strains used. Our experiments do not differentiate between Hda1 and Tup1 physically binding, or whether they simply bind adjacent DNA sequences, but it is important to note Hda1 and Tup1 were previously shown to physically associate [36], and that the interaction observed by sequential ChIP is enhanced by GCN5 deletion.


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 can inhibit Hda1-Tup1 associations at some promoters. (A) Sequential ChIP was used to observe Hda1-Tup1 physical interactions at specific promoters. WT, hda1Δ and gcn5Δ hda1Δ cells expressing combinations of GALpro-HDA1-HA and CUP1pro-TUP1-GST were induced using 4% galactose for 5 hours and 0.4 mM CuSO4 for 3 hours. ChIP reactions were first performed with antibodies against HA. Bound proteins were eluted from beads using 10 mM DTT for 30 minutes at 37°C. The eluted proteins were then incubated with anti-GST antibodies. The immune complexes were isolated again, cross links were reversed, and "end point" PCR was performed using the recovered DNA as template. (B) Two independent experiments were performed and processed using ImageJ. The means and standard errors are shown. (C) Westerns showing expression of the proteins used in the sequential ChIP experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Gcn5 can inhibit Hda1-Tup1 associations at some promoters. (A) Sequential ChIP was used to observe Hda1-Tup1 physical interactions at specific promoters. WT, hda1Δ and gcn5Δ hda1Δ cells expressing combinations of GALpro-HDA1-HA and CUP1pro-TUP1-GST were induced using 4% galactose for 5 hours and 0.4 mM CuSO4 for 3 hours. ChIP reactions were first performed with antibodies against HA. Bound proteins were eluted from beads using 10 mM DTT for 30 minutes at 37°C. The eluted proteins were then incubated with anti-GST antibodies. The immune complexes were isolated again, cross links were reversed, and "end point" PCR was performed using the recovered DNA as template. (B) Two independent experiments were performed and processed using ImageJ. The means and standard errors are shown. (C) Westerns showing expression of the proteins used in the sequential ChIP experiment.
Mentions: Others have also shown Tup1 and Hda1 functionally interact to repress gene transcription [36,41,68], and to associate in vitro [36], but not necessarily in vivo [46]. To investigate whether Tup1 and Hda1 do function together, we asked if Tup1 and Hda1 can physically interact at promoters, and if Gcn5 can influence this. To do so we performed sequential ChIP in cells expressing a combination of Hda1-HA and/or GST-Tup1. ChIP was first performed using antibodies against HA. Bound proteins were released, recovered, and incubated with antibodies against GST. Bound protein/DNA complexes were again isolated and PCR was performed using primers against the test promoters. The results show that in cells expressing either Hda1-HA or GST-Tup1, no bound DNA was recovered (Figure 7A and 7B). However, in cells co-expressing the plasmids, PCR fragments were obtained for all promoters tested. This supports the idea that Tup1 and Hda1 can associate in vivo at specific promoters. Nonetheless, this could also reflect close, but independent Hda1 and Tup1 binding on the same promoter. In gcn5Δ cells co-expressing the plasmids, putative complex formation was again observed, and was visibly increased at CDC20, PDS1, and BCY1 promoters, suggesting Gcn5 may negatively impact this interaction. Figure 7C shows that the proteins were expressed similarly in the strains used. Our experiments do not differentiate between Hda1 and Tup1 physically binding, or whether they simply bind adjacent DNA sequences, but it is important to note Hda1 and Tup1 were previously shown to physically associate [36], and that the interaction observed by sequential ChIP is enhanced by GCN5 deletion.

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