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N-terminal domain of nuclear IL-1α shows structural similarity to the C-terminal domain of Snf1 and binds to the HAT/core module of the SAGA complex.

Zamostna B, Novak J, Vopalensky V, Masek T, Burysek L, Pospisek M - PLoS ONE (2012)

Bottom Line: Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes.We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively.Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Prague, Czech Republic.

ABSTRACT
Interleukin-1α (IL-1α) is a proinflammatory cytokine and a key player in host immune responses in higher eukaryotes. IL-1α has pleiotropic effects on a wide range of cell types, and it has been extensively studied for its ability to contribute to various autoimmune and inflammation-linked disorders, including rheumatoid arthritis, Alzheimer's disease, systemic sclerosis and cardiovascular disorders. Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes. Despite the importance of IL-1α, little is known regarding its binding targets and functions in the nucleus. We took advantage of the histone acetyltransferase (HAT) complexes being evolutionarily conserved from yeast to humans and the yeast SAGA complex serving as an epitome of the eukaryotic HAT complexes. Using gene knock-out technique and co-immunoprecipitation of the IL-1α precursor with TAP-tagged subunits of the yeast HAT complexes, we mapped the IL-1α-binding site to the HAT/Core module of the SAGA complex. We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively. This finding is further supported with the ability of the IL-1α precursor to partially rescue growth defects of snf1Δ yeast strains on media containing 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of His3. Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly.

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Related in: MedlinePlus

A model suggesting a mutually exclusive role for Ahc1 and Spt7 in SAGA complex assembly.Co-IP experiments showed that pre-IL-1α binds to the HAT/Core of both the ADA and SAGA complexes. In the TAP/Spt7,ahc1Δ strain, only rarely weak co-precipitation of Spt7-TAP and pre-IL-1α was observed. Ahc1 thus may operate as an exchange factor that facilitates Spt7 binding to the ADA HAT, bringing various non-canonical co-activators and accessory proteins (e.g., IL-1α) and providing the resulting complex with DNA-binding abilities that give rise to a fully functional SAGA complex. Therefore, at least from the point of IL-1α function, ADA might not represent a real HAT complex but rather an intermediate protein complex that is however necessary for the assembly and proper function of the SAGA HAT complex.
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pone-0041801-g007: A model suggesting a mutually exclusive role for Ahc1 and Spt7 in SAGA complex assembly.Co-IP experiments showed that pre-IL-1α binds to the HAT/Core of both the ADA and SAGA complexes. In the TAP/Spt7,ahc1Δ strain, only rarely weak co-precipitation of Spt7-TAP and pre-IL-1α was observed. Ahc1 thus may operate as an exchange factor that facilitates Spt7 binding to the ADA HAT, bringing various non-canonical co-activators and accessory proteins (e.g., IL-1α) and providing the resulting complex with DNA-binding abilities that give rise to a fully functional SAGA complex. Therefore, at least from the point of IL-1α function, ADA might not represent a real HAT complex but rather an intermediate protein complex that is however necessary for the assembly and proper function of the SAGA HAT complex.

Mentions: Analysis of selected TAP-tagged yeast strains carrying deletion of the SPT7 and AHC1 genes, which are believed to maintain the integrity of the SAGA and ADA complex, respectively [7], [26], revealed that although the loss of Spt7 from the cell leads to the disintegration of the SAGA but not the ADA complex, as expected, the loss of Ahc1 from the cell did not allow for the efficient co-immunoprecipitation of the IL-1α precursor with Spt7-TAP. However, an ahc1 mutation did not affect the co-immunoprecipitation of the IL-1α precursor and Gcn5, a constituent of the HAT/Core, and Spt8, an exclusive member of the SAGA complex (Figure 6). To exclude that the results are influenced by disturbances either in the Spt7 protein levels in the ahc1Δ strain or in the experimental procedure, we repeated the experiment several times and confirmed by western blotting that ahc1 deletion does not significantly reduce intracellular Spt7 protein levels and also immunoprecipitation of the IL-1α precursor from the corresponding yeast cell lysates worked well (Figure S2). These results are unexpected for several reasons. First, Spt7 is the core SAGA subunit that is present even in the absence of Spt3, Spt8, Spt20, Gcn5 and Ada1 [64]. In the absence of Spt7, the SAGA HAT complex is disrupted, and this disruption severely affects transcriptional activation [7], [65]. Spt7 also regulates the levels of certain SAGA subunits and plays a central role in the SAGA complex formation [64]. Thus, the reason why Spt7 should be absent in a protein complex co-immunoprecipitated with pre-IL-1α in the ahc1 deletion strain is not obvious unless the ADA complex is the complex responsible. Furthermore, Spt8 is believed to be closely associated with Spt7, and its interaction with SAGA has been reported to be dependent on Spt7 [10], [64]. These results may be explained by a new model of Ahc1 function in yeast cells in which Ahc1 contributes to the association of Spt7 with SAGA. We speculate that Ahc1 functions as an exchange factor that is not exclusively required for but facilitates the association of Spt7 and perhaps other factors with the ADA HAT complex, resulting in a fully functional SAGA complex that is capable of interacting with various general and non-canonical transcriptional co-activators and accessory proteins, such as pre-IL-1α and AMP-activated protein kinase. Therefore, ADA would, at least for some of the cellular regulatory loops, not represent a HAT complex but rather an intermediate and/or reserve protein complex that is associated with non-canonical co-activators and other accessory proteins that may be necessary for the proper assembly of the SAGA complex with all of its co-activators. It should be noted that the purification of the SAGA complex has also been reported from the ahc1Δ strain [26]. However, the proposed model does not exclude ADA-independent SAGA assembly but rather suggests that some co-activators or accessory proteins may be brought to the SAGA complex only via the ADA complex, which may also have a regulatory function in the control of gene expression (Figure 7). This model is supported by recent data presented by Lee and co-workers, who dissected SAGA and ADA complexes using systematic gene knock-out and TAP-mediated protein complex purification approaches. They demonstrated that using Ada2-TAP, they could, as one would expect, purify most of the SAGA or ADA complex subunits in different knock-out strains with the exception of TAP-Ada2/spt20Δ and TAP-Ada2/ahc2Δ. In the first case, only the ADA complex was present, and in the latter case, only the HAT/Core module could be precipitated using a TAP tag. Lee and co-workers suggested that Ahc2 is responsible for tethering Ahc1 into the Ada complex [10]. Their TAP-Ada2/ahc2Δ result would be in agreement with the results of our experiments in which Spt7 did not efficiently co-purify with pre-IL-1α in the ahc1Δ strain. However, in our hands, pre-IL-1α readily co-immunoprecipitated with Gcn5-TAP, Spt8-TAP and Spt7-TAP in the corresponding ahc2Δ strains. There can be more explanations of this result, including that we tested different TAP-tagged proteins, used yeast with a different genetic background and namely immunoprecipitated the HAT complexes via pre-IL-1α, which may also have some, yet unknown function in the HAT complex assembly. Should be stressed, that just few our and Washburn’s group experiments, together concerning the Ahc2 function, provided the first results about this only recently annotated protein and more work focused on elucidation of Ahc2 role in the ADA complex assembly and activity will have to be done. To the best of our knowledge, the enzymatic activities and substrate specificity of the ADA complex have only been determined in vitro. Moreover, both Lee and co-workers [10] and Eberharter and co-workers [26] obtained different results, which may be explained by different methods of ADA complex purification. On the other hand, we and both of these groups demonstrated that the knock-out of the AHC1 gene, which is specific for the ADA complex, cannot rescue the Gal4BD-VP16- or Gal4BD-IL-1αNTP-mediated toxicity [10], [26], [40], suggesting a low direct transactivation potential of the ADA complex.


N-terminal domain of nuclear IL-1α shows structural similarity to the C-terminal domain of Snf1 and binds to the HAT/core module of the SAGA complex.

Zamostna B, Novak J, Vopalensky V, Masek T, Burysek L, Pospisek M - PLoS ONE (2012)

A model suggesting a mutually exclusive role for Ahc1 and Spt7 in SAGA complex assembly.Co-IP experiments showed that pre-IL-1α binds to the HAT/Core of both the ADA and SAGA complexes. In the TAP/Spt7,ahc1Δ strain, only rarely weak co-precipitation of Spt7-TAP and pre-IL-1α was observed. Ahc1 thus may operate as an exchange factor that facilitates Spt7 binding to the ADA HAT, bringing various non-canonical co-activators and accessory proteins (e.g., IL-1α) and providing the resulting complex with DNA-binding abilities that give rise to a fully functional SAGA complex. Therefore, at least from the point of IL-1α function, ADA might not represent a real HAT complex but rather an intermediate protein complex that is however necessary for the assembly and proper function of the SAGA HAT complex.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412866&req=5

pone-0041801-g007: A model suggesting a mutually exclusive role for Ahc1 and Spt7 in SAGA complex assembly.Co-IP experiments showed that pre-IL-1α binds to the HAT/Core of both the ADA and SAGA complexes. In the TAP/Spt7,ahc1Δ strain, only rarely weak co-precipitation of Spt7-TAP and pre-IL-1α was observed. Ahc1 thus may operate as an exchange factor that facilitates Spt7 binding to the ADA HAT, bringing various non-canonical co-activators and accessory proteins (e.g., IL-1α) and providing the resulting complex with DNA-binding abilities that give rise to a fully functional SAGA complex. Therefore, at least from the point of IL-1α function, ADA might not represent a real HAT complex but rather an intermediate protein complex that is however necessary for the assembly and proper function of the SAGA HAT complex.
Mentions: Analysis of selected TAP-tagged yeast strains carrying deletion of the SPT7 and AHC1 genes, which are believed to maintain the integrity of the SAGA and ADA complex, respectively [7], [26], revealed that although the loss of Spt7 from the cell leads to the disintegration of the SAGA but not the ADA complex, as expected, the loss of Ahc1 from the cell did not allow for the efficient co-immunoprecipitation of the IL-1α precursor with Spt7-TAP. However, an ahc1 mutation did not affect the co-immunoprecipitation of the IL-1α precursor and Gcn5, a constituent of the HAT/Core, and Spt8, an exclusive member of the SAGA complex (Figure 6). To exclude that the results are influenced by disturbances either in the Spt7 protein levels in the ahc1Δ strain or in the experimental procedure, we repeated the experiment several times and confirmed by western blotting that ahc1 deletion does not significantly reduce intracellular Spt7 protein levels and also immunoprecipitation of the IL-1α precursor from the corresponding yeast cell lysates worked well (Figure S2). These results are unexpected for several reasons. First, Spt7 is the core SAGA subunit that is present even in the absence of Spt3, Spt8, Spt20, Gcn5 and Ada1 [64]. In the absence of Spt7, the SAGA HAT complex is disrupted, and this disruption severely affects transcriptional activation [7], [65]. Spt7 also regulates the levels of certain SAGA subunits and plays a central role in the SAGA complex formation [64]. Thus, the reason why Spt7 should be absent in a protein complex co-immunoprecipitated with pre-IL-1α in the ahc1 deletion strain is not obvious unless the ADA complex is the complex responsible. Furthermore, Spt8 is believed to be closely associated with Spt7, and its interaction with SAGA has been reported to be dependent on Spt7 [10], [64]. These results may be explained by a new model of Ahc1 function in yeast cells in which Ahc1 contributes to the association of Spt7 with SAGA. We speculate that Ahc1 functions as an exchange factor that is not exclusively required for but facilitates the association of Spt7 and perhaps other factors with the ADA HAT complex, resulting in a fully functional SAGA complex that is capable of interacting with various general and non-canonical transcriptional co-activators and accessory proteins, such as pre-IL-1α and AMP-activated protein kinase. Therefore, ADA would, at least for some of the cellular regulatory loops, not represent a HAT complex but rather an intermediate and/or reserve protein complex that is associated with non-canonical co-activators and other accessory proteins that may be necessary for the proper assembly of the SAGA complex with all of its co-activators. It should be noted that the purification of the SAGA complex has also been reported from the ahc1Δ strain [26]. However, the proposed model does not exclude ADA-independent SAGA assembly but rather suggests that some co-activators or accessory proteins may be brought to the SAGA complex only via the ADA complex, which may also have a regulatory function in the control of gene expression (Figure 7). This model is supported by recent data presented by Lee and co-workers, who dissected SAGA and ADA complexes using systematic gene knock-out and TAP-mediated protein complex purification approaches. They demonstrated that using Ada2-TAP, they could, as one would expect, purify most of the SAGA or ADA complex subunits in different knock-out strains with the exception of TAP-Ada2/spt20Δ and TAP-Ada2/ahc2Δ. In the first case, only the ADA complex was present, and in the latter case, only the HAT/Core module could be precipitated using a TAP tag. Lee and co-workers suggested that Ahc2 is responsible for tethering Ahc1 into the Ada complex [10]. Their TAP-Ada2/ahc2Δ result would be in agreement with the results of our experiments in which Spt7 did not efficiently co-purify with pre-IL-1α in the ahc1Δ strain. However, in our hands, pre-IL-1α readily co-immunoprecipitated with Gcn5-TAP, Spt8-TAP and Spt7-TAP in the corresponding ahc2Δ strains. There can be more explanations of this result, including that we tested different TAP-tagged proteins, used yeast with a different genetic background and namely immunoprecipitated the HAT complexes via pre-IL-1α, which may also have some, yet unknown function in the HAT complex assembly. Should be stressed, that just few our and Washburn’s group experiments, together concerning the Ahc2 function, provided the first results about this only recently annotated protein and more work focused on elucidation of Ahc2 role in the ADA complex assembly and activity will have to be done. To the best of our knowledge, the enzymatic activities and substrate specificity of the ADA complex have only been determined in vitro. Moreover, both Lee and co-workers [10] and Eberharter and co-workers [26] obtained different results, which may be explained by different methods of ADA complex purification. On the other hand, we and both of these groups demonstrated that the knock-out of the AHC1 gene, which is specific for the ADA complex, cannot rescue the Gal4BD-VP16- or Gal4BD-IL-1αNTP-mediated toxicity [10], [26], [40], suggesting a low direct transactivation potential of the ADA complex.

Bottom Line: Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes.We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively.Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Prague, Czech Republic.

ABSTRACT
Interleukin-1α (IL-1α) is a proinflammatory cytokine and a key player in host immune responses in higher eukaryotes. IL-1α has pleiotropic effects on a wide range of cell types, and it has been extensively studied for its ability to contribute to various autoimmune and inflammation-linked disorders, including rheumatoid arthritis, Alzheimer's disease, systemic sclerosis and cardiovascular disorders. Interestingly, a significant proportion of IL-1α is translocated to the cell nucleus, in which it interacts with histone acetyltransferase complexes. Despite the importance of IL-1α, little is known regarding its binding targets and functions in the nucleus. We took advantage of the histone acetyltransferase (HAT) complexes being evolutionarily conserved from yeast to humans and the yeast SAGA complex serving as an epitome of the eukaryotic HAT complexes. Using gene knock-out technique and co-immunoprecipitation of the IL-1α precursor with TAP-tagged subunits of the yeast HAT complexes, we mapped the IL-1α-binding site to the HAT/Core module of the SAGA complex. We also predicted the 3-D structure of the IL-1α N-terminal domain, and by employing structure similarity searches, we found a similar structure in the C-terminal regulatory region of the catalytic subunit of the AMP-activated/Snf1 protein kinases, which interact with HAT complexes both in mammals and yeast, respectively. This finding is further supported with the ability of the IL-1α precursor to partially rescue growth defects of snf1Δ yeast strains on media containing 3-Amino-1,2,4-triazole (3-AT), a competitive inhibitor of His3. Finally, the careful evaluation of our data together with other published data in the field allows us to hypothesize a new function for the ADA complex in SAGA complex assembly.

Show MeSH
Related in: MedlinePlus