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AtEAF1 is a potential platform protein for Arabidopsis NuA4 acetyltransferase complex.

Bieluszewski T, Galganski L, Sura W, Bieluszewska A, Abram M, Ludwikow A, Ziolkowski PA, Sadowski J - BMC Plant Biol. (2015)

Bottom Line: Plants carrying a T-DNA insertion in one of the genes encoding AtEAF1 showed decreased FLC expression and early flowering, similarly to Atyaf9 mutants.Chromatin immunoprecipitation analyses of the single mutant Ateaf1b-2 and artificial miRNA knock-down Ateaf1 lines showed decreased levels of H4K5 acetylation in the promoter regions of major flowering regulator genes, further supporting the role of AtEAF1 as a subunit of the plant NuA4 complex.Growing evidence suggests that the molecular functions of the NuA4 and SWR1 complexes are conserved in plants and contribute significantly to plant development and physiology.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Histone acetyltransferase complex NuA4 and histone variant exchanging complex SWR1 are two chromatin modifying complexes which act cooperatively in yeast and share some intriguing structural similarities. Protein subunits of NuA4 and SWR1-C are highly conserved across eukaryotes, but form different multiprotein arrangements. For example, the human TIP60-p400 complex consists of homologues of both yeast NuA4 and SWR1-C subunits, combining subunits necessary for histone acetylation and histone variant exchange. It is currently not known what protein complexes are formed by the plant homologues of NuA4 and SWR1-C subunits.

Results: We report on the identification and molecular characterization of AtEAF1, a new subunit of Arabidopsis NuA4 complex which shows many similarities to the platform protein of the yeast NuA4 complex. AtEAF1 copurifies with Arabidopsis homologues of NuA4 and SWR1-C subunits ARP4 and SWC4 and interacts physically with AtYAF9A and AtYAF9B, homologues of the YAF9 subunit. Plants carrying a T-DNA insertion in one of the genes encoding AtEAF1 showed decreased FLC expression and early flowering, similarly to Atyaf9 mutants. Chromatin immunoprecipitation analyses of the single mutant Ateaf1b-2 and artificial miRNA knock-down Ateaf1 lines showed decreased levels of H4K5 acetylation in the promoter regions of major flowering regulator genes, further supporting the role of AtEAF1 as a subunit of the plant NuA4 complex.

Conclusions: Growing evidence suggests that the molecular functions of the NuA4 and SWR1 complexes are conserved in plants and contribute significantly to plant development and physiology. Our work provides evidence for the existence of a yeast-like EAF1 platform protein in A. thaliana, filling an important gap in the knowledge about the subunit organization of the plant NuA4 complex.

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Ateaf1b-2andAtyaf9a-1 Atyaf9b-2mutants gain increased resistance to TSA. (a) 12 day-old seedlings grown in the presence of TSA or mock. All images are in the same scale. (b) Comparison of average fresh weight between plants treated with TSA or mock. Error bars represent standard deviation of 4 biological replicates. Double asterisks indicate a p-value < 0.01 (t-test).
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Fig4: Ateaf1b-2andAtyaf9a-1 Atyaf9b-2mutants gain increased resistance to TSA. (a) 12 day-old seedlings grown in the presence of TSA or mock. All images are in the same scale. (b) Comparison of average fresh weight between plants treated with TSA or mock. Error bars represent standard deviation of 4 biological replicates. Double asterisks indicate a p-value < 0.01 (t-test).

Mentions: Published experimental data support involvement of Arabidopsis homologues of NuA4 subunits YAF9 and ESA1 (HAM1 and HAM2) in the acetylation of lysine 5 of histone H4 [13]. Therefore, if AtEAF1 is a functional subunit of the Arabidopsis NuA4 complex, partial loss of its function should lead to changes in H4K5 acetylation levels. As an initial test of the influence of AtEAF1 on H4K5 acetylation, we grew Ateaf1b-2 and Atyaf9a-1 Atyaf9b-2 seedlings for 12 days on MS medium supplemented with 1% sucrose or 1% sucrose and 12.5 μM Trichostatin A (TSA) (Figure 4). TSA is a specific inhibitor of histone deacetylases and has a strong negative effect on plant growth, coinciding with dramatic accumulation of acetylated histones [23,24]. We reasoned that impaired function of an important histone acetylatransferase such as NuA4 could prevent abnormal accumulation of acetylated histones and give mutant plants an advantage over WT plants under TSA challenge. As expected, average fresh weight of Atyaf9a-1 Atyaf9b-2 and Ateaf1b-2 mutant plants grown on plates containing TSA was significantly larger than those of WT plants grown in the same conditions, whereas no significant differences where observed under control conditions (Figure 4b). In order to verify if the observed effect can be attributed to differences in global H4K5 acetylation levels, we tested the abundance of histone H4 acetylated on lysine 5 by Western Blot (Additional file 7). Only the double mutant Atyaf9a-1 Atyaf9b-2 displayed decreased levels of acetylated H4K5 relative to WT which indicates that the increased resistance of Ateaf1b-2 plants to TSA cannot be due to a global loss of H4K5 acetylation. This observation could be explained if AtEAF1 had a specialized function in the Arabidopsis NuA4 complex. In fact, in yeast eaf1 mutant a strong decrease in histone H4 acetylation was observed in the promoter region of the PHO5 gene, but no decrease in bulk histone H4 acetylation was reported [5]. Therefore we decided to test if specific genomic targets of histone acetyltraferases are affected in plants with impaired function of AtEAF1. We focused on major regulators of flowering transition FLC, FT, CONSTANS (CO) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) as histone acetylation in these genes was found to be deregulated by various H4 acetylation mutants in previous studies [15-17]. Our observations of flowering timing in Ateaf1b-2 mutant, presented above, further justified that choice.Figure 4


AtEAF1 is a potential platform protein for Arabidopsis NuA4 acetyltransferase complex.

Bieluszewski T, Galganski L, Sura W, Bieluszewska A, Abram M, Ludwikow A, Ziolkowski PA, Sadowski J - BMC Plant Biol. (2015)

Ateaf1b-2andAtyaf9a-1 Atyaf9b-2mutants gain increased resistance to TSA. (a) 12 day-old seedlings grown in the presence of TSA or mock. All images are in the same scale. (b) Comparison of average fresh weight between plants treated with TSA or mock. Error bars represent standard deviation of 4 biological replicates. Double asterisks indicate a p-value < 0.01 (t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4358907&req=5

Fig4: Ateaf1b-2andAtyaf9a-1 Atyaf9b-2mutants gain increased resistance to TSA. (a) 12 day-old seedlings grown in the presence of TSA or mock. All images are in the same scale. (b) Comparison of average fresh weight between plants treated with TSA or mock. Error bars represent standard deviation of 4 biological replicates. Double asterisks indicate a p-value < 0.01 (t-test).
Mentions: Published experimental data support involvement of Arabidopsis homologues of NuA4 subunits YAF9 and ESA1 (HAM1 and HAM2) in the acetylation of lysine 5 of histone H4 [13]. Therefore, if AtEAF1 is a functional subunit of the Arabidopsis NuA4 complex, partial loss of its function should lead to changes in H4K5 acetylation levels. As an initial test of the influence of AtEAF1 on H4K5 acetylation, we grew Ateaf1b-2 and Atyaf9a-1 Atyaf9b-2 seedlings for 12 days on MS medium supplemented with 1% sucrose or 1% sucrose and 12.5 μM Trichostatin A (TSA) (Figure 4). TSA is a specific inhibitor of histone deacetylases and has a strong negative effect on plant growth, coinciding with dramatic accumulation of acetylated histones [23,24]. We reasoned that impaired function of an important histone acetylatransferase such as NuA4 could prevent abnormal accumulation of acetylated histones and give mutant plants an advantage over WT plants under TSA challenge. As expected, average fresh weight of Atyaf9a-1 Atyaf9b-2 and Ateaf1b-2 mutant plants grown on plates containing TSA was significantly larger than those of WT plants grown in the same conditions, whereas no significant differences where observed under control conditions (Figure 4b). In order to verify if the observed effect can be attributed to differences in global H4K5 acetylation levels, we tested the abundance of histone H4 acetylated on lysine 5 by Western Blot (Additional file 7). Only the double mutant Atyaf9a-1 Atyaf9b-2 displayed decreased levels of acetylated H4K5 relative to WT which indicates that the increased resistance of Ateaf1b-2 plants to TSA cannot be due to a global loss of H4K5 acetylation. This observation could be explained if AtEAF1 had a specialized function in the Arabidopsis NuA4 complex. In fact, in yeast eaf1 mutant a strong decrease in histone H4 acetylation was observed in the promoter region of the PHO5 gene, but no decrease in bulk histone H4 acetylation was reported [5]. Therefore we decided to test if specific genomic targets of histone acetyltraferases are affected in plants with impaired function of AtEAF1. We focused on major regulators of flowering transition FLC, FT, CONSTANS (CO) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) as histone acetylation in these genes was found to be deregulated by various H4 acetylation mutants in previous studies [15-17]. Our observations of flowering timing in Ateaf1b-2 mutant, presented above, further justified that choice.Figure 4

Bottom Line: Plants carrying a T-DNA insertion in one of the genes encoding AtEAF1 showed decreased FLC expression and early flowering, similarly to Atyaf9 mutants.Chromatin immunoprecipitation analyses of the single mutant Ateaf1b-2 and artificial miRNA knock-down Ateaf1 lines showed decreased levels of H4K5 acetylation in the promoter regions of major flowering regulator genes, further supporting the role of AtEAF1 as a subunit of the plant NuA4 complex.Growing evidence suggests that the molecular functions of the NuA4 and SWR1 complexes are conserved in plants and contribute significantly to plant development and physiology.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Histone acetyltransferase complex NuA4 and histone variant exchanging complex SWR1 are two chromatin modifying complexes which act cooperatively in yeast and share some intriguing structural similarities. Protein subunits of NuA4 and SWR1-C are highly conserved across eukaryotes, but form different multiprotein arrangements. For example, the human TIP60-p400 complex consists of homologues of both yeast NuA4 and SWR1-C subunits, combining subunits necessary for histone acetylation and histone variant exchange. It is currently not known what protein complexes are formed by the plant homologues of NuA4 and SWR1-C subunits.

Results: We report on the identification and molecular characterization of AtEAF1, a new subunit of Arabidopsis NuA4 complex which shows many similarities to the platform protein of the yeast NuA4 complex. AtEAF1 copurifies with Arabidopsis homologues of NuA4 and SWR1-C subunits ARP4 and SWC4 and interacts physically with AtYAF9A and AtYAF9B, homologues of the YAF9 subunit. Plants carrying a T-DNA insertion in one of the genes encoding AtEAF1 showed decreased FLC expression and early flowering, similarly to Atyaf9 mutants. Chromatin immunoprecipitation analyses of the single mutant Ateaf1b-2 and artificial miRNA knock-down Ateaf1 lines showed decreased levels of H4K5 acetylation in the promoter regions of major flowering regulator genes, further supporting the role of AtEAF1 as a subunit of the plant NuA4 complex.

Conclusions: Growing evidence suggests that the molecular functions of the NuA4 and SWR1 complexes are conserved in plants and contribute significantly to plant development and physiology. Our work provides evidence for the existence of a yeast-like EAF1 platform protein in A. thaliana, filling an important gap in the knowledge about the subunit organization of the plant NuA4 complex.

Show MeSH