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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-2, Atyaf9a-1 Atyaf9b-2andAtEAF1-amiRNA lines display reduced H4K5 acetylation but different activity ofFLCandFT.(a, b) Acetylation levels in different parts of the FLC and FT genes normalized to H3 presented as fold change over WT Col-0. (c) Expression levels of FLC and FT relative to WT Col-0. Asterisks in (a, b, c) indicate a p-value < 0.05 or p-value < 0.01 (double asterisk) (t-test).
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Fig6: Ateaf1b-2, Atyaf9a-1 Atyaf9b-2andAtEAF1-amiRNA lines display reduced H4K5 acetylation but different activity ofFLCandFT.(a, b) Acetylation levels in different parts of the FLC and FT genes normalized to H3 presented as fold change over WT Col-0. (c) Expression levels of FLC and FT relative to WT Col-0. Asterisks in (a, b, c) indicate a p-value < 0.05 or p-value < 0.01 (double asterisk) (t-test).

Mentions: Our next step was to characterize differences in histone H4K5 acetylation levels over FLC and FT genes between WT, Ateaf1b-2, 2.29, 2.39 and Atyaf9a-1 Atyaf9b-2 plants by chromatin immunoprecipitation (ChIP). We carried out the experiments on 10-day old seedlings grown under long day conditions, collected at the end of the day. For the amplification of the DNA fragments obtained from ChIP we used five pairs of PCR primers for each gene, corresponding to various functional elements of the gene (Figure 6a, b, Additional file 2). We observed a moderate but consistent decrease in H4K5 acetylation over both genes. Interestingly, we observed a stronger reduction in acetylation levels near the 5′ end of the genes, especially in the FLC locus. Following this observation, we tested the acetylation levels in the promoter regions of two other major flowering regulators, CO and SOC1. We observed a significant and consistent drop of H4K5 acetylation in the promoter of CO but little change in SOC1, except for the Atyaf9a-1 Atyaf9b-2 line, which showed significantly lower acetylation at both loci (Figure 7a).Figure 6


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-2, Atyaf9a-1 Atyaf9b-2andAtEAF1-amiRNA lines display reduced H4K5 acetylation but different activity ofFLCandFT.(a, b) Acetylation levels in different parts of the FLC and FT genes normalized to H3 presented as fold change over WT Col-0. (c) Expression levels of FLC and FT relative to WT Col-0. Asterisks in (a, b, c) indicate a p-value < 0.05 or p-value < 0.01 (double asterisk) (t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Ateaf1b-2, Atyaf9a-1 Atyaf9b-2andAtEAF1-amiRNA lines display reduced H4K5 acetylation but different activity ofFLCandFT.(a, b) Acetylation levels in different parts of the FLC and FT genes normalized to H3 presented as fold change over WT Col-0. (c) Expression levels of FLC and FT relative to WT Col-0. Asterisks in (a, b, c) indicate a p-value < 0.05 or p-value < 0.01 (double asterisk) (t-test).
Mentions: Our next step was to characterize differences in histone H4K5 acetylation levels over FLC and FT genes between WT, Ateaf1b-2, 2.29, 2.39 and Atyaf9a-1 Atyaf9b-2 plants by chromatin immunoprecipitation (ChIP). We carried out the experiments on 10-day old seedlings grown under long day conditions, collected at the end of the day. For the amplification of the DNA fragments obtained from ChIP we used five pairs of PCR primers for each gene, corresponding to various functional elements of the gene (Figure 6a, b, Additional file 2). We observed a moderate but consistent decrease in H4K5 acetylation over both genes. Interestingly, we observed a stronger reduction in acetylation levels near the 5′ end of the genes, especially in the FLC locus. Following this observation, we tested the acetylation levels in the promoter regions of two other major flowering regulators, CO and SOC1. We observed a significant and consistent drop of H4K5 acetylation in the promoter of CO but little change in SOC1, except for the Atyaf9a-1 Atyaf9b-2 line, which showed significantly lower acetylation at both loci (Figure 7a).Figure 6

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