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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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AtYAF9A and AtYAF9B interact with AtSWC4 and the HSA-containing fragment of AtEAF1. (a) Coimmunoprecipitation test of interaction between AtYAF9A and AtYAF9B with the HSA-containing fragments of PIE1, AtEAF1 and AtINO80. Yaf9:nEYFP fusion proteins were detected with antibodies against GFP. Bands above the 55 kDa bars are nonspecific signals from immunoglobulin heavy chains. For additional experimental controls, see Additional file 5. (b) BiFC assay in Arabidopsis mesophyll protoplasts. Each co-transfection consisted of a pair of complementary BiFC constructs (nEYFP and cEYFP fusion, right panels, yellow dots indicate fluorescence complementation in the nuclei) and an ECFP construct as an internal transfection control (left panels, cyan). AtYAF9Bsv is the short splice variant of AtYAF9B, lacking the putative C-terminal SWC4-binding domain. (c) Enlarged images of single protoplasts showing nuclear localization of the EYFP fluorescence, indicating interaction between AtSWC4 and AtYAF9A (upper image) or AtYAF9B (lower image). Chloroplast autofluorescence is shown in red and ECFP fluorescence in cyan. Scale bar: 10 μm. Contrast and brightness were enhanced in all micrographs in (b) and (c) to improve clarity.
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Fig2: AtYAF9A and AtYAF9B interact with AtSWC4 and the HSA-containing fragment of AtEAF1. (a) Coimmunoprecipitation test of interaction between AtYAF9A and AtYAF9B with the HSA-containing fragments of PIE1, AtEAF1 and AtINO80. Yaf9:nEYFP fusion proteins were detected with antibodies against GFP. Bands above the 55 kDa bars are nonspecific signals from immunoglobulin heavy chains. For additional experimental controls, see Additional file 5. (b) BiFC assay in Arabidopsis mesophyll protoplasts. Each co-transfection consisted of a pair of complementary BiFC constructs (nEYFP and cEYFP fusion, right panels, yellow dots indicate fluorescence complementation in the nuclei) and an ECFP construct as an internal transfection control (left panels, cyan). AtYAF9Bsv is the short splice variant of AtYAF9B, lacking the putative C-terminal SWC4-binding domain. (c) Enlarged images of single protoplasts showing nuclear localization of the EYFP fluorescence, indicating interaction between AtSWC4 and AtYAF9A (upper image) or AtYAF9B (lower image). Chloroplast autofluorescence is shown in red and ECFP fluorescence in cyan. Scale bar: 10 μm. Contrast and brightness were enhanced in all micrographs in (b) and (c) to improve clarity.

Mentions: As shown above, we found evidence for physical association of AtARP4 and AtSWC4 with AtEAF1, a domain relative of yeast NuA4 subunit EAF1. We assumed that AtARP4 and AtSWC4 associated with AtEAF1 through interaction with the latter’s HSA domain. Cooperative binding of ARP4 and SWC4 to the N-terminal region of SWR1, containing the HSA domain, requires YAF9 in yeast [7]. One of the two Arabidopsis homologues of YAF9 was shown recently to be required for histone H4 acetylation in the FLC locus, in line with its role as a plant NuA4 subunit [16]. To see whether the HSA domain of AtEAF1 can recruit A. thaliana YAF9, we transiently coexpressed nEYFP-tagged AtYAF9A or AtYAF9B with the HSA-containing fragment of AtEAF1 fused to the Flag-tag in Arabidopsis mesophyll protoplasts. Coimmunoprecipitation showed that AtYAF9A and AtYAF9B do indeed interact with this fragment of AtEAF1 (Figure 2a, Additional file 5). We also tested HSA-containing fragments of PIE1 and AtINO80 fused to Flag-tag, but no interaction was observed.Figure 2


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)

AtYAF9A and AtYAF9B interact with AtSWC4 and the HSA-containing fragment of AtEAF1. (a) Coimmunoprecipitation test of interaction between AtYAF9A and AtYAF9B with the HSA-containing fragments of PIE1, AtEAF1 and AtINO80. Yaf9:nEYFP fusion proteins were detected with antibodies against GFP. Bands above the 55 kDa bars are nonspecific signals from immunoglobulin heavy chains. For additional experimental controls, see Additional file 5. (b) BiFC assay in Arabidopsis mesophyll protoplasts. Each co-transfection consisted of a pair of complementary BiFC constructs (nEYFP and cEYFP fusion, right panels, yellow dots indicate fluorescence complementation in the nuclei) and an ECFP construct as an internal transfection control (left panels, cyan). AtYAF9Bsv is the short splice variant of AtYAF9B, lacking the putative C-terminal SWC4-binding domain. (c) Enlarged images of single protoplasts showing nuclear localization of the EYFP fluorescence, indicating interaction between AtSWC4 and AtYAF9A (upper image) or AtYAF9B (lower image). Chloroplast autofluorescence is shown in red and ECFP fluorescence in cyan. Scale bar: 10 μm. Contrast and brightness were enhanced in all micrographs in (b) and (c) to improve clarity.
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Fig2: AtYAF9A and AtYAF9B interact with AtSWC4 and the HSA-containing fragment of AtEAF1. (a) Coimmunoprecipitation test of interaction between AtYAF9A and AtYAF9B with the HSA-containing fragments of PIE1, AtEAF1 and AtINO80. Yaf9:nEYFP fusion proteins were detected with antibodies against GFP. Bands above the 55 kDa bars are nonspecific signals from immunoglobulin heavy chains. For additional experimental controls, see Additional file 5. (b) BiFC assay in Arabidopsis mesophyll protoplasts. Each co-transfection consisted of a pair of complementary BiFC constructs (nEYFP and cEYFP fusion, right panels, yellow dots indicate fluorescence complementation in the nuclei) and an ECFP construct as an internal transfection control (left panels, cyan). AtYAF9Bsv is the short splice variant of AtYAF9B, lacking the putative C-terminal SWC4-binding domain. (c) Enlarged images of single protoplasts showing nuclear localization of the EYFP fluorescence, indicating interaction between AtSWC4 and AtYAF9A (upper image) or AtYAF9B (lower image). Chloroplast autofluorescence is shown in red and ECFP fluorescence in cyan. Scale bar: 10 μm. Contrast and brightness were enhanced in all micrographs in (b) and (c) to improve clarity.
Mentions: As shown above, we found evidence for physical association of AtARP4 and AtSWC4 with AtEAF1, a domain relative of yeast NuA4 subunit EAF1. We assumed that AtARP4 and AtSWC4 associated with AtEAF1 through interaction with the latter’s HSA domain. Cooperative binding of ARP4 and SWC4 to the N-terminal region of SWR1, containing the HSA domain, requires YAF9 in yeast [7]. One of the two Arabidopsis homologues of YAF9 was shown recently to be required for histone H4 acetylation in the FLC locus, in line with its role as a plant NuA4 subunit [16]. To see whether the HSA domain of AtEAF1 can recruit A. thaliana YAF9, we transiently coexpressed nEYFP-tagged AtYAF9A or AtYAF9B with the HSA-containing fragment of AtEAF1 fused to the Flag-tag in Arabidopsis mesophyll protoplasts. Coimmunoprecipitation showed that AtYAF9A and AtYAF9B do indeed interact with this fragment of AtEAF1 (Figure 2a, Additional file 5). We also tested HSA-containing fragments of PIE1 and AtINO80 fused to Flag-tag, but no interaction was observed.Figure 2

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
Related in: MedlinePlus