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Light availability promotes distinct responses of plastid protein acetylation marks directed from the N-acetyltransferase GNAT2

ABSTRACT: Acetylation of amino groups is an important and recurrent protein modification in all eukaryotes. In plants, both lysine and N-terminal acetylation have additional important roles in protein regulation in chloroplasts. However, it is yet unclear how acetylation patterns respond to environmental stresses, and whether lysine or N-terminal modifications similarly contribute to the associated perturbations. A new family of plastid acetyltransferase, called GNATs, was recently discovered, which consists of closely related enzymes featuring both lysine- and N-terminal acetyltransferase activities often on the same polypeptide targets. Here, we take advantage of this unique characteristic of Arabidopsis GNAT2 to obtain a holistic multi-omics acetylation-dependent view during the acclimation of plants to short term changes in light. No substantial difference in transcriptome or adenylate energy charge oscillations were observed between WT and gnat2 knockout mutant lines when they were submitted for two hours to high-light or dark growth conditions. A detailed characterization of the N-terminal acetylome reveals that both its yield and coverage remain unchanged upon different light conditions in both genotypes. Unlike the N-terminal acetylome, the GNAT2-associated lysine acetylome is sensitive to the different light conditions used. In addition, a strong reduction in both types of acetylations on several plastid proteins was observed upon GNAT2 inactivation under all light conditions. Our data suggests that the lysine acetylome marks on proteins more rapidly fluctuate following acclimation to the environmental condition, while N-terminal acetylation changes are associated to longer term responses, like those promoted in the gnat2 background. Taken together, our data revealed unique strategies of plant acclimation to the different applied treatments involving specific PTMs and emphasizes distinct timescale responses of plastid lysine and N-terminal acetylomes to environmental changes.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Rosette

SUBMITTER: Carmela Giglione

LAB HEAD: Carmela GIGLIONE

PROVIDER: PXD040235 | Pride | 2024-08-22

REPOSITORIES: Pride

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			Action	DRS
	20200706_SCX_PM_d3NAc_Athaliana_WT_HighLight_RepBio1_10Fracs.rov	Other
	20200706_SCX_PM_d3NAc_Athaliana_WT_HighLight_RepBio1_10Fracs.xml	Xml
	20200706_SCX_PM_d3NAc_Athaliana_WT_HighLight_RepBio1_Frac_02.raw	Raw
	20200706_SCX_PM_d3NAc_Athaliana_WT_HighLight_RepBio1_Frac_02.rov	Other
	20200706_SCX_PM_d3NAc_Athaliana_WT_HighLight_RepBio1_Frac_03.raw	Raw

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Project description:Aims: To map histone modifications with unprecedented resolution both globally and locus-specifically, and to link modification patterns to gene expression. Materials & methods: Using correlations between quantitative mass spectrometry and chromatin immunoprecipitation/microarray analyses, we have mapped histone post-translational modifications in fission yeast (Schizosaccharomyces pombe). Results: Acetylations at lysine 9, 18 and 27 of histone H3 give the best positive correlations with gene expression in this organism. Using clustering analysis and gene ontology search tools, we identified promoter histone modification patterns that characterize several classes of gene function. For example, gene promoters of genes involved in cytokinesis have high H3K36me2 and low H3K4me2, whereas the converse pattern is found ar promoters of gene involved in positive regulation of the cell cycle. We detected acetylation of H4 preferentially at lysine 16 followed by lysine 12, 8 and 5. Our analysis shows that this H4 acetylation bias in the coding regions is dependent upon gene length and linked to gene expression. Our analysis also reveals a role for H3K36 methylation at gene promoters where it functions in a crosstalk between the histone methyltransferase Set2KMT3 and the histone deacetylase Clr6, which removes H3K27ac leading to repression of transcription. Conclusion: Histone modification patterns could be linked to gene expression in fission yeast. The combined microarray strategy in this study was performed essentially as outlined in Wiren et al, 2005. We used the S. pombe spotted microarrays (Eurogentec, Belgium). For histone modification maps, ChIP-CHIP method was essentially used according to Robyr and Grunstein (2003). Antibodies against H3K9Ac, H3K14Ac, H3K18Ac, H3K23Ac, H3K27Ac, H3K56Ac , H4K5Ac, H4K8Ac, H4K12Ac, H4K16Ac (Suka, Suka et al. 2001; Xu, Zhang et al. 2007) and H3K36Me2 (Millipore) were used. The histone ?H3cter? antibody (Upstate) was used for ChIP according to Wiren et al, 2005. Spotted microarrays were hybridized using Cy3 and Cy5. For normalization of the data (in Series supplementary file) all the different modification channels were divided by average input value and then divided by ?H3cter? value to correct the histone loss calculations followed by 50th percentile normalization. For our cluster analysis we have used variance normalization method. To reduce the variations between different sample preparations we used same extracts for all IPs. The standard S.pombe laboratory strain Hu303(972h-) was used for this study. For Wt, set1D and set2D expression studies we have used Affymetrix genechip yeast Genome 2.0 microarray. Wt, set1D and set2D cells were grown to mid logarithmic phase (5*10 power 6 cells/ml) in rich medium. RNA was extracted and hybridized according to Affymetrix protocol. For each expression profile at least two independent cultures were analyzed with microarray experiments.

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Light availability promotes distinct responses of plastid protein acetylation marks directed from the N-acetyltransferase GNAT2

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