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. 2018 Oct;17(10):1922-1936.
doi: 10.1074/mcp.RA118.000640. Epub 2018 Jul 18.

Global Involvement of Lysine Crotonylation in Protein Modification and Transcription Regulation in Rice

Shuai Liu  1 , Chao Xue  1 , Yuan Fang  2 , Gang Chen  1 , Xiaojun Peng  3 , Yong Zhou  1 , Chen Chen  1 , Guanqing Liu  1 , Minghong Gu  1 , Kai Wang  4 , Wenli Zhang  2 , Yufeng Wu  5 , Zhiyun Gong  6
Affiliations

Global Involvement of Lysine Crotonylation in Protein Modification and Transcription Regulation in Rice

Shuai Liu et al. Mol Cell Proteomics. 2018 Oct.

Abstract

Lysine crotonylation (Kcr) is a newly discovered posttranslational modification (PTM) existing in mammals. A global crotonylome analysis was undertaken in rice (Oryza sativa L. japonica) using high accuracy nano-LC-MS/MS in combination with crotonylated peptide enrichment. A total of 1,265 lysine crotonylation sites were identified on 690 proteins in rice seedlings. Subcellular localization analysis revealed that 51% of the crotonylated proteins identified were localized in chloroplasts. The photosynthesis-associated proteins were also mostly enriched in total crotonylated proteins. In addition, a genomic localization analysis of histone Kcr by ChIP-seq was performed to assess the relevance between histone Kcr and the genome. Of the 10,923 identified peak regions, the majority (86.7%) of the enriched peaks were located in gene body, especially exons. Furthermore, the degree of histone Kcr modification was positively correlated with gene expression in genic regions. Compared with other published histone modification data, the Kcr was co-located with the active histone modifications. Interestingly, histone Kcr-facilitated expression of genes with existing active histone modifications. In addition, 77% of histone Kcr modifications overlapped with DNase hypersensitive sites (DHSs) in intergenic regions of the rice genome and might mark other cis-regulatory DNA elements that are different from IPA1, a transcription activator in rice seedlings. Overall, our results provide a comprehensive understanding of the biological functions of the crotonylome and new active histone modification in transcriptional regulation in plants.

Keywords: Epigenetics; Histones; Oryza sativa; Photosynthesis; Protein Modification; Transcriptional Regulation; crotonyl lysine; histone lysine crotonylation; post-translational modification.

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Conflict of interest statement

The authors declare that they have no conflicts of interest

Figures

Fig. 1.
Fig. 1.
An overview of Kcr modifications in rice. (A) Crotonylation was detected in the nucleus and cytoplasm of two-week-old rice root by immunofluorescence using an anti-Kcr antibody (green), and nuclei were stained with DAPI (red). Scale bars: 5 μm. (B) Western blot analysis of the total protein content of rice seedling leaves showing duplicates. (C) Western blot analysis of histones of rice seedling leaves. (D) Randomly selected crotonylpeptide DLV-(crotonyl)K-ETIATL with the crotonylation site at K1099 in the elongation factor. (E) Dot-spot assay of validated crotonylated proteins. The numbers 1, 3, 5, 7, and 9 represent crotonyl peptides TIMP-(crotonyl)K-DIQLA, IQGIT-(crotonyl)K-PAIR, LEV-(crotonyl)K-EIAEIM, LAEEG-(crotonyl)K-VAIR, and DLV-(crotonyl)K-ETIATL, respectively. The numbers 2, 4, 6, 8, and 10 represent unmodified peptides TIMP-K-DIQLA, IQGIT-K-PAIR, LEV-K-EIAEIM, LAEEG-K-VAIR, and DLV-K-ETIATL, respectively.
Fig. 2.
Fig. 2.
Motif analysis of lysine crotonylation peptides. (A) Heat map of the amino acid compositions of the crotonylation sites showing the frequency of the different of amino acids around the Kcr; +1, and −1 represent the position around the Kcr. (B) Crotonylated peptide motifs and conservation of Kcr sites. The height of each letter corresponds to the frequency of that amino acid residue at that position. The 0 position K refers to the crotonylated sites. (C) Number of identified peptides containing Kcr in each motif.
Fig. 3.
Fig. 3.
Gene ontology functional classification of crotonylated proteins. Gene ontology functional classification of the identified crotonylated proteins based on (A) subcellular localization, (B) biological processes, (C) molecular function, and (D) cellular component.
Fig. 4.
Fig. 4.
Crotonylated proteins in enriched in carbon assimilation and photosynthesis pathways in C3 plants. The identified crotonylated proteins are highlighted in blue.
Fig. 5.
Fig. 5.
Top three clusters of highly interconnected lysine-crotonylated protein networks. The network of lysine-crotonylated protein interactions (listed in protein ID names) was analyzed using the Cytoscape software (version 3.3.0). (A) Ribosome; (B) photosynthesis; (C) glycolysis/gluconeogenesis. The size of the balls represents the numbers of Kcr modifications in each figure.
Fig. 6.
Fig. 6.
The Kcr sites identified in human histones and rice histones (H3, H4 in this study). In humans, seven H3 Kcr PTM sites (H3K4/K9/K18/K23/K27/K56/K122) and three H4 Kcr PTM sites (H4K5/K8/K122) were identified. In rice, four H3 Kcr PTM sites (H3K14/K56/K79/K122) and two H4 Kcr PTM sites (H4K31/K79) were identified. The data for human Kcr sites were used from Suzuki et al. (55) and Tan et al. (26).
Fig. 7.
Fig. 7.
The genomic distribution of histone Kcr-enriched regions. (A) Visualization of the Kcr seq locus on Chr1:407100–409600. (B) Genome-wide distribution of histone Kcr in the rice genome. The promoter was defined as the 1 kb sequence upstream of the gene transcription start site. (C) Distribution of Kcr density around differentially expressed genes. The Kcr modification was calculated by the number of reads per kilobase of the mapped genomic region. The arrow indicates the direction of transcription from the transcription start site. The rice genes were divided into five categories based the expression level from the top 20% to the bottom 20% based on published RNA-seq data from same rice seedlings at the same stage of development (49). (D) Expression comparisons of genes associated with different combinations of histone modifications. The nontranscription element gene expression values (FPKM) of each combination are indicated by box plots. All: all rice genes. Kcr: genes with Kcr modification. H3K9ac+Kcr: genes with both H3K9ac and Kcr. H3K9ac-Kcr: Genes with H3K9ac but not Kcr; the rest may be deduced by analogy. *** indicates significant differences between the two combinations (p < 2.2e-16, Kolmogorov-Smirnov test).
Fig. 8.
Fig. 8.
Association of histone Kcr and gene transcription in rice genome. (A) A representative region showing histone modifications (Kcr, H3K14cr, H3K9ac, H4K12ac, H3K4me2, H3K36me3, and H3K27me3) in the rice genome. The gray blocks indicate the Kcr-enriched genomic regions. Y indicates H3K14cr-enriched regions, while X indicates H3K14cr nonenriched regions in the gray block. (B) Heat map of histone Kcr and five histone modifications in generic regions and intergenic regions. The regions 1 kb upstream and downstream of the transcription start site (indicated by an arrow, for the generic regions) and the middle of Kcr regions (for the intergenic regions) were clustered based on density of corresponding histone modifications. Density was calculated by the number of reads per kilobase region per million mapped reads. (C) The percentage of histone Kcr-enriched regions overlapping with regions enriched with other histone modifications (H3K4me3, H3K4me2, H3K9ac, H3K36me3, H3K27me3, and H3K14cr). (D) The percentage of histone Kcr-enriched regions overlapping with regions enriched with functional regions (DNase I hypersensitive sites (DHSs), and IPA1-binding regions). The promoter was defined as the 1 kb sequence upstream of the transcriptional start site and the intergenic region indicates the nongeneric and nonpromoter regions.
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