Genome-wide Profiling of Histone Lysine Butyrylation Reveals its Role in the Positive Regulation of Gene Transcription in Rice
Histone modifications play important roles in growth and development of rice (Oryza sativa L.). Lysine butyrylation (Kbu) with a four-carbon chain is a newly-discovered histone acylation modification in rice.
In this study, we performed chromatin immunoprecipitation sequencing (ChIP-seq) analyses, the result showed that major enrichment of histone Kbu located in genebody regions of rice genome, especially in exons. The enrichment level of Kbu histone modification is positively correlated with gene expression. Furthermore, we compared Kbu with DNase-seq and other histone modifications in rice. We found that 60.06% Kub enriched region co-located with DHSs in intergenic regions. The similar profiles were detected among Kbu and several acetylation modifications such as H3K4ac, H3K9ac, and H3K23ac, indicating that Kbu modification is an active signal of transcription. Genes with both histone Kbu and one other acetylation also had significantly increased expression compared with genes with only one acetylation. Gene Ontology (GO) enrichment analysis revealed that these genes with histone Kbu can regulate multiple metabolic process in different rice varieties.
Our study showed that the lysine butyrylation modificaiton may promote gene expression as histone acetylation and will provide resources for futher studies on histone Kbu and other epigenetic modifications in plants.
KeywordsOryza sativa Histone modification Lysine butyrylation (Kbu) Transcriptional regulation ChIP-seq
Bromodomain testis-specific gene
Chromatin immunoprecipitation sequencing
DNase I hypersensitive sites
Double plant homeodomain finger
Monocytic leukemia zinc-finger protein-related factor
Epigenetic regulation of gene expression is an intricate process that does not involve a change in DNA sequence. Epigenetic marks, such as DNA methylation, histone modification and non-coding RNA have significant effects on regulating transcription. Among these, post-translational modifications (PTMs) of histones that may transform the chromatin state are essential for gene expression. With the development of new biotechnique and updates to PDBs (protein data banks), increasing numbers of histone PTMs have been identified; for example, lysine acylations modifications have various forms, such as acetylation (Tropberger et al. 2013), butyrylation (Zhang et al. 2009), propionylation (Zhao and Jensen 2009), crotonylation (Tan et al. 2011), methylation (Peach et al. 2012), malonylation (Xie et al. 2012), succinylation (Zhang et al. 2011), 2-hydroxyisobutyrylation (Dai et al. 2014), and β-hydroxybutyrylation (Liu et al. 2019). These acylation modifications always mark lysine with different hydrocarbon chain lengths and hydrophobicity or charge (Azevedo and Saiardi 2015).
Lysine acetylation, which is one of the most studied modifications, is essential for the control of gene expression. Previous research has indicated that there are complex interactions between lysine acetylation and transcription factors, such as enhancers, silencers, and promoters (Perillo et al. 2008; Bannister and Kouzarides 2011). The level of histone acetylation is maintained by histone acetyl-transferases (HAT) and histone-deacetylases (HDAC) (Zhao et al. 2014). Also, histone acylation homeostasis can be maintained by different histone complexes associated with these two enzymes, such as crotonylation, butyrylation, and propionylation (Ogryzko et al. 1996; Chen et al. 2007; Kaczmarska et al. 2017; Sabari et al. 2018). Human sirtuins, which have homology to the yeast sir2 histone deacetylase, have both deacetylase and deacylase activities (Ogryzko et al. 1996; Cheng et al. 2009; Sabari et al. 2017).
Lysine butyrylation (Kbu) is a novel PTM that is found widely in histone and non-histone proteins (Chen et al. 2007). Kbu has been identified in multiple cell types in animals, plants, and fungi, suggesting that Kbu is evolutionarily conserved (Goudarzi et al. 2016; Lu et al. 2018). Butyrylation is an acylation modification similar to crotonylation, with a four-carbon chain in the planar orientation (Flynn et al. 2015). Interestingly, the histone Kbu marks active gene TSSs and directly stimulates transcription (Goudarzi et al. 2016). In addition, H4K5bu can prevent binding of the bromodomain testis-specific gene (BRDT) (Goudarzi et al. 2016). Moreover, H4K5bu and H4K8bu are related to delayed histone removal in spermatogenic cells (Goudarzi et al. 2016). Recently, the double plant homeodomain finger (DPF) of the lysine acetyltransferase MORF (the monocytic leukemia zinc-finger protein-related factor) was shown to be a reader of global histone H3K14 acylation that can bind H3K14bu to form a recruitment and stabilization MORF-DPF-H3K14bu complex at promoters of target genes (Klein et al. 2017). Because they are photoautotrophic organisms, plants are significantly different from mammals with respect to their primary metabolic processes. The relationships between butyrylation and gene expression in the interactions with primary metabolism in plants are less known at present.
Rice (Oryza sativa L.) is a model monocot species that plays a fundamental role in plant genome research (Shi et al. 2015). Several protein modifications have been identified in rice, such as methylation (Cheng et al. 2018), acetylation (Xue et al. 2018), and crotonylation (Liu et al. 2018). Recently, butyrylation, was identified by Lu et al. (2018) as an active modification mark that regulates gene expression in the rice cultivar ‘DongJin’ (DJ) (Lu et al. 2018). Therefore, we performed additional experiments and a combined public data analysis to identify histone Kbu in the japonica rice cultivar ‘Nipponbare’.
We confirmed that Kbu is present in histones and non-histone proteins in rice using biological experiments. We also profiled the genome-wide distribution of the Kbu modification by ChIP-seq analysis with a pan anti-Kbu antibody. In addition, we compared Kbu with 12 other histone modifications and DHS in rice. In brief, our research will enlarge the discovery of the biological functions of histone lysine butyrylation in rice.
Genome-wide Profiling of Histone Kbu in Rice
Summary of ChIP-seq data
Kbu(pan-antibody) replicate 1
Kbu(pan-antibody) replicate 2
Confirmation of Kbu peak sites and non-peak sites by quantitative PCR
Histone Kbu is Related to Gene Expression in Rice
Summary of Kbu-related genes and peaks of two rice varieties (Nipponbare and DongJin)
Number of peaks
Common Kbu-related genes
Specific Kbu-related genes
Kbu-related expressed gene
Common expressed gene
We next investigated the relationship between gene expression and histone Kbu. Among total 39,045 non-TE genes in rice, 14,808 genes (37.93%) are marked by histone Kbu. In these genes, 80.93% genes (11,984) are expressed, with FPKM> = 1 (Wu et al. 2011). For example, the peaks of Kbu ChIP-seq located at expressed gene LOC_Os02g14720, rather than non-expression gene LOC_Os02g14700 (Fig. 2b). The results suggested that the sites of histone Kbu were enriched principally in expressed genes. In addition, we found genes with generally higher expression levels associated with higher histone Kbu density (Fig. 2c) In Dongjin (Lu et al. 2018), the peaks (27,665) were identifed in 19,355 genes, in which 14,151 genes (73.11%) are expressed in Dongjin, with FPKM> = 1, while 11,984 genes (80.93%) are expressed in Nipponbare. Nine thousand nine hundred sixty-one genes are shared between two cultivars (Table 3). These results show that histone Kbu has a high correlation with gene expression in different cultivars of rice.
Concurrence among Histone Kbu, other Histone Modifications, and DNase-hypersensitive (DH) Sites
In the mouse, 35% H3K14bu peaks cover the promoter-TSS regions (Kebede et al. 2017). Here, we found 23.87% histone Kbu modifications were enriched in promoter and intergenic regions in rice. We next investigated the relationship between histone Kbu and DNase I hypersensitive sites (DHSs), because these sites harbor cis-regulatory elements in open chromatin. More than half of the histone Kbu peaks overlapped with DHSs in the rice genome (Zhang et al. 2012), and 60.06% of the peaks in intergenic regions co-located with DHSs (Figs. 4b and 5). These results indicate that histone Kbu can be an active mark and may recruit transcriptional regulators to facilitate gene transcription.
Histone Kbu Combined with Histone Acetylation Facilitates Transcription
Putative Functions of Genes Associated with Histone Kbu
Compared to the Kbu-related genes in DongJin (Lu et al. 2018), although there are highly similarly between Nipponbare and DongJin, there are still many different genes. We further conduct the GO analysis on 2249 and 6796 specific Kbu-related genes in Nipponbare and Dongjin, respectively (Table 3). Genes with histone Kbu in Nipponbare are enriched in translation, transport and localization process, while in Dongjin, gene with such histone modification participates in transcription, binding process and stress-response. Nevertheless, both in Nipponbare and Dongjin, these genes can regulate multiple metabolic processes. Hence Kbu is important for growth and development of rice.
Recently, Lu et al. (2018) identified four rice histone lysine butyrylation sites (H3K14bu, H4K12bu, H2BK42bu, and H2BK134bu) using LS-MS/MS in the rice cultivarDongJin. Their results showed that Kbu is enriched in the 5′ regions of expressed genes, and 26,769 Kbu-marked genes were identified. In this study, 21,202 histone Kbu-marked peaks also appeared to be mainly in the 5′ regions, TSS regions, and exons. The peaks covered a large proportion of the genebody regions, especially in exons. Most of the Kbu peaks in the exons were mapped to the coding sequences and the 5′-UTR. This distribution is similar to that of histone Kbu in Dongjin (Lu et al. 2018). Meanwhile, it is similar to that of histone H3K4me2/3 in rice (Du et al. 2013). However, in mouse, H3K14bu is mostly enriched in introns and promoter-TSS regions, and more than two-thirds of the peaks covered these regions (Kebede et al. 2017). These results suggest that histone Kbu may regulate gene expression via different mechanisms in plants and mammals. In addition, we found genes with generally higher expression levels associated with higher histone Kbu density, as was found for H3K14bu in mouse and Kbu in other rice variety (Kebede et al. 2017; Lu et al. 2018). These results show that histone Kbu has a high correlation with gene expression in eukaryote.
Lu et al. (2018) showed that Kbu is an active mark, but data for only six histone modifications was used to compare with Kbu. In addition, histone Kbu seems to contribute to the H3K9ac-marked active chromatin state and to balance genes under stress. In this study, we also found that the enrichment level of Kbu is proportional to gene expression. Moreover, we integrated our data with 12 public histone modification data, including H3K4ac, H3K9ac, H3K23ac, H3K27ac, H4K12ac, H4K16ac, H3K4me2/3, H3K36me3, H3K9me1/3, and H3K27me3 (He et al. 2010; Zhang et al. 2012; Lu et al. 2015; Fang et al. 2016b) and also verified the large proportion of Kbu sites overlapping with H3K9ac. These results abundantly showed that histone lysine butyrylation is consistent with active histone modifications such as H3K4ac, H3K9ac, H3K23ac, H3K27ac, H4K12ac, H4K16ac, H3K4me2/3, and H3K36me3. Interestingly, H3K36me3 is similar only to Kbu and H3K4me3, which suggests that H3K36me3 may have similar functions to Kbu and H3K4me3. In addition, 60% of the sites overlap with DHSs, suggesting that Kbu is related to cis-regulatory DNA elements in rice. Our analysis will enlarge a general understanding of epigenetic regulation of transcription via histone Kbu modification, and will enable investigation into the crosstalk between different histone modifications in plants.
It is important to identify and characterize histone modification enzyme systems in order to understand how histone modification is regulated. Some histone acylation is known to be associated with histone acetyltransferase and histone deacetyltransferase (Ogryzko et al. 1996; Chen et al. 2007; Kaczmarska et al. 2017; Sabari et al. 2018). Lu et al. (2018) confirmed that OsSRT2 possesses decrotonylase activity, but not debutyrylase activity. However, the p300 protein, which is an important histone acetyltransferase, also catalyzes histone butyrylation in human (Chen et al. 2007). In our study, we found that histone Kbu tends to co-localize with multiple active histone modifications, especially Kac. In a previous study, three p300 homologous genes were identified in a phylogenetic analysis in rice (Liu et al. 2018). However, it is presently unknown whether p300 catalyzes histone Kbu and, if so, how acetylation and butyrylation is regulated in rice. Additionally, the role of histone Kbu in the regulation of histone structure and function in rice requires further investigation.
Materials and Methods
Rice (Oryza sativa) cultivar ‘Nipponbare’ plants were germinated and grown in water without hormones under a 12 h/12 h photoperiod at 28 °C day/25 °C night with 70% humidity. Leaves and stem tissues of 14-day-old rice seedlings were used for histone protein extraction and ChIP-DNA isolation.
Histone Protein Extraction
Histone proteins were extracted based on a previous method (Liu et al. 2018). Leaves and stem tissues of 14-day-old rice seedling were first ground to a powder in a mortar in the presence of liquid nitrogen. The powdered tissue was then mixed with extraction buffer and centrifuged. The pellet was mixed with nuclei lysis buffer for 30 min on ice, centrifuged again, and the supernatant was removed. The pellet was resuspended in 0.2 M HCl and incubated on ice for 1 h. The proteins were precipitated by addition of 100% TCA, recovered by centrifugation and washed with cold acetone. The sediment was redissolved in Protein Lysis Buffer by sonication and stored at − 80 °C.
Western Blotting and Immunofluorescence Analyses
Western blotting was performed as previously described (Liu et al. 2018). Immunofluorescence analysis was performed using the method described by Gong et al. (Gong et al. 2009). The rice histone proteins were separated electophoretically in denaturing gels by SDS-PAGE (5%/12%). The antibodies used in this study were rabbit pan anti-Kbu antibody (1:5000; PTM BioLabs, HangZhou China, PTM-301) and rabbit anti-H3 antibody (1:10,000; PTM BioLabs, HangZhou China, PTM-1001); the goat secondary anti-rabbit antibody is conjugated with Alexa 488 (Invitrogen, A11008). Chromosomes were counterstained with DAPI dye (Vector Laboratories, H-1200).
ChIP, ChIP-seq, and qPCR
ChIP experiments were performed using a pan anti-Kbu antibody (PTM BioLabs, PTM-301) following a published protocol (Nagaki et al. 2003). Chromatin fragments were obtained by incubation overnight with MNase and protein A-coated beads (GE17–1279-01; Sigma Aldrich). The ChIP-DNA fragments were used for library construction with the Illumina protocol and were then sequenced on the Illumina HiSeq 2500 instrument. ChIP-qPCR was performed using SYBR qPCR Master Mix (Vazyme, Q311–02/03) according to the procedure described by Mukhopadhyay et al. (Mukhopadhyay et al. 2008). Input-DNA was set as the control and the following thermocycling conditions were used: initial denaturation at 95 °C for 600 s, three-step amplification comprising 35 cycles of 94 °C for 15 s to 60 °C for 15 s to 72 °C for 30s. qPCR2 values were acquired by subtracting ChIP-DNA C(t) from Input-DNA C(t), while the threshold cycles of Input-DNA (qPCR1) were set to 0. Input DNAs were used for normalization in ChIP-qPCR. The primers used in ChIP-qPCR are given in Table 2.
ChIP-seq Data Analysis
Raw data were cleaned by cutadapt v2.1 with illumina TruSeq adapter. Bowtie2 v2.3.5 was used for mapping clean data to rice reference genome Tigr 7 (Langmead and Salzberg 2012; Kawahara et al. 2013). Only unique mapped reads without mismatch were retain for further analysis. Aligned bam files were converted to bigwig format using in-house script and visualized with IGV v2.4.5. MACS2 v2.1.2 was used to call peak with parameter ‘callpeak -g 3.8e8 --broad’ (Zhang et al. 2008). BEDTools was then used to merge replicates and identify shared peaks among different histone modification (Quinlan and Hall 2010). Genes contain Kbu peaks were regarded as Kbu related genes.
Based on expression level, all genes were divided into 6 groups. 1 kb upstream and downstream of each gene TSS regions were split into 20 bp bins to plot histone Kbu profile. Percentage of shared peaks of histone modifications was drawn by R package pheatmap. Heatmaps of all histone modifications were plotted using ngsplot v2.6.3 (Shen et al. 2014). All data processing and analysis were performed by python or R.
Gene Ontology (GO) Analysis
One thousand nine hundred thirty-six peaks identified by macs2 with fold enrichment greater than 10 and -log10 (qvalue) greater than 100 were regarded as high confident peaks. We selected 1480 high-confidence histone Kbu-associated genes for gene ontology analysis using the agriGO v2 database (http://systemsbiology.cau.edu.cn/agriGOv2/) (Tian et al. 2017). The significance of a particular GO assignment was calculated using the Fisher test and corrected by FDR with a 0.05 significance level.
SL, CX, XC, LY and ZG performed research; SL, GL, PC and ZG wrote the paper; GL, TZ, and ZQ analyzed data; and ZQ, TZ and ZG designed research. All authors read and approved the final manuscript.
This work was supported by the National Natural Science Foundation of China (31571266, 31871232), the Fund of Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Jiangsu Agricultural Science and Technology Independent Innovation Fund (CX(18)2014), and Postgraduate Education Reform Project of Jiangsu Province (grant number XKYCX17_051).
Ethics Approval and Consent to Participate
Consent for Publication
The authors declare that they have no competing interests.
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