Characteristic Analysis Of Histone Kcr/Kbu And Functional Analysis Of HDACs In Rice

Histone modification,an important part of epigenetic regulation,plays crucial roles in the growth and development and response to stress in plants.Histone acetylation,one of the most widely studied modifications,is catalyzed by histone acetyltransferases and histone deacetylases.Recent studies have shown that histone acetyltransferase and deacetylase can also regulate other new acylation in mammals,such as histone crotonylation(Kcr)and butyrylation(Kbu).However,the mechanism of histone acylation to participate in the regulation of transcriptional activity and response to environment stress need to be further improved in rice(Oryza sativa L.).In this study,we mainly analyzed the whole genome distribution characteristics of crotonylation and butyrylation in rice;The regulation of H3K14ac/cr distributed on rice under cold stress was analyzed combined with the change of transcriptome level.The functions of genes coding histone deacetylation RPD3/HDA1 were researched in rice.The main work of this study includes:1.Global proteome analysis of rice lysine crotonylationIn this study,IF and WB analysis showed that crotonylation was widely distributed in Oryza sativa.A total of 1,265 lysine sites were identified on 690 proteins in rice by LC-MS/MS.Six conserved motifs(Kcr*V,Kcr*I,Kcr*L,KcrD,DKcr,and EKcr)were identified in Kcr modified peptides.These proteins are mainly involved in protein synthesis,photosynthesis and glycolysis.Subcellular localization analysis revealed that 51%of the crotonylated proteins identified were localized in chloroplasts.The protein interaction network analysis showed that Kcr modified protein was mainly related to ribosome,photosynthesis and glycolysis.In addition,the results from WB showed the concentration of Kcr in albino was significantly lower than that in green seedlings.It suggested that Kcr plays an important role in plant photosynthesis.2.Characterization of rice histone lysine crotonylation(Kcr)modificationIn addition,seven histone crotonylated sites were identified in rice.With ChIP-seq analysis,the majority(86.7%)of the Kcr enriched peaks were located in gene body,especially exons Furthermore,the enrichment of Kcr modification was positively correlated with gene expression in genic regions.Compared with other published ChIP-seq data(H3K9ac,H4K12ac,H3K4me2,H3K36me3,and H3K27me3),the Kcr was co-located with the active histone modifications.Interestingly,histone Kcr facilitated expression of genes with existing active histone modifications.We explored the histone Kcr frequency in the binding regions of the transcription activator ideal plant architecturel(IPA1),as a representative of a category of active enhancers in rice seedlings.Previous studies have indicated that 88%of IPA1-binding regions show DNase I hypersensitivity sites(DHSs),which mark cis-regulatory DNA elements.Interestingly,we found 77%of histone Kcr regions overlapped with DHSs in intergenic regions of the rice genome,while only 6%overlapped with IPA1-binding regions.3.Characterization of rice histone lysine buryty lation(Kbu)modificationLysine butyrylation(Kbu)with a four-carbon chain is a newly-discovered histone acylation modification in rice.In this study,ChIP-seq analyses showed that Kbu mainly enriched in genebody regions of rice genome,especially in exons.The enrichment level of Kbu 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%Kbu enrichedin region co-located with DHSs in intergenic regions.Similar profiles were detected among Kbu and other acetylation modifications such as H3K4ac,H3K9ac,and H3K23ac.It was indicated that Kbu modification is an active signal of transcription.Genes with both Kbu and another acetylation also had significantly increased expression.Gene Ontology(GO)enrichment analysis revealed that these genes with Kbu can regulate multiple metabolic process in rice,such as transcription,binding and stress response.4.Co-location analysis of Kcr and KbuThe above analysis shows that histone Kcr and Kbu are two new acylation modifications in rice.Both of them can promote gene expression.Therefore,the co location analysis of the two modifications was carried out.In this study,the number of genes marked by both Kcr and Kbu is 8913.GO functional annotation analysis showed that these genes are mainly related to the synthesis and metabolism of biomacromolecules,and also participate in gene expression and translation.In cell components,these genes are related to the synthesis and metabolism of biomacromolecules,such as nucleolus,ribosome,Golgi body.It is suggested that these two modifications may be involved in the synthesis and metabolism of biomacromolecules.The number of peaks enriched by both Kcr and Kbu is 9574.265 specific motifs were obtained in these peaks with bioinformatics analysis,which were related to 992 transcription factor binding sites.These results indicate that both new acylation modifications play an important role in plant growth and development.5.Distribution characteristics analysis of H3K14ac/cr in rice under cold stress.Histone modification is an important epigenetic regulation of plant response to stress.By Western blot analysis,we found that the modification levels of H3K14ac,H3K36ac,and H3K14cr in rice seedlings treated with 4℃ for 10 hours were significantly up-regulated,while the level of Kbu was decreased.The H3K14ac and H3K14cr were selected to study the regulatory relationship between histone modification and the regulation of cold stress response genes.Transcriptome analysis and GO functional enrichment analysis showed that differentially expressed genes(DEGs)were mainly enriched in stress response,metabolic process and photosynthesis.The results of ChIP-seq analysis showed that the number of differentially expressed genes modified by acetylation was larger than that modified by crotonylation.The expression of DREB1A with higher two histone mofications level was promoted under cold stress.Through further correlation analysis,it was found that there was a positive correlation between H3K14ac modification level and gene expression level under cold stress in rice.GO analysis showed that genes with up-regulated H3K14ac/cr level and up-regulated expression were enriched in the stress response process.6.Functional analysis of RPD3/HDA1 family genes in riceIn yeast,RPD3 and HDA1 families have obvious deacetylase activity.RPD3/HDA1 family homologous with RPD3 and HDA1 is the largest HDACs subfamily in plants.The histone deacetylase RPD3/HDA1 family is the largest HDACs subfamily in plants,which contains the histone deacetylation domain.Through homologous sequence alignment and phylogenetic tree analysis,it was found that RPD3/HDA1 family proteins in rice.In this study,the deacetylase OsHDA701、OsHDA702 and OsHDA706 were used as the research objects.The transformation vectors of OsHDA701,OsHDA702,OsHDA706 were constructed by CRISPR/Cas9 system and integrated into rice variety ZH11 or Nipponbare to get the mutant and transgenic plants.OsHDA701 was mainly expressed in ear and root.By investigating the agronomic traits of OsHDA701 mutant,we found that the plant height was significantly reduced in Yangzhou,while it was significantly increased in Hainan.Under different environmental conditions,the seed length and grain thickness of the mutant became longer and thinner,but the thousand seed weight and grain width of the plant did not change significantly.WB analysis showed that some acetylation modification level in OsHDA 701 mutant was significantly increased,including H3K36ac,H3K27ac,H3K14ac and H4K8ac,especially H3K14ac,while H3K18ac and H4K5ac did not change significantly.WB analysis of crotonylation showed that H3K18cr did not change significantly,while H3K14cr modification level decreased significantly.These indicated that OsHDA701 could specifically erase acetylation and regulate the crotonylation of H3K14 in rice.Interestingly,the head date of Oshda701 was delayed under day 25℃/night 20℃,while advanced under day 35℃/night 28℃.These results suggest that OsHDA 701 may be involved in the epigenetic regulation of rice growth related genes controlled under different temperature conditions.OsHDA702 mutant lines could not enter into reproductive growth stage and eventually died.It indicates that OsHDA702 is the key gene for the transition from vegetative growth to reproductive growth in rice.WB analysis showed that only H3K14cr modification was increased in OsHDA702 mutant,while H3K14ac level was significantly reduced.This indicated that OsHDA 702 could have the function of decrotonylase and affect the enrichment of acetylation modification.By investigating the agronomic traits of OsHDA 706 mutant,we found that the plant height was significantly reduced,the spike length was shorter,the number of tillers was significantly lower,the seed setting rate was poor,and the plant appeared premature senescence.WB analysis of histone modification in wild type and mutant showed that the modification level of H3K36ac,H3K27ac and H4K8ac were decreased.The results showed that OsHDA706 was very important for rice growth and development.OsHDA706 mutant would affect the enrichment of acetylation,but not crotonylation.The above studies preliminarily showed that RPD3/HDA1 family gene of histone deacetylase family had an important effect on the growth and development of rice,but it may not be completely the function of removing histone acetylation or crotonylation.The results lay an important foundation for further analysis of the biological functions of related genes in rice.