Studies On Rice Gene Expression Changes And Epigenetic Characteristics Of Dna Double-strand Break Sites Under ⁶⁰Co-? Irradiation Treatment

In response to abiotic stress,epigenetic changes such as gene transcription levels,histone modifications,and chromatin structure in the plant will occur in varying degrees,indicating that epigenetic changes are closely related to plant stress responses.As one of abiotic stresses,ionizing radiation can induce DNA damage such as double-strand break(DSB)in plants.So far,there are still few related studies on DNA damage response and DSB related genetic and epigenetic characteristics in plants.In addition,the production and repair of DNA damage is the molecular basis of plant mutation breeding.How to make effective use of DSB for plant mutation breeding service is one of the hot issues in the basic research of DNA damage application in plants.Therefore,analysis of DNA damage response and DSB-associated genetic and epigenetic features are both conducive to elucidating the molecular mechanisms of genetics and epigenetics of DNA damage,and are also useful in providing effective screening sites informations for artificial mutation breeding in crop,which can further improve the efficiency of artificial mutation breeding.In this study,7-day-old sub-cultured rice seedlings(Oryza sativa.subsp.Keng.(cv.Nipponbare))were used as experimental materials.Firstly,mRNA-seq and ChIP-seq techniques were used to study the effect of 60Co-? irradiation on gene expression and histone modifications in the rice genome.Secondly,DSB-seq technology was used to identify DSB sites in the rice genome,and accordingly analyzed the DNA sequence characteristics of DSB sites.Finally,various omics data sets such as mRNA-seq,ChIP-seq,and DNase-seq were combined to analyze the chromatin structural features in association with DSBs,including histone modifications and open chromatin.The main findings are as follows:1.Chromatin states function in regulation of differentially expressed genes(DEGs)induced by 60Co-? irradiationIn this study,we collected the leaf tissue from the plants with OGy and 50Gy/30min,and extracted total RNA for RNA-seq.A total of 3,839 DEGs were identified by mRNA-seq analysis,including 2,127 up-regulated genes and 1,712 down-regulated genes.Gene ontology(GO)term enrichment analysis showed that the up-regulated genes showed a strong overrepresentation of GO terms functioning in biological metabolisms,response to cell death and protein modification,etc;Whereas the down-regulated genes were mainly responsible for DNA replication and gene expression,etc.We performed ChIP experiments coupled with ChIP-sequencing using antibodies against seven histone marks,including H3K4me3,H3K36me3,H3K27me3,H3K4ac,H3K27ac,H3K4mel and H4K12ac.ChIP-seq analyses demonstrate that 60Co-? ray induced global histone modification changes mainly occur at the promoters,exons and intergenic regions.In addition,according to the combined distribution of seven histone marks,we divided the whole genome chromatin states(CSs)into 15 classes,and then analyzed the relationship between DEGs expression levels and these 15 CSs by combining mRNA-seq and ChIP-seq data.We found that DEGs induced by 60Co-? ray treatment are regulated by chromatin states,and the expression of up-regulated genes may be regulated by the chromatin status composed of single or multiple histone modifications,while the expression of down-regulated genes is mainly regulated by the chromatin status composed of multiple histone modifications.2.Epigenetic characterization of DNA Double-strand Breaking(DSB)Sites in rice genomicIn this study,we collected the leaf tissue from the plants with OGy and 50Gy/30min,and isolation nuclei for DSB-seq.We identified 19,248 DSBs,which included 2,000 DSB hotspots that defined by the first top of 2,000 DSB sites with high reads densities.In the whole genome of rice,DNA double-strand breaks damage is more likely occurred on euchromatin under 60Co-? irradiation treatment,DSB sites are mainly distributed in the gene body region,especially in the promoter region and there is a significant positive correlation between the DSB sites and gene expression.Two typical motifs were identified from the DNA sequences of DSB hotspots in rice,namely AAAAAAAAAA and CCYCYCYCYCTC(Y represents a T or C base),and they were the binding site of the C2H2 zinc finger factors.Combined with DNase-seq and ChIP-seq data analysed,it was found that activated histone modifications are mainly distributed on both sides of the DSB sites,and their enrichment levels are related to chromatin states;Similarly,the distribution pattern of the phosphorylated H2A.X nucleosomes(?-H2A.X)is related to the chromatin states,y-H2A.X mainly distributed surround both sides of DSB sites when the chromatin is open,while the chromatin is in a "closed",tightly concentrated state,it is mainly distributed on the DSB sites.Taken together,this study found that 60Co-? ray induced DEGs are regulated by chromatin states,which help to gain insights into the apparent mechanism of gene expression regulation related to plant DNA damage response.DSB-seq technology was used for the first time to identify DSB sites and DSB hotspots at the whole genome level of rice,and to analyze the basis molecular mechanisms of genetics and epigenetics of DNA damage in plants,which will help improve the efficiency of artificial mutation breeding in plants,and promote the modern crop breeding process.