Constructing Plant Chromatin State Database And Studying Epigenetic Regulatory Mechanisms Under Dark Stress

With the development of plant epigenetics,publicly available plant epigenomic data sets are emerging quickly.It is necessary to integrate and analyze multiple epigenomic data effectively,which will help to discover functional elements hidden in chromatin,and further analyze the epigenetic regulatory mechanisms.However,compared human genome,the integration and analysis platform for plant is still limited,and it is a challenge on choosing the efficient combination of tools for plant data types and constructing analysis workflow in data integration process.Hence,it is necessary to construct a plant epigenomic data integration and mining platform.In this study,chromatin states combined with self-organization mapping(SOM)maps were used to integrate,visualize,and analyze plant epigenomic data to explore coordinated relationships between epigenetic marks and regulation of gene expression.First,I integrated 216 public and in-house epigenomic data in Arabidopsis,and applied Hidden Markov Model(ChromHMM)to define 36 chromatin states,including 290,553 segments across the whole genome in Arabidopsis thaliana.I annotated each chromatin state based on combinations of epigenetic marks and enriched feature regions,and used different colors to mark these chromatin states.In the meanwhile,I used SOM to cluster the Arabidopsis genomic segments based on epigenomic data in a 30 x 45 map.I constructed a Plant Chromatin State Database(PCSD,http://systemsbiology.cau.edu.cn/chromstates),combining the chromatin states,SOM map,and features of epigenetic marks of Arabidopsis thaliana,Oryza sativa and Zea mays.The SOM maps and UCSC genome browser were used as visualization tools to display integrated epigenomic data.I also integrated motif and GO analysis tools in the PCSD database for functional analysis of interested DNA fragments and related genes,respectively.In addition,PCSD provides analysis tools of epigenomic data for discovery of chromatin states and comparative analysis of SOM maps.For PCSD,I tested its validity and practicability in many ways,including conservation analysis of chromatin states for homologue genes,searching the binding sites of transcriptional factors on the upstream of genes,and so on.In addition to discover functional elements hidden in chromatin,the chromatin state platform is useful to further analyze the epigenetic regulatory mechanisms during plant growth and stress responses.Dark stress leads to complex traits such as leaf senescence,elongation of hypocotyls and petiole,and early flowering,etc.Recently,large-scale transcriptomic data showed that the expression levels of senescence-associated genes,including transcription factors,significantly changed after dark treatment.However,studies on epigenetic regulation and the cooperative effects of different epigenetic marks under dark stress have not been reported.To study the characteristics and dynamic changes of epigenetic marks under dark stress,I analyzed high-throughput sequencing of DHSs,histone modifications,and small RNA,respectively,to compare the differences between these epigenetic marks under dark stress and normal light conditions.I also combined transcriptomic data to mine key genes under dark stress and construct regulatory pathways and networks.In the meanwhile,I use SOM maps in PCSD to integrated analyze various epigenomic data under dark stress.I analyzed DNase-seq data and studied the changes in chromatin accessibility and its relationship with gene expression under dark stress in Arabidopsis.I found that many DHSs were diminished after dark treatment in euchromatic regions,but the DHS-increased regions were most located on LTR/Gypsy retrotransposons in the heterochromatin flanking the centromeres.These results suggested euchromatin regions might become closed,but heterochromatin regions become open after dark treatment.In the meanwhile,I analyzed the relationship between DHSs and RNA-seq based gene expression.I found that the DHS-diminished and down-regulated genes were significantly associated with photosynthetic processes.Through TF and motif analysis,I found these photosynthesis-associated genes were regulated by GLKI,a key regulator in retrograde signaling.In addition,I found some genes involved in RdDM pathway were DHS-diminished and down-regulated after dark stress I analyzed TE-associated siRNA using siRNA-seq.I found that the expression of siRNA was consistent with DHSs' changes.The results suggested that DHSs might impact transcriptional activity of TE under dark stress.Furthermore,I analyzed ChIP-seq data to investigate dynamics changes of H3K4me3 in response to darkness in Arabidopsis.The H3K4me3-changed genes were mainly H3K4me3-increased genes after dark treatment.Combined with RNA-seq,I identified 474 H3K4me3-increased and up-regulated genes after dark treatment,and found these genes were related to senescence and autophagy through GO and GSEA analysis.It suggested that H3K4me3 might be associated with senescence and autophagy under dark stress.Furthermore,the changes of H3K4me3 exists cross-talk and difference between dark-induced and age-triggered senescence.In the meanwhile,I found many TFs which were involved in development and response to stress,including MADS,WRKY and MYB families,and miRNAs had H3K27me3 changes under dark stress.In addition,many miRNAs changed expression level under dark stress,including some miRNAs involved in leaf development and flower time.Finally,I used the SOM map in PCSD platform to integrate the epigenomic data analysis.I found that dark stress caused major changes in DHSs,H3K4me3,and H3K27me3 in the regions where they located.The major change of DHSs and H3K27me3 is diminished under darkness,and the major change of H3K4me3 is increased under darkness.Through analyzing the related genes in changed SOM units,I found that genes with different epigenetic marks' changes were different.For example,DHSs'changes regulated photosynthesis-associated genes,and H3K4me3's changes regulated senescence-and autophagy-associated genes.These results suggested that these epigenetic marks including DHSs,H3K4m3,H3K27me3 and small RNA,might perform their functions,and coordinately regulate the gene expression in response to darkness,resulting in phenotypic changes such as leaf senescence,hypocotyl and petiole growth,and early flowering,etc.Through analyzing epigenetic marks under dark stress,the dynamic changes and coordinated regulation of various epigenetic marks under dark stress were visually demonstrated,and the validity and practicality of the PCSD platform were also tested.In summary,I collected different public and in-house epigenomic datasets in Arabidopsis thaliana,and integrated these datasets into chromatin states together with SOM maps.Then I constructed an integration platform for epigenomic data analysis and the visualization,i.e.the Plant Chromatin State Database(PCSD),to facilitate the annotation of functional elements on the genome and exploration of the cooperative epigenetics regulatory mechanisms on gene expression.Next,with the integrative analysis of multiple epigenomic data,I studied the epigenetic regulatory mechanisms under extended darkness in Arabidopsis thaliana.I analyzed various epigenomic data such as DNase-seq,ChIP-seq and sRNA-seq together with RNA-seq data,to explore changes in epigenetic marks,and combined with chromatin states and SOM maps to explore the cooperative epigenetic regulation,which might reveal the epigenetic regulatory mechanisms in response to dark stress.