| Open chromatin plays an extremely important regulatory role during the growth and development of eukaryotes,which is generally considered to be nucleosome depleted or with low nucleosome abundance in previous studies.However,increasing studies have shown that the promoter of eukaryotes is occupied by “fragile nucleosome”,which are easily destroyed by micrococcal nuclease,and are co-distributed with DHSs,enhancers and transcription factor binding sites.“Fragile nucleosome” are found in species such as humans,mice,yeast and maize,but the biological implication of “fragile nucleosome” associated with open chromatin is still unclear,especially in plants.At present,the widely used technololgy to identify the genome-wide nucleosome map is MNase-seq.In order to digest naked DNA completely,a high concentration of MNase needs to be used to lyse nucleus.Under the condition of high MNase concentration,some “fragile nucleosome” sensitive to MNase will also be digested,making the identification of “fragile nucleosome” more difficult.In order to identify the relationship between open chromatin and “fragile nucleosome”in maize,in this study,we conducted MH-seq,LMNase-seq under the same low MNase concentration system,and HMNase-seq under the high MNase concentration,to identify open chromatin and “fragile nucleosome” integrally,combined with RNA-seq,TF-Ch IP-seq,DAP-seq,BS-seq and CAGE-seq,to study the interaction between open chromatin,“fragile nucleosome”,CAGE TSS and transcription factor binding sites in maize.The main findings are as follows:1.According to the relative positional relationship with “fragile nucleosome”,open chromatin can be divided into two different types in maize: MHSs+Nu-and MHSs+FNs.MHSs+Nu-are typial nucleosome free,MHSs+FNs and “fragile nucleosome” are codistributed.MHSs+FNs are more numerous than MHSs+Nu-and more open than the latter.Both types of MHSs can promote gene expression.2.The core DNA of nucleosome identified based on high concentration of MNase has higher G/C dinucleotide frequencies than nucleosome identified at low concentration MNase,while nucleosomes identified at low concentration MNase have higher A/T dinucleotide frequencies in the linker DNA.The core DNA of “fragile nucleosome” co-distributed with open chromatin has higher G/C dinucleotide frequencies and lower A/T dinucleotide frequencies,which are different from canonical nucleosomes.3.Both MHSs+Nu-and MHSs+FNs have the potential to be bound by transcription factors.Both have a common transcription factor and some unique transcription factors that only bind to one of the MHSs.Both MHSs can function as promoters or enhancers in the protoplastbased transient assays.4.The +1 "fragile nucleosome" abundance of the corresponding genes of MHSs+Nu-and MHSs+FNs are positively correlated with gene expression.The-1 "fragile nucleosome" of MHSs+Nu--related genes is negatively correlated with gene expression,and the-1 "fragile nucleosome" of MHSs+FNs-related genes is positively correlated with gene expression.MHSs+Nu-can affect the localization of nucleosomes that are farther upstream and downstream,while MHSs+FNs can only affect the localization of nucleosomes that are relatively short upstream and downstream.5.When combined with CAGE TSS,the expression of MHSs+FNs associated genes is higher than MHSs+Nu-associated genes.When any types of MHSs combined with broad CTSSs,the expression of associated genes is higher than when combined with sharp CTSSs.When broad CTSSs and sharp CTSSs are combined with MHSs+Nu-and MHSs+FNs,the RNA polymerase II occupancy level of the corresponding genes of sharp CTSSs is always higher.6.MHSs+Nu-associated genes are more inclined to express in tissue-specific way,whereas MHSs+FNs associated genes are more inclined to be constitutive expression.MHSs+Nuassociated genes are more enriched in translation,protein metabolic process and protein binding than MHSs+FNs associated genes,while MHSs+FNs associated genes are more enriched in thylakoid,photosynthesis,and response to abiotic stimulus than MHSs+Nuassociated genes.7.MHSs located in genic region have lower CG and CHG methylation level and higher CHH methylation level than MHSs located in the intergenic region;both the CG,CHG and CHH methylation level of MHSs+FNs are all lower than MHSs+Nu-.Broad CTSS-related genes have lower CHG methylation levels within downstream 1kb of TSS.When combined with different types of MHSs,there is almost no difference in the three context of methylation level downstream of the TSS between the broad CTSSs and sharp CTSSs.In conclusion,this study identified nucleosomes by digesting chromatin with high and low concentration MNase and identifying open chromatin with low concentration MNase,provide evidence that there are two types of open chromatin in maize genome,the DNA sequence and influence on gene expression of two types of open chromatin were analyzed,which is helpful to expand understanding of the relationship between open chromatin and nucleosomes in plant genomes,and enrich the functional research of open chromatin. |
