Integrative Analysis Of Haplotype-Resolved Epigenomes And 3D Genomes In An Elite Rice Hybrid

Epigenetic modifications and the three-dimensional(3D)chromatin structures play an important role in transcriptional regulation.The distal regulatory elements that marked with specific chromatin features usually exert their function of modulating gene expression by coming into proximity with gene promoters and forming chromatin loops in 3D space.Therefore,it is of great significance to develop efficient epigenomic and 3D genomic techniques,identify distal regulatory elements,and explore the regulating mechanisms of3 D genome organizations on gene expression and plant phenotypic variations.Certain epigenomic research projects such as the Encyclopedia of DNA Elements(ENCODE)has identified a large number of transcriptional regulatory elements in human cells,such as promoters,enhancers,silencers and insulators.These transcriptional regulatory elements have some specific epigenetic marks.For example,the promoters of transcription genes usually show high enrichment of histone mark H3K4me3;enhancers are significantly associated with H3K27 ac,H3K4me1 and open chromatin;and insulators usually align with the binding sites of the insulator protein CTCF.Compared to the studies of enhancers,the genome-wide identification of silencers is relatively lagging behind.Recently,many findings have shown that histone modification H3K27me3 is related to silencers,and some studies treat H3K27me3 as an epigenetic mark of silencers to identify the silencing elements in the whole genome.However,there are few reports on the genomewide identification of enhancers,silencers and other regulatory elements in plants.In recent years,many single-cell epigenomic techniques based on chromatin immunocleavage(ChIC)have been developed and applied to profile histone modifications and transcription factor occupancy in animal cells.Compared with theChIP-seq technology,theChIC-based technique is a rapid and efficient method that base on the principle of antibody-assisted chromatin in-situ tagmentation around the target protein by nuclease or the transposase Tn5.It is sonication-free and immunoprecipitation-free and is scalable for profiling chromatin features at single cell level.Studies have shown that there are allelic differences in chromatin states,3D genome structures,and transcriptional activities in diploid and polyploid species.Hi-C and other Hi-C derivates are widely used in the study of plant 3D genome architecture.However,due to the low resolution of Hi-C technology,there are no reports of haplotype-resolved 3D genome maps in plants.It is difficult to explore the molecular regulation mechanisms of allele-specific expression(ASE)from the perspective of allele differential chromatin structures with Hi-C data.In this study,the haplotype-resolved epigenomes and 3D genomes of an elite rice hybrid were mapped.Based on the 3D genome maps,a chromosomal-level 3D genome model was reconstructed.The relationships between allele differential histone modification,allele differential chromatin interaction,and ASE were comprehensively investigated.The super silencers(SS)were systematically identified along the whole rice genome.A rapid and efficient ChIC-based epigenomic technology,nucleus CUT&Tag,and the singlenucleus CUT&Tag,were established in plants.The relevant findings are as follows:(1)Haplotype-resolved epigenomes of an elite rice hybrid were mapped.The results showed that allelic preference of active chromatin features(such as H3K4me3,H3K4me1,H3K27 ac and RNAPII)was positively correlated with the preference of ASE,while the allelic preference of repressive histone modifications(such as H3K27me3 and H3K9me2)is negatively correlated with the preference of ASE.(2)Haplotype mapping the chromatin interactions found extensive allele differential chromatin interactions in an elite rice hybrid,and revealed the molecular regulation mechanism of allele-specific chromatin interactions on ASE.(3)Super silencers of rice have been systematically identified.A chromatin loop between a silencer and the panicle-related gene FZP was identified,suggesting that distal silencers could regulate gene expression and genetic traits by forming chromatin loops and coming into proximity with gene promoters.Extensive SS-associated chromatin interactions were detected.Some SS were involved in allele differential chromatin conformation.In rice seedlings,9 MADS-box genes were modified by SS,and many other MADS-box genes were modified by typical H3K27me3 peaks.These MADS-box genes are co-localized,co-modified,and co-suppressed in the nucleus of rice seedlings and other tissues such as roots and mature leaves.However,the co-modification is erased or partially erased;the co-localization disappeared;and the co-suppression is activated and converted into co-suppression,in rice panicles.The coordinated expression shifting patterns of MADS-box gene family indicate that they may be synergistically involved in the development of rice floral organs and young panicles.(4)A 3D genome structure model of rice was reconstructed at chromosomal level.The spatial distance between each two of the 24 chromosomes in rice were calculated based on the chromatin interaction intensity.And then,a chromosomal-level 3D genome structure was remodeled based on the spatial distance matrix.The results showed that the rice nucleus is fold into a spherical configuration.The gene-rich and activelytranscribed chromosome 1 is located in the central area of the nucleus and has higher chromatin interaction intensity with other chromosomes,while other chromosomes are distributed on the peripheral regions in the nucleus.(5)A nucleus CUT&Tag(nCUT&Tag)method was developed in plants.nCUT&Tag is a ChIC-based method that eliminates the requirement of sonication and immunoprecipitation.It takes only 1 day to complete the entire process and can be used for chromatin profiling with very low-input materials(0.01 g of plant tissue).nCUT&Tag can be used to analyze active and repressive histone modifications in different tissues(formaldehyde-crosslinked or non-crosslinked)of the monocot rice and the dicot rapeseed.(6)Based on nCUT&Tag protocol,certain epigenomic techniques such as ATAC-seq,Stacc-seq,and single-nucleus CUT&Tag(snCUT&Tag)were developed in rice,providing useful tools for plant single-cell epigenomic and three-dimensional genomic studies.Taken together,in this study,the haplotype-resolved epigenomes and 3D genomes at single-base resolution were mapped in rice at the first time,and the molecular regulation mechanisms of allele-specific expression were elucidated.Super silencers and super silencer-associated chromatin interactions were globally identified in the rice genome.A chromosomal-resolution 3D genome model was reconstructed,and some useful epigenomic techniques such as nCUT&Tag and snCUT&Tag were established.These results provide useful tools for plant single-cell/nucleus epigenomic and 3D genomic research,and provide new insights for the understanding of the molecular mechanism of transcription regulation.