RESUMEN
Photoperiods integrate with the circadian clock to coordinate gene expression rhythms and thus ensure plant fitness to the environment. Genome-wide characterization and comparison of rhythmic genes under different light conditions revealed delayed phase under constant darkness (DD) and reduced amplitude under constant light (LL) in rice. Interestingly, ChIP-seq and RNA-seq profiling of rhythmic genes exhibit synchronous circadian oscillation in H3K9ac modifications at their loci and long non-coding RNAs (lncRNAs) expression at proximal loci. To investigate how gene expression rhythm is regulated in rice, we profiled the open chromatin regions and transcription factor (TF) footprints by time-series ATAC-seq. Although open chromatin regions did not show circadian change, a significant number of TFs were identified to rhythmically associate with chromatin and drive gene expression in a time-dependent manner. Further transcriptional regulatory networks mapping uncovered significant correlation between core clock genes and transcription factors involved in light/temperature signaling. In situ Hi-C of ZT8-specific expressed genes displayed highly connected chromatin association at the same time, whereas this ZT8 chromatin connection network dissociates at ZT20, suggesting the circadian control of gene expression by dynamic spatial chromatin conformation. These findings together implicate the existence of a synchronization mechanism between circadian H3K9ac modifications, chromatin association of TF and gene expression, and provides insights into circadian dynamics of spatial chromatin conformation that associate with gene expression rhythms.
Asunto(s)
Ritmo Circadiano , Oryza , Cromatina/genética , Relojes Circadianos/genética , Ritmo Circadiano/genética , Epigenoma , Perfilación de la Expresión Génica , Oryza/genética , Oryza/fisiología , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Epigenetic modifications that exhibit circadian oscillations also promote circadian oscillations of gene expression. Brassica napus is a heterozygous polyploid species that has undergone distant hybridization and genome doubling events and has a young and distinct species origin. Studies incorporating circadian rhythm analysis of epigenetic modifications can offer new insights into differences in diurnal oscillation behavior among subgenomes and the regulation of diverse expressions of homologous gene rhythms in biological clocks. RESULTS: In this study, we created a high-resolution and multioscillatory gene expression dataset, active histone modification (H3K4me3, H3K9ac), and RNAPII recruitment in Brassica napus. We also conducted the pioneering characterization of the diurnal rhythm of transcription and epigenetic modifications in an allopolyploid species. We compared the evolution of diurnal rhythms between subgenomes and observed that the Cn subgenome had higher diurnal oscillation activity in both transcription and active histone modifications than the An subgenome. Compared to the A subgenome in Brassica rapa, the An subgenome of Brassica napus displayed significant changes in diurnal oscillation characteristics of transcription. Homologous gene pairs exhibited a higher proportion of diurnal oscillation in transcription than subgenome-specific genes, attributed to higher chromatin accessibility and abundance of active epigenetic modification types. We found that the diurnal expression of homologous genes displayed diversity, and the redundancy of the circadian system resulted in extensive changes in the diurnal rhythm characteristics of clock genes after distant hybridization and genome duplication events. Epigenetic modifications influenced the differences in the diurnal rhythm of homologous gene expression, and the diurnal oscillation of homologous gene expression was affected by the combination of multiple histone modifications. CONCLUSIONS: Herein, we presented, for the first time, a characterization of the diurnal rhythm characteristics of gene expression and its epigenetic modifications in an allopolyploid species. Our discoveries shed light on the epigenetic factors responsible for the diurnal oscillation activity imbalance between subgenomes and homologous genes' rhythmic expression differences. The comprehensive time-series dataset we generated for gene expression and epigenetic modifications provides a valuable resource for future investigations into the regulatory mechanisms of protein-coding genes in Brassica napus.
Asunto(s)
Brassica napus , Brassica napus/genética , Poliploidía , Ritmo Circadiano/genética , Genoma de PlantaRESUMEN
DNA methylation plays an important role in gene regulation and genomic stability. However, large DNA hypomethylated regions known as DNA methylation valleys (DMVs) or canyons have also been suggested to serve unique regulatory functions, largely unknown in rice (Oryza sativa). Here, we describe the DMVs in rice seedlings, which were highly enriched with developmental and transcription regulatory genes. Further detailed analysis indicated that grand DMVs (gDMVs) might be derived from nuclear integrants of organelle DNA (NORGs). Furthermore, Domains Rearranged Methylase 2 (OsDRM2) maintained DNA methylation at short DMV (sDMV) shores. Epigenetic maps indicated that sDMVs were marked with H3K4me3 and/or H3K27me3, although the loss of DNA methylation had a negligible effect on histone modification within these regions. In addition, we constructed H3K27me3-associated interaction maps for homozygous T-DNA insertion mutant of the gene (osdrm2) and wild type (WT). From a global perspective, most (90%) compartments were stable between osdrm2 and WT plants. At a high resolution, we observed a dramatic loss of long-range chromatin loops in osdrm2, which suffered an extensive loss of non-CG (CHG and CHH, H = A, T, or C) methylation. From another viewpoint, the loss of non-CG methylation at sDMV shores in osdrm2 could disrupt H3K27me3-mediated chromatin interaction networks. Overall, our results demonstrated that DMVs are a key genomic feature in rice and are precisely regulated by epigenetic modifications, including DNA methylation and histone modifications. OsDRM2 maintained DNA methylation at sDMV shores, while OsDRM2 deficiency strongly affected three-dimensional (3D) genome architectures.
Asunto(s)
Metilación de ADN , Oryza , Metilación de ADN/genética , Cromatina/genética , Histonas/genética , Histonas/metabolismo , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Metiltransferasas/genética , ADN , Regulación de la Expresión Génica de las Plantas/genéticaRESUMEN
Histone post-translational modifications and transcription factors (TFs) play vital roles in regulating gene expression. A comprehensive understanding of transcriptional regulation requires genome-wide mapping of chromatin features such as histone modifications and TF binding sites. Here, we describe a detailed nucleus CUT&Tag (Cleavage Under Targets and Tagmentation) protocol, which is an antibody-guided in situ protein-DNA interaction mapping method using protein A/G fused Tn5 transposase. Compared with regular ChIP-seq in plants, nucleus CUT&Tag (nCUT&Tag) omits many steps such as sonication and immunoprecipitation, thus saving much time and making it possible to efficiently profile chromatin features from low-input and even single cells with higher signal-to-noise ratio.
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Cromatina , ADN , Cromatina/genética , Núcleo Celular , Anticuerpos , Sitios de UniónRESUMEN
Hierarchical chromatin structures are critical for transcriptional regulation and many biological processes. It has been widely known that the linear genome of many plants and animals is partitioned into various chromatin interacting domains or gene regulatory modules with specific chromatin features, such as H3K4me3-related active interacting domains, H3K27me3 or Polycomb-related repressive domains, and H3K9me2-related heterochromatin domains. ChIA-PET, which combines chromatin immunoprecipitation (ChIP) assay with proximity ligation, can detect gene contact networks that are connected by co-regulated genes by pulling down specific chromatin complexes using an antibody of interest. Here, we describe a detailed, long-read ChIA-PET protocol for mapping promoter-centered active gene modules in plants.
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Cromatina , Oryza , Animales , Cromatina/genética , Oryza/genética , Secuenciación de Inmunoprecipitación de Cromatina , Heterocromatina , Inmunoprecipitación de CromatinaRESUMEN
Histone modification H3K27me3 is an important chromatin mark that plays vital roles in repressing expression of developmental genes. Here, we construct high-resolution 3D genome maps using long-read chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) and characterize H3K27me3-associated chromatin interactions in an elite rice hybrid, Shanyou 63. We find that many H3K27me3-marked regions may function as silencer-like regulatory elements. The silencer-like elements can come into proximity with distal target genes via forming chromatin loops in 3D space of the nuclei, regulating gene silencing and plant traits. Natural and induced deletion of silencers upregulate expression of distal connected genes. Furthermore, we identify extensive allele-specific chromatin loops. We find that genetic variations alter allelic chromatin topology, thus modulating allelic gene imprinting in rice hybrids. In conclusion, the characterization of silencer-like regulatory elements and haplotype-resolved chromatin interaction maps provide insights into the understanding of molecular mechanisms underlying allelic gene silencing and plant trait controlling.
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Cromatina , Oryza , Cromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Oryza/genética , Haplotipos , Silenciador del GenRESUMEN
Characterizing genome-wide histone posttranscriptional modifications and transcriptional factor occupancy is crucial for deciphering their biological functions. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful method for genome-wide profiling of histone modifications and transcriptional factor-binding sites. However, the current ChIP-seq experimental procedure in plants requires significant material and several days for completion. CUT&Tag is an alternative method of ChIP-seq for low-sample and single-cell epigenomic profiling using protein A-Tn5 transposase fusion proteins (PAT). In this study, we developed a nucleus CUT&Tag (nCUT&Tag) protocol based on the live-cell CUT&Tag technology. Our results indicate that nCUT&Tag could be used for histone modifications profiling in both monocot rice and dicot rapeseed using crosslinked or fresh tissues. In addition, both active and repressive histone marks such as H3K4me3 and H3K9me2 can be identified using our nCUT&Tag. More importantly, all the steps in nCUT&Tag can be finished in only 1 day, and the assay can be performed with as little as 0.01 g of plant tissue as starting materials. Therefore, our results demonstrate that nCUT&Tag is an efficient alternative strategy for plant epigenomic studies.
RESUMEN
The complexity of the epigenome landscape and transcriptional regulation is significantly increased during plant polyploidization, which drives genome evolution and contributes to the increased adaptability to diverse environments. However, a comprehensive epigenome map of Brassica napus is still unavailable. In this study, we performed integrative analysis of five histone modifications, RNA polymerase II occupancy, DNA methylation, and transcriptomes in two B. napus lines (2063A and B409), and established global maps of regulatory elements, chromatin states, and their dynamics for the whole genome (including the An and Cn subgenomes) in four tissue types (young leaf, flower bud, silique, and root) of these two lines. Approximately 65.8% of the genome was annotated with different epigenomic signals. Compared with the Cn subgenome, the An subgenome possesses a higher level of active epigenetic marks and lower level of repressive epigenetic marks. Genes from subgenome-unique regions contribute to the major differences between the An and Cn subgenomes. Asymmetric histone modifications between homeologous gene pairs reflect their biased expression patterns. We identified a novel bivalent chromatin state (with H3K4me1 and H3K27me3) in B. napus that is associated with tissue-specific gene expression. Furthermore, we observed that different types of duplicated genes have discrepant patterns of histone modification and DNA methylation levels. Collectively, our findings provide a valuable epigenetic resource for allopolyploid plants.
Asunto(s)
Brassica napus/genética , Epigenoma/genética , Genoma de Planta/genética , Secuenciación de Inmunoprecipitación de Cromatina , Regulación de la Expresión Génica de las Plantas/genéticaRESUMEN
The eukaryotic genome has a hierarchical three-dimensional (3D) organization with functional implications for DNA replication, DNA repair, and transcriptional regulation. Over the past decade, scientists have endeavored to elucidate the spatial characteristics and functions of plant genome architecture using high-throughput chromatin conformation capturing technologies such as Hi-C, ChIA-PET, and HiChIP. Here, we systematically review current understanding of chromatin organization in plants at multiple scales. We also discuss the emerging opinions and concepts in 3D genome research, focusing on state-of-the-art 3D genome techniques, RNA-chromatin interactions, liquid-liquid phase separation, and dynamic chromatin alterations. We propose the application of single-cell/single-molecule multi-omics, multiway (DNA-DNA, DNA-RNA, and RNA-RNA interactions) chromatin conformation capturing methods, and proximity ligation-independent 3D genome-mapping technologies to explore chromatin organization structure and function in plants. Such methods could reveal the spatial interactions between trait-related SNPs and their target genes at various spatiotemporal resolutions, and elucidate the molecular mechanisms of the interactions among DNA elements, RNA molecules, and protein factors during the formation of key traits in plants.
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Genoma de Planta , Imagenología Tridimensional , Plantas/genética , Cromatina/química , Cromatina/genética , Conformación de Ácido Nucleico , ARN de Planta/genética , ARN de Planta/metabolismoRESUMEN
The linear genome of eukaryotes is partitioned into diverse chromatin states and packaged into a three-dimensional (3D) structure, which has functional implications in DNA replication, DNA repair, and transcriptional regulation. Over the past decades, research on plant functional genomics and epigenomics has made great progress, with thousands of genes cloned and molecular mechanisms of diverse biological processes elucidated. Recently, 3D genome research has gradually attracted great attention of many plant researchers. Herein, we briefly review the progress in genomic and epigenomic research in plants, with a focus on Arabidopsis and rice, and summarize the currently used technologies and advances in plant 3D genome organization studies. We also discuss the relationships between one-dimensional linear genome sequences, epigenomic states, and the 3D chromatin architecture. This review provides basis for future research on plant 3D genomics.
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Cromatina/genética , Epigenoma/genética , Genoma de Planta/genética , Genómica , Arabidopsis/genética , Cromatina/ultraestructura , Epigénesis Genética , Regulación de la Expresión Génica/genética , Oryza/genéticaRESUMEN
Epigenomic modifications are instrumental for transcriptional regulation, but comprehensive reference epigenomes remain unexplored in rice. Here, we develop an enhanced chromatin immunoprecipitation (eChIP) approach for plants, and generate genome-wide profiling of five histone modifications and RNA polymerase II occupancy with it. By integrating chromatin accessibility, DNA methylation, and transcriptome datasets, we construct comprehensive epigenome landscapes across various tissues in 20 representative rice varieties. Approximately 81.8% of rice genomes are annotated with different epigenomic properties. Refinement of promoter regions using open chromatin and H3K4me3-marked regions provides insight into transcriptional regulation. We identify extensive enhancer-like promoters with potential enhancer function on transcriptional regulation through chromatin interactions. Active and repressive histone modifications and the predicted enhancers vary largely across tissues, whereas inactive chromatin states are relatively stable. Together, these datasets constitute a valuable resource for functional element annotation in rice and indicate the central role of epigenomic information in understanding transcriptional regulation.
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Epigenoma , Epigenómica , Regulación de la Expresión Génica , Oryza/genética , Cromatina/metabolismo , Genoma de Planta , Código de Histonas/genética , Histonas/metabolismo , Anotación de Secuencia Molecular/métodos , Regiones Promotoras Genéticas , RNA-Seq , Secuencias Reguladoras de Ácidos NucleicosRESUMEN
Insight into high-resolution three-dimensional genome organization and its effect on transcription remains largely elusive in plants. Here, using a long-read ChIA-PET approach, we map H3K4me3- and RNA polymerase II (RNAPII)-associated promoter-promoter interactions and H3K9me2-marked heterochromatin interactions at nucleotide/gene resolution in rice. The chromatin architecture is separated into different independent spatial interacting modules with distinct transcriptional potential and covers approximately 82% of the genome. Compared to inactive modules, active modules possess the majority of active loop genes with higher density and contribute to most of the transcriptional activity in rice. In addition, promoter-promoter interacting genes tend to be transcribed cooperatively. In contrast, the heterochromatin-mediated loops form relative stable structure domains in chromatin configuration. Furthermore, we examine the impact of genetic variation on chromatin interactions and transcription and identify a spatial correlation between the genetic regulation of eQTLs and e-traits. Thus, our results reveal hierarchical and modular 3D genome architecture for transcriptional regulation in rice.
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Cromatina/metabolismo , Genoma de Planta , Heterocromatina/metabolismo , Oryza/genética , Proteínas de Plantas/metabolismo , Cromatina/genética , Regulación de la Expresión Génica de las Plantas , Heterocromatina/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Chromatin loops connect regulatory elements to their target genes. They serve as bridges between transcriptional regulation and phenotypic variation in mammals. However, spatial organization of regulatory elements and its impact on gene expression in plants remain unclear. Here, we characterize epigenetic features of active promoter proximal regions and candidate distal regulatory elements to construct high-resolution chromatin interaction maps for maize via long-read chromatin interaction analysis by paired-end tag sequencing (ChIA-PET). The maps indicate that chromatin loops are formed between regulatory elements, and that gene pairs between promoter proximal regions tend to be co-expressed. The maps also demonstrated the topological basis of quantitative trait loci which influence gene expression and phenotype. Many promoter proximal regions are involved in chromatin loops with distal regulatory elements, which regulate important agronomic traits. Collectively, these maps provide a high-resolution view of 3D maize genome architecture, and its role in gene expression and phenotypic variation.
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Cromatina/genética , Regulación de la Expresión Génica de las Plantas , Redes Reguladoras de Genes/genética , Sitios de Carácter Cuantitativo/genética , Zea mays/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Mapeo Cromosómico , Producción de Cultivos , Elementos de Facilitación Genéticos/genética , Epigénesis Genética , Epigenómica/métodos , Genoma de Planta/genética , Estudio de Asociación del Genoma Completo , Mutación , Fenotipo , Regiones Promotoras Genéticas/genéticaRESUMEN
Auxin response factors (ARFs) are an important family of proteins in auxin-mediated response, with key roles in various physiological and biochemical processes. To date, a genome-wide overview of the ARF gene family in citrus was not available. A systematic analysis of this gene family in citrus was begun by carrying out a genome-wide search for the homologs of ARFs. A total of 19 nonredundant ARF genes (CiARF) were found and validated from the sweet orange. A comprehensive overview of the CiARFs was undertaken, including the gene structures, phylogenetic analysis, chromosome locations, conserved motifs of proteins, and cis-elements in promoters of CiARF. Furthermore, expression profiling using real-time PCR revealed many CiARF genes, albeit with different patterns depending on types of tissues and/or developmental stages. Comprehensive expression analysis of these genes was also performed under two hormone treatments using real-time PCR. Indole-3-acetic acid (IAA) and N-1-napthylphthalamic acid (NPA) treatment experiments revealed differential up-regulation and down-regulation, respectively, of the 19 citrus ARF genes in the callus of sweet orange. Our comprehensive analysis of ARF genes further elucidates the roles of CiARF family members during citrus growth and development process.