The eukaryotic genome has a hierarchicalthree-dimensional(3D)organization with functional implications for DNA replication,DNA repair,and transcriptional regulation.Over the past decade,scientists have endeavored to e...The eukaryotic genome has a hierarchicalthree-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,ChlA-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 mecha-nisms of the interactions among DNA elements,RNA molecules,and protein factors during the formation of key traits in plants.展开更多
In plants,the genome structure of hybrids changes compared with their parents,but the effects of these changes in hybrids remain elusive.Comparing reciprocal crosses between Col×C24 and C24×Col in Arabidopsi...In plants,the genome structure of hybrids changes compared with their parents,but the effects of these changes in hybrids remain elusive.Comparing reciprocal crosses between Col×C24 and C24×Col in Arabidopsis using high-throughput chromosome conformation capture assay(Hi-C)analysis,we found that hybrid three-dimensional(3D)chromatin organization had more long-distance interactions relative to parents,and this was mainly located in promoter regions and enriched in genes with heterosis-related pathways.The interactions between euchromatin and heterochromatin were increased,and the compartment strength decreased in hybrids.In compartment domain(CD)boundaries,the distal interactions were more in hybrids than their parents.In the hybrids of CURLY LEAF(clf)mutants clfCol×clfC24and clfC24×clfCol,the heterosis phenotype was damaged,and the long-distance interactions in hybrids were fewer than in their parents with lower H3K27me3.ChIP-seq data revealed higher levels of H3K27me3 in the region adjacent to the CD boundary and the same interactional homo-trans sites in the wild-type(WT)hybrids,which may have led to more long-distance interactions.In addition,the differentially expressed genes(DEGs)located in the boundaries of CDs and loop regions changed obviously in WT,and the functional enrichment for DEGs was different between WT and clf in the longdistance interactions and loop regions.Our findings may therefore propose a new epigenetic explanation of heterosis in the Arabidopsis hybrids and provide new insights into crop breeding and yield increase.展开更多
The distribution of repetitive DNAs along chromosomes is one of the crucial elements for understanding the organization and the evolution of plant genomes. Using a modified genomic in situ hybridization （GISH） proce...The distribution of repetitive DNAs along chromosomes is one of the crucial elements for understanding the organization and the evolution of plant genomes. Using a modified genomic in situ hybridization （GISH） procedure, fluorescence in situ hybridization （FISH） with genomic DNA to their own chromosomes （called self-genomic in situ hybridization, self-GISH） was carried out in six selected plant species with different genome size and amount of repetitive DNA. Nonuniform distribution of the fluorescent labeled probe DNA was observed on the chromosomes of all the species that were tested. The signal patterns varied among species and were related to the genome size. The chromosomes of the small Arabidopsis genome were labeled almost only in the pericentromeric regions and the nucleolus organizer regions （NORs）. The signals in the relatively small genomes, rice, sorghum, and Brassica oleracea var. capitata L., were dispersed along the chromosome lengths, with a predominant distribution in the pericentromeric or proximal regions and some heterochromatic arms. All chromosomes of the large genomes, maize and barley, were densely labeled with strongly labeled regions and weakly labeled or unlabeled regions being arranged alternatively throughout the lengths. In addition, enhanced signal bands were shown in all pericentromeres and the NORs in B. oleracea var. capitata, and in all pericentromeric regions and certain intercalary sites in barley. The enhanced signal band pattern in barley was found consistent with the N-banding pattern of this species. The GISH with self-genomic DNA was compared with FISH with Cot-1 DNA in rice, and their signal patterns are found to be basically consistent. Our results showed that the self-GISH signals actually reflected the hybridization of genomic repetitive DNAs to the chromosomes, thus the self-GISH technique would be useful for revealing the distribution of the regions where repetitive DNAs concentrate along chromosomes and some chromatin differentiation associated with 展开更多
Understanding the regulatory networks for germ cell fate specification is necessary to developing strategies for improving the efficiency of germ cell production in vitro.In this study,we developed a coupled screening...Understanding the regulatory networks for germ cell fate specification is necessary to developing strategies for improving the efficiency of germ cell production in vitro.In this study,we developed a coupled screening strategy that took advantage of an arrayed bi-molecular fluorescence complementation(BiFC)platform for protein-protein interaction screens and epiblast-like cell(EpiLC)-induction assays using reporter mouse embryonic stem cells(mESCs).Investigation of candidate interaction partners of core human pluripotent factors OCT4,NANOG,KLF4 and SOX2 in EpiLC differentiation assays identified novel primordial germ cell(PGC)-inducing factors including BEN-domain(BEND/Bend)family members.Through RNA-seq,ChIP-seq,and ATAC-seq analyses,we showed that Bend5 worked together with Bend4 and helped mark chromatin boundaries to promote EpiLC induction in vitro.Our findings suggest that BEND/Bend proteins represent a new family of transcriptional modulators and chromatin boundary factors that participate in gene expression regulation during early germline development.展开更多
Recent progress in understanding higher-order chromatin organization in the nucleus has been considerable. From single gene to chromosome territory, realistic biophysical models can now accurately predict some of the ...Recent progress in understanding higher-order chromatin organization in the nucleus has been considerable. From single gene to chromosome territory, realistic biophysical models can now accurately predict some of the structural feature of cell nuclei. Despite growing evidence of a deterministic nuclear organization, the physiological consequence of spatial genome organization is still unclear. In the simple eukaryotic model, Saccharomyces cerevisiae, clear correlation between gene position and transcription has been established. In this review, we will focus on higher-order chromatin organization in yeast with respect to the nuclear envelope and nucleolus. In Arabidopsis thaliana, a model plant for which we have a complete genome sequence, chromosome territory （CT） arrangement and somatic homologous pairing in interphase nuclei seem to occur randomly. Since chromosomes containing nucleolar organizer regions associate more frequently to form a single nucleolar structure, as in yeast, the nucleolus seems to play a major role in organizing nuclear space. Recent findings have begun to elucidate how plant regulatory factors, such as chromatin remodeling or histone chaperones, affect the chromatin state of ribosomal DNA genes located in two distinct CTarrangements in the nucleus. The functional outcome of yeast nuclear organization allowed us to propose how nuclear organization might contribute to a novel type of epigenetic regulation： the spatial regulation of transcription.展开更多
The evolutionary conserved Polycomb Group(PcG)repressive system comprises two central protein complexes,PcG repressive complex 1(PRC1)and PRC2.These complexes,through the incorporation of histone modifications on chro...The evolutionary conserved Polycomb Group(PcG)repressive system comprises two central protein complexes,PcG repressive complex 1(PRC1)and PRC2.These complexes,through the incorporation of histone modifications on chromatin,have an essential role in the normal development of eukaryotes.In recent years,a significant effort has been made to characterize these complexes in the different kingdoms,and despite there being remarkable functional and mechanistic conservation,some key molecular principles have diverged.In this review,we discuss current views on the function of plant PcG complexes.We compare the composition of PcG complexes between animals and plants,highlight the role of recently identified plant PcG accessory proteins,and discuss newly revealed roles of known PcG partners.We also examine the mechanisms by which the repression is achieved and how these complexes are recruited to target genes.Finally,we consider the possible role of some plant PcG proteins in mediating local and long-range chromatin interactions and,thus,shaping chromatin 3D architecture.展开更多
Activation of regeneration upon tissue damages requires the activation of many developmental genes responsible for cell proliferation,migration,differentiation,and tissue patterning.Ample evidence revealed that the re...Activation of regeneration upon tissue damages requires the activation of many developmental genes responsible for cell proliferation,migration,differentiation,and tissue patterning.Ample evidence revealed that the regulation of chromatin organization functions as a crucial mechanism for establishing and maintaining cellular identity through precise control of gene transcription.The alteration of chromatin organization can lead to changes in chromatin accessibility and/or enhancer-promoter interactions.Like embryogenesis,each stage of tissue regeneration is accompanied by dynamic changes of chromatin organization in regeneration-responsive cells.In the past decade,many studies have been conducted to investigate the contribution of chromatin organization during regeneration in various tissues,organs,and organisms.A collection of chromatin regulators were demonstrated to play critical roles in regeneration.In this review,we will summarize the progress in the understanding of chromatin organization during regeneration in different research organisms and discuss potential common mechanisms responsible for the activation of regeneration response program.展开更多
Cell fate transition is a fascinating process involving complex dynamics of three-dimensional(3D)chromatin organization and phase separation,which play an essential role in cell fate decision by regulating gene expres...Cell fate transition is a fascinating process involving complex dynamics of three-dimensional(3D)chromatin organization and phase separation,which play an essential role in cell fate decision by regulating gene expression.Phase separation is increasingly being considered a driving force of chromatin folding.In this review,we have summarized the dynamic features of 3D chromatin and phase separation during physiological and pathological cell fate transitions and systematically analyzed recent evidence of phase separation facilitating the chromatin structure.In addition,we discuss current advances in understanding how phase separation contributes to physical and functional enhancerpromoter contacts.We highlight the functional roles of 3D chromatin organization and phase separation in cell fate transitions,and more explorations are required to study the regulatory relationship between 3D chromatin organization and phase separation.展开更多
In the nucleus, chromatin is folded into hierarchical architecture that is tightly linked to various nuclear functions. However, the underlying molecular mechanisms that confer these architectures remain incompletely ...In the nucleus, chromatin is folded into hierarchical architecture that is tightly linked to various nuclear functions. However, the underlying molecular mechanisms that confer these architectures remain incompletely understood. Here, we investigated the functional roles of H3 lysine 9 dimethylation(H3 K9 me2), one of the abundant histone modifications, in three-dimensional(3 D)genome organization. Unlike in mouse embryonic stem cells, inhibition of methyltransferases G9 a and GLP in differentiated cells eliminated H3 K9 me2 predominantly at A-type(active) genomic compartments, and the level of residual H3 K9 me2 modifications was strongly associated with B-type(inactive) genomic compartments. Furthermore, chemical inhibition of G9 a/GLP in mouse hepatocytes led to decreased chromatin-nuclear lamina interactions mainly at G9 a/GLP-sensitive regions, increased degree of genomic compartmentalization, and up-regulation of hundreds of genes that were associated with alterations of the 3 D chromatin. Collectively, our data demonstrated essential roles of H3 K9 me2 in 3 D genome organization.展开更多
Development of ChiP-chip and ChlP-seq technologies has allowed genome-wide high-resolution profiling of chromatin-associated marks and binding sites for epigenetic regulators. However, signals for directing epigenetic...Development of ChiP-chip and ChlP-seq technologies has allowed genome-wide high-resolution profiling of chromatin-associated marks and binding sites for epigenetic regulators. However, signals for directing epigenetic modifiers to their target sites are not understood. In this paper, we tested the hypothesis that genome location can affect the involvement of epigenetic regulators using Chromatin Charting （CC） Lines, which have an identical transgene construct inserted at different locations in the Arabidopsis genome. Four CC lines that showed evidence for epigenetic silencing of the luciferase reporter gene were transformed with RNAi vectors individually targeting epigenetic regulators LHP1, MOM1, CMT3, DRD1, DRM2, SUVH2, CLF, and HD1. Involvement of a particular epigenetic regulator in silencing the transgene locus in a CC line was determined by significant alterations in luciferase expression after suppression of the regulator＇s expression. Our results suggest that the targeting of epigenetic regulators can be influenced by genome location as well as sequence context. In addition, the relative importance of an epigenetic regulator can be influenced by tissue identity. We also report a novel approach to predict interactions between epigenetic regulators through clustering analysis of the regulators using alterations in gene expression of putative downstream targets, including endogenous loci and transgenes, in epigenetic mutants or RNAi lines. Our data support the existence of a complex and dynamic network of epigenetic regulators that serves to coordinate and control global gene expression in higher plants.展开更多
基金the National Natural Science Foundation of China(no.31771422 to X.L.,no.31771402 to G.L.)the National Key Research and Development Program of China(no.2016YFD0100904 to X.L.)the open funds of the National Key Laboratory of Crop Genetic Improvement(no.ZK201906 to X.L.).
文摘The eukaryotic genome has a hierarchicalthree-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,ChlA-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 mecha-nisms of the interactions among DNA elements,RNA molecules,and protein factors during the formation of key traits in plants.
基金supported by the Key Program of National Natural Science Foundation of China(32230006 to D.X.W.)the Shandong Development Fund of Science&Technology+2 种基金the Award of Natural Science Foundation of Shandong Province(ZR2021ZD30)China Postdoctoral Science Foundation(8206300443)Boya Postdoctoral Program of Peking University。
文摘In plants,the genome structure of hybrids changes compared with their parents,but the effects of these changes in hybrids remain elusive.Comparing reciprocal crosses between Col×C24 and C24×Col in Arabidopsis using high-throughput chromosome conformation capture assay(Hi-C)analysis,we found that hybrid three-dimensional(3D)chromatin organization had more long-distance interactions relative to parents,and this was mainly located in promoter regions and enriched in genes with heterosis-related pathways.The interactions between euchromatin and heterochromatin were increased,and the compartment strength decreased in hybrids.In compartment domain(CD)boundaries,the distal interactions were more in hybrids than their parents.In the hybrids of CURLY LEAF(clf)mutants clfCol×clfC24and clfC24×clfCol,the heterosis phenotype was damaged,and the long-distance interactions in hybrids were fewer than in their parents with lower H3K27me3.ChIP-seq data revealed higher levels of H3K27me3 in the region adjacent to the CD boundary and the same interactional homo-trans sites in the wild-type(WT)hybrids,which may have led to more long-distance interactions.In addition,the differentially expressed genes(DEGs)located in the boundaries of CDs and loop regions changed obviously in WT,and the functional enrichment for DEGs was different between WT and clf in the longdistance interactions and loop regions.Our findings may therefore propose a new epigenetic explanation of heterosis in the Arabidopsis hybrids and provide new insights into crop breeding and yield increase.
基金This work was supported by the National Natural Sciences Foundation of China (No. 39870423).
文摘The distribution of repetitive DNAs along chromosomes is one of the crucial elements for understanding the organization and the evolution of plant genomes. Using a modified genomic in situ hybridization （GISH） procedure, fluorescence in situ hybridization （FISH） with genomic DNA to their own chromosomes （called self-genomic in situ hybridization, self-GISH） was carried out in six selected plant species with different genome size and amount of repetitive DNA. Nonuniform distribution of the fluorescent labeled probe DNA was observed on the chromosomes of all the species that were tested. The signal patterns varied among species and were related to the genome size. The chromosomes of the small Arabidopsis genome were labeled almost only in the pericentromeric regions and the nucleolus organizer regions （NORs）. The signals in the relatively small genomes, rice, sorghum, and Brassica oleracea var. capitata L., were dispersed along the chromosome lengths, with a predominant distribution in the pericentromeric or proximal regions and some heterochromatic arms. All chromosomes of the large genomes, maize and barley, were densely labeled with strongly labeled regions and weakly labeled or unlabeled regions being arranged alternatively throughout the lengths. In addition, enhanced signal bands were shown in all pericentromeres and the NORs in B. oleracea var. capitata, and in all pericentromeric regions and certain intercalary sites in barley. The enhanced signal band pattern in barley was found consistent with the N-banding pattern of this species. The GISH with self-genomic DNA was compared with FISH with Cot-1 DNA in rice, and their signal patterns are found to be basically consistent. Our results showed that the self-GISH signals actually reflected the hybridization of genomic repetitive DNAs to the chromosomes, thus the self-GISH technique would be useful for revealing the distribution of the regions where repetitive DNAs concentrate along chromosomes and some chromatin differentiation associated with
基金the National Key R&D Program of China(2017YFA0102801)The National Natural Science Foundation of China(Grant Nos.31930058,31671540,32170802,and 31301082)+1 种基金Natural Science Foundation of Guangdong Province(2015B020228002,2017A030313093)Guangdong Basic and Applied Basic Research Foundation(2019A1515011422,2021A1515010759).
文摘Understanding the regulatory networks for germ cell fate specification is necessary to developing strategies for improving the efficiency of germ cell production in vitro.In this study,we developed a coupled screening strategy that took advantage of an arrayed bi-molecular fluorescence complementation(BiFC)platform for protein-protein interaction screens and epiblast-like cell(EpiLC)-induction assays using reporter mouse embryonic stem cells(mESCs).Investigation of candidate interaction partners of core human pluripotent factors OCT4,NANOG,KLF4 and SOX2 in EpiLC differentiation assays identified novel primordial germ cell(PGC)-inducing factors including BEN-domain(BEND/Bend)family members.Through RNA-seq,ChIP-seq,and ATAC-seq analyses,we showed that Bend5 worked together with Bend4 and helped mark chromatin boundaries to promote EpiLC induction in vitro.Our findings suggest that BEND/Bend proteins represent a new family of transcriptional modulators and chromatin boundary factors that participate in gene expression regulation during early germline development.
文摘Recent progress in understanding higher-order chromatin organization in the nucleus has been considerable. From single gene to chromosome territory, realistic biophysical models can now accurately predict some of the structural feature of cell nuclei. Despite growing evidence of a deterministic nuclear organization, the physiological consequence of spatial genome organization is still unclear. In the simple eukaryotic model, Saccharomyces cerevisiae, clear correlation between gene position and transcription has been established. In this review, we will focus on higher-order chromatin organization in yeast with respect to the nuclear envelope and nucleolus. In Arabidopsis thaliana, a model plant for which we have a complete genome sequence, chromosome territory （CT） arrangement and somatic homologous pairing in interphase nuclei seem to occur randomly. Since chromosomes containing nucleolar organizer regions associate more frequently to form a single nucleolar structure, as in yeast, the nucleolus seems to play a major role in organizing nuclear space. Recent findings have begun to elucidate how plant regulatory factors, such as chromatin remodeling or histone chaperones, affect the chromatin state of ribosomal DNA genes located in two distinct CTarrangements in the nucleus. The functional outcome of yeast nuclear organization allowed us to propose how nuclear organization might contribute to a novel type of epigenetic regulation： the spatial regulation of transcription.
基金This work was supported by a PID2019-106664GB-I00 grant from the Spanish Ministry of Science and Innovation.
文摘The evolutionary conserved Polycomb Group(PcG)repressive system comprises two central protein complexes,PcG repressive complex 1(PRC1)and PRC2.These complexes,through the incorporation of histone modifications on chromatin,have an essential role in the normal development of eukaryotes.In recent years,a significant effort has been made to characterize these complexes in the different kingdoms,and despite there being remarkable functional and mechanistic conservation,some key molecular principles have diverged.In this review,we discuss current views on the function of plant PcG complexes.We compare the composition of PcG complexes between animals and plants,highlight the role of recently identified plant PcG accessory proteins,and discuss newly revealed roles of known PcG partners.We also examine the mechanisms by which the repression is achieved and how these complexes are recruited to target genes.Finally,we consider the possible role of some plant PcG proteins in mediating local and long-range chromatin interactions and,thus,shaping chromatin 3D architecture.
基金supported by National Natural Science Foundation of China(W.W.,32270891).
文摘Activation of regeneration upon tissue damages requires the activation of many developmental genes responsible for cell proliferation,migration,differentiation,and tissue patterning.Ample evidence revealed that the regulation of chromatin organization functions as a crucial mechanism for establishing and maintaining cellular identity through precise control of gene transcription.The alteration of chromatin organization can lead to changes in chromatin accessibility and/or enhancer-promoter interactions.Like embryogenesis,each stage of tissue regeneration is accompanied by dynamic changes of chromatin organization in regeneration-responsive cells.In the past decade,many studies have been conducted to investigate the contribution of chromatin organization during regeneration in various tissues,organs,and organisms.A collection of chromatin regulators were demonstrated to play critical roles in regeneration.In this review,we will summarize the progress in the understanding of chromatin organization during regeneration in different research organisms and discuss potential common mechanisms responsible for the activation of regeneration response program.
基金This work was supported by grants from the National Natural Science Foundation of China(Grant Nos.31970811,31771639 and 32170798)the Guangdong Regenerative Medicine and Health of Guangdong Laboratory Frontier Exploration Project(2018GZR110105007)+6 种基金the Guangdong Innovative and Entrepreneurial Research Team Program(2016ZT06S029)Guangdong Basic and Applied Basic Research Foundation(2011B1515120063)to J.D.the Fundamental Research Funds for the Central Universities of Jinan University(Natural Science)(2162004)China Postdoctoral Science Foundation(2021M701441)China Postdoctoral Special Grant Foundation(2022T150269)Guangdong Basic and Applied Basic Research Foundation(2021A1515)Guangzhou Basic and Applied Basic Research Foundation(202201010961)to L.F..
文摘Cell fate transition is a fascinating process involving complex dynamics of three-dimensional(3D)chromatin organization and phase separation,which play an essential role in cell fate decision by regulating gene expression.Phase separation is increasingly being considered a driving force of chromatin folding.In this review,we have summarized the dynamic features of 3D chromatin and phase separation during physiological and pathological cell fate transitions and systematically analyzed recent evidence of phase separation facilitating the chromatin structure.In addition,we discuss current advances in understanding how phase separation contributes to physical and functional enhancerpromoter contacts.We highlight the functional roles of 3D chromatin organization and phase separation in cell fate transitions,and more explorations are required to study the regulatory relationship between 3D chromatin organization and phase separation.
基金supported by the National Key R&D Program of China(Grant No.2018YFC1003500 awarded to BW)the National Natural Science Foundation of China(Grant No.31771435 awarded to BW)
文摘In the nucleus, chromatin is folded into hierarchical architecture that is tightly linked to various nuclear functions. However, the underlying molecular mechanisms that confer these architectures remain incompletely understood. Here, we investigated the functional roles of H3 lysine 9 dimethylation(H3 K9 me2), one of the abundant histone modifications, in three-dimensional(3 D)genome organization. Unlike in mouse embryonic stem cells, inhibition of methyltransferases G9 a and GLP in differentiated cells eliminated H3 K9 me2 predominantly at A-type(active) genomic compartments, and the level of residual H3 K9 me2 modifications was strongly associated with B-type(inactive) genomic compartments. Furthermore, chemical inhibition of G9 a/GLP in mouse hepatocytes led to decreased chromatin-nuclear lamina interactions mainly at G9 a/GLP-sensitive regions, increased degree of genomic compartmentalization, and up-regulation of hundreds of genes that were associated with alterations of the 3 D chromatin. Collectively, our data demonstrated essential roles of H3 K9 me2 in 3 D genome organization.
文摘Development of ChiP-chip and ChlP-seq technologies has allowed genome-wide high-resolution profiling of chromatin-associated marks and binding sites for epigenetic regulators. However, signals for directing epigenetic modifiers to their target sites are not understood. In this paper, we tested the hypothesis that genome location can affect the involvement of epigenetic regulators using Chromatin Charting （CC） Lines, which have an identical transgene construct inserted at different locations in the Arabidopsis genome. Four CC lines that showed evidence for epigenetic silencing of the luciferase reporter gene were transformed with RNAi vectors individually targeting epigenetic regulators LHP1, MOM1, CMT3, DRD1, DRM2, SUVH2, CLF, and HD1. Involvement of a particular epigenetic regulator in silencing the transgene locus in a CC line was determined by significant alterations in luciferase expression after suppression of the regulator＇s expression. Our results suggest that the targeting of epigenetic regulators can be influenced by genome location as well as sequence context. In addition, the relative importance of an epigenetic regulator can be influenced by tissue identity. We also report a novel approach to predict interactions between epigenetic regulators through clustering analysis of the regulators using alterations in gene expression of putative downstream targets, including endogenous loci and transgenes, in epigenetic mutants or RNAi lines. Our data support the existence of a complex and dynamic network of epigenetic regulators that serves to coordinate and control global gene expression in higher plants.
