It has been reported that rice chromosome 4 has eight major heterochromatic knobs within the heterochromatic half and that this organization correlates with chromosomal-level transcriptional activity. To better unders...It has been reported that rice chromosome 4 has eight major heterochromatic knobs within the heterochromatic half and that this organization correlates with chromosomal-level transcriptional activity. To better understand this chromosomal organization, we created a model based on the statistical distribution of various types of gene models to divide chromosome 4 into 17 euchromatic and heterochromatic regions that correspond with the cytological staining. Fluorescence in-situ hybridization （FISH） experiments using a set of bacterial artificial chromosome （BAC） clones from chromosome 4 placed all 18 clones in the region predicted by the model. Elevated levels of H3K4 di- and tri-methylation detected by chromatin-immunoprecipitation （CHIP） on chip were correlated with euchromatic regions whereas lower levels of these two modifications were detected in heterochromatic regions. Small RNAs were more abundant in the heterochromatic regions. To validate these findings, H3K4 trimethylation, H3K9 acetylation, H4K12 acetylation, and H3K9 di- and tri-methylation of 19 individual genes were measured by ChlP-PCR. Genes in heterochromatic regions had elevated H3K9 di- and tri-methylation while genes in euchromatic regions had elevated levels of the other three modifications. We also assayed cytosine methylation of these genes using the restriction enzymes McrBC, Hapll, and Msp I. This analysis indicated that cytosines of transposable elements and some genes located in heterochromatic regions were methylated while cytosines of the other genes were unmethylated. These results suggest that local transcriptional activity may reflect the organization of the corresponding part of the chromosome. They also indicate that epigenetic regulation plays an important role in correlating chromosomal organization with transcriptional activity.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
Spatial organization of DNA within the nucleus is important for controlling DNA replication and repair,genetic recombination,and gene expression.Here,we present CRISPR-PIN,a CRISPR/dCas9-based tool that allows control...Spatial organization of DNA within the nucleus is important for controlling DNA replication and repair,genetic recombination,and gene expression.Here,we present CRISPR-PIN,a CRISPR/dCas9-based tool that allows control of gene Position in the Nucleus for the yeast Saccharomyces cerevisiae.This approach utilizes a cohesindockerin interaction between dCas9 and a perinuclear protein.In doing so,we demonstrate that a single gRNA can enable programmable interaction of nuclear DNA with the nuclear periphery.We demonstrate the utility of this approach for two applications:the controlled segregation of an acentric plasmid and the re-localization of five endogenous loci.In both cases,we obtain results on par with prior reports using traditional,more cumbersome genetic systems.Thus,CRISPR-PIN offers the opportunity for future studies of chromosome biology and gene localization.展开更多
文摘It has been reported that rice chromosome 4 has eight major heterochromatic knobs within the heterochromatic half and that this organization correlates with chromosomal-level transcriptional activity. To better understand this chromosomal organization, we created a model based on the statistical distribution of various types of gene models to divide chromosome 4 into 17 euchromatic and heterochromatic regions that correspond with the cytological staining. Fluorescence in-situ hybridization （FISH） experiments using a set of bacterial artificial chromosome （BAC） clones from chromosome 4 placed all 18 clones in the region predicted by the model. Elevated levels of H3K4 di- and tri-methylation detected by chromatin-immunoprecipitation （CHIP） on chip were correlated with euchromatic regions whereas lower levels of these two modifications were detected in heterochromatic regions. Small RNAs were more abundant in the heterochromatic regions. To validate these findings, H3K4 trimethylation, H3K9 acetylation, H4K12 acetylation, and H3K9 di- and tri-methylation of 19 individual genes were measured by ChlP-PCR. Genes in heterochromatic regions had elevated H3K9 di- and tri-methylation while genes in euchromatic regions had elevated levels of the other three modifications. We also assayed cytosine methylation of these genes using the restriction enzymes McrBC, Hapll, and Msp I. This analysis indicated that cytosines of transposable elements and some genes located in heterochromatic regions were methylated while cytosines of the other genes were unmethylated. These results suggest that local transcriptional activity may reflect the organization of the corresponding part of the chromosome. They also indicate that epigenetic regulation plays an important role in correlating chromosomal organization with transcriptional activity.
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.
基金This work was supported by the Camille Dreyfus Teacher-ScholarAward.
文摘Spatial organization of DNA within the nucleus is important for controlling DNA replication and repair,genetic recombination,and gene expression.Here,we present CRISPR-PIN,a CRISPR/dCas9-based tool that allows control of gene Position in the Nucleus for the yeast Saccharomyces cerevisiae.This approach utilizes a cohesindockerin interaction between dCas9 and a perinuclear protein.In doing so,we demonstrate that a single gRNA can enable programmable interaction of nuclear DNA with the nuclear periphery.We demonstrate the utility of this approach for two applications:the controlled segregation of an acentric plasmid and the re-localization of five endogenous loci.In both cases,we obtain results on par with prior reports using traditional,more cumbersome genetic systems.Thus,CRISPR-PIN offers the opportunity for future studies of chromosome biology and gene localization.
