Genetic dissection of gene silencing mediated by H3K27 methylation
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ABSTRACT:
ORGANISM(S): Fusarium graminearum
PROVIDER: GSE131036 | GEO | 2020/01/01
REPOSITORIES: GEO
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Project description:Phenotype-driven forward genetic experiments are among the most powerful approaches for linking biology and disease to genomic elements. Although widely used in a range of model organisms, positional cloning of causal variants is still a very laborious process. Here, we describe a novel universal approach, named fast forward genetics that combines traditional bulk segregant techniques with next-generation sequencing technology and targeted genomic enrichment, to dramatically improve the process of mapping and cloning multiple mutants in a single experiment. In a two-step procedure the mutation is first roughly mapped by ‘light’ sequencing of the bulk segregant pool, followed by genomic enrichment and deep-sequencing of the mutant pool for the linked genomic region. The latter step allows for simultaneous fine-mapping and mutation discovery. We successfully applied this approach to three Arabidopsis mutants, but the method can in principle be applied to any model organism of interest and is largely independent of the genome size. Moreover, we show that both steps can be performed in multiplex using barcoded samples, thereby increasing efficiency enormously.
2010-12-31 | GSE24511 | GEO
Project description:Here we utilize a forward genetics approach in Neurospora crassa to identify novel effectors of Polycomb repression. We recovered several mutant alleles of a gene (NCU07505),which we deemed effector of Polycomb repression 1 (epr-1), encoding a protein with a bromo-adjacent homology (BAH) domain and plant homeodomain (PHD) finger. Although epr-1mutants display phenotypic and gene expression changes similar to strains lacking PRC2 components, H3K27 methylation is notably unaffected. We demonstrate that EPR-1 forms nuclear foci, reminiscent of Polycomb bodies {Pirrotta:2012br}, and its genomic distribution is limited to and dependent upon H3K27-methylated chromatin, which it recognizes through its BAH domain. Finally, we discover that EPR-1 homologs are widely distributed across eukaryotes, contrary to previous reports {LopezGonzalez:2014id, Li:2018kg}, suggesting an ancient role of EPR-1 homologs in Polycomb repression that was secondarily lost in select lineages.
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Project description:The aim of this study was to minimize the number of candidate genes responsible for salt tolerance between a pair of rice varieties (CSR27 and MI48) with contrasting level of salt tolerance by bulked segregant analysis of their recombinant inbred lines. Microarray analysis of RNA extracted from the tolerant and susceptible parents without and with stress showed 798 and 2407 differentially expressed genes, respectively. The number of differentially expressed genes was drastically reduced to 70 and 30, by pooling the RNAs from ten extreme tolerant and ten extreme susceptible RILs due to normalization of irrelevant differentially expressed genes between the parents.
2010-04-20 | GSE16108 | GEO
Project description:Genetic dissection of gene silencing mediated by H3K27 methylation
| PRJNA542356 | ENA
Project description:Constitutive heterochromatin in Arabidopsis thaliana is marked by repressive chromatin modifications including DNA methylation, histone 3 dimethylation at lysine 9 (H3K9me2), and monomethylation at lysine 27 (H3K27me1). The enzymes catalyzing DNA methylation and H3K9me2 have been identified and mutations in these proteins lead to the reactivation of silenced heterochromatic elements. The enzymes responsible for heterochromatic H3K27me1, in contrast, remain unknown. Here we show that the divergent SET-domain proteins ARABIDOPSIS TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATXR6 exhibit H3K27 monomethyltransferase activity and double mutants have reduced H3K27me1 in vivo and show partial heterochromatin decondensation. atxr5 atxr6 plants also show transcriptional activation of repressed heterochromatic elements. Interestingly, H3K9me2 and DNA methylation are unaffected in the double mutant. These results indicate that ATXR5 and ATXR6 form a novel class of H3K27 methyltransferases and that H3K27me1 represents a new pathway required for transcriptional repression in Arabidopsis. Comparison of methylation in wild type and ATXR6/ATXR6 mutants
2009-05-31 | E-GEOD-16071 | biostudies-arrayexpress
Project description:Polycomb complexes regulate cell-type specific gene expression programs through heritable silencing of target genes. Trimethylation of histone H3 lysine 27 (H3K27me3) is essential for this process. Perturbation of H3K36 is thought to interfere with H3K27me3. We show that mutants of Drosophila replication-dependent (H3.2K36R) or -independent (H3.3K36R) histone H3 genes generally maintain Polycomb silencing and reach later stages of development. In contrast, combined (H3.3K36RH3.2K36R) mutants display widespread Hox gene misexpression and fail to develop past the first larval instar. Chromatin profiling showed that the H3.2K36R mutation disrupts H3K27me3 levels broadly and significantly throughout silenced domains, whereas these regions are mostly unaffected in H3.3K36R animals. Analysis of H3.3 distributions showed that this histone is enriched at presumptive PREs (Polycomb Response Elements) located outside of silenced domains but relatively depleted from those inside. We conclude that H3.2 and H3.3 K36 residues collaborate to repress Hox genes via distinct genomic subcompartments and mechanisms.
2023-02-06 | GSE215017 | GEO
Project description:Phenotype-driven forward genetic experiments are among the most powerful approaches for linking biology and disease to genomic elements. Although widely used in a range of model organisms, positional cloning of causal variants is still a very laborious process. Here, we describe a novel universal approach, named fast forward genetics that combines traditional bulk segregant techniques with next-generation sequencing technology and targeted genomic enrichment, to dramatically improve the process of mapping and cloning multiple mutants in a single experiment. In a two-step procedure the mutation is first roughly mapped by ‘light’ sequencing of the bulk segregant pool, followed by genomic enrichment and deep-sequencing of the mutant pool for the linked genomic region. The latter step allows for simultaneous fine-mapping and mutation discovery. We successfully applied this approach to three Arabidopsis mutants, but the method can in principle be applied to any model organism of interest and is largely independent of the genome size. Moreover, we show that both steps can be performed in multiplex using barcoded samples, thereby increasing efficiency enormously. Inducible overexpression of the RETINOBLASTOMA-RELATED (RBR-OE) gene in Arabidopsis roots causes the complete differentiation of stem cells and premature differentiation of daughter cells, leading to a full exhaustion of the primary root meristem. In order to identify regulators of RBR function in cell differentiation, RBR-OE plants in the Columbia background (Col0) were treated with EMS mutagenesis and a set of genetic suppressors of RBR-OE, which restores root growth capacity, were isolated. In this study, we used one the identified suppressor lines, which segregated as a recessive mutation. Mapping populations were generated by outcrossing to Ler ecotype. Seedlings from the F2 population were grown for 15 days post germination (dpg). A pool of 60 seedlings each with a clear suppressor phenotype (homozygous for suppressor mutation) and of 60 seedlings showing RBOE phenotype (Heterozygous for the suppressor mutation) were prepared and genomic DNA was isolated with the RNeasy Plant Mini Kit from QIAGEN according to manufacturer's protocol. The other two, mutants 136 and 193 were obtained in fluorescence based mutant screen and a QCmarker based mutagenesis, respectively. Mutants were generated by chemical mutagenesis (EMS) in Colombia (Col) genetic background. Mutants were subsequently crossed to the Landsberg (Ler) ecotype to create the mapping populations. Bulk-segregant pools of about 200 mutant as well as wild-type plants were generated for every mutant line.
2010-12-31 | E-GEOD-24511 | biostudies-arrayexpress
Project description:af09_lignin - dml6 - Transcriptome analysis of lignin mutants and UV stress effect on secondary wall synthesis - DML6 vs S DML6+UV vs S+UV S vs S+UV DML6 vs DML6+UV Keywords: normal vs disease comparison,treated vs untreated comparison
2007-06-20 | GSE8094 | GEO
Project description:Trimethylation of histone H3 lysine 27 (H3K27me3) plays a critical role in regulating gene expression during plant and animal development. We characterized the genome-wide distribution of H3K27me3 in five developmentally distinct tissues in maize plants of two genetic backgrounds, B73 and Mo17, representatives of two distinct heterotic groups. There are numerous differences in the distribution of H3K27me3 among tissues. In contrast, we found the distribution of H3K27me3 among the two genetic backgrounds to be quite similar. The tissue-specific patterns of H3K27me3 are often associated with differences in gene expression among the tissues and most of the imprinted genes that are expressed solely from the paternal allele in endosperm are targets of H3K27me3. Many maize genes with important developmental roles, including numerous genes encoding putative transcription factors, are modified with H3K27me3 in at least one of the tissues that were profiled. A comparison of the H3K27me3 targets in rice, maize, and Arabidopsis provided evidence for conservation of the H3K27me3 targets among plant species. However, there was limited evidence for conserved targeting of H3K27me3 in the two maize subgenomes derived from whole genome duplication. Genomic profiling of H3K27me3 in loss-of-function mutant stocks for Mez2 and Mez3, two of the three putative H3K27me3 methyltranferases present in the maize genome, suggests partial redundancy of this gene family for maintaining H3K27me3 patterns. Only a portion of the targets of H3K27me3 requires Mez2 and/or Mez3 and there was limited evidence for functional consequences of H3K27me3 at these targets. This study provides a catalogue of 6,337 genes that are marked by H3K27me3 in five maize tissues. B73 tissues for h3k27 analysis: Samples are replicated in triplicate with antibody pulldown (IP) and control genomic DNA (input) channels. histone modification mutants for h3k27 analysis: Samples are replicated in triplicate with antibody pulldown (IP) and control genomic DNA (input) channels. Mo17 tissues for h3k27 analysis: Samples contain antibody pulldown (IP) and control genomic DNA (input) channels.
2013-02-20 | E-GEOD-39456 | biostudies-arrayexpress
Project description:The aim of this study was to minimize the number of candidate genes responsible for salt tolerance between a pair of rice varieties (CSR27 and MI48) with contrasting level of salt tolerance by bulked segregant analysis of their recombinant inbred lines. Microarray analysis of RNA extracted from the tolerant and susceptible parents without and with stress showed 798 and 2407 differentially expressed genes, respectively. The number of differentially expressed genes was drastically reduced to 70 and 30, by pooling the RNAs from ten extreme tolerant and ten extreme susceptible RILs due to normalization of irrelevant differentially expressed genes between the parents. RNA from CSR27, MI48, tolerant bulk and susceptible bulk grown under control and salt stress conditions were analysed in two different biological replications (A and B) making total sixteen samples
2010-04-28 | E-GEOD-16108 | biostudies-arrayexpress