Genome-wide profiling of SWI/SNF chromatin remodeler BRAHMA and Histone H3 lysine demethyalse Relative of Early Flowering 6 (REF6) in Arabidopsis [ChIP-Seq]
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ABSTRACT: BRAHMA (BRM) is a conserved SWI/SNF-type chromatin remodeling ATPase implicated in many key nuclear events. Histone H3 Lysine 27 (H3K27) demethylases specifically remove the repressive histone mark, trimethylation of H3K27 (H3K27me3). Both proteins are thought to play active roles in regulating gene activities at the chromatin level, but their genome-wide coordination remains to be determined. In Arabidopsis thaliana, RELATIVE OF EARLY FLOWERING 6 (REF6, also known as JMJ12) is the first identified plant H3K27 demethylase. Here, genome-wide analyses revealed that REF6 targets to thousands of genes across the Arabidopsis genome and co-localizes with BRM at more than 1,000 genes, many of which are genes involved in response to various stimuli, especially plant hormones. Loss of REF6 activity results in decreased BRM occupancy at hundreds of BRM-REF6 co-targets, indicating that REF6 is required for the recruitment of BRM to chromatin. Further, REF6 targets to genomic loci that contains the CTCTGTTT motif in vivo Examination of BRM occupancy in 14-day-old wt and ref6-1 seedlings. Examination of REF6 occupancy in 14-day-old wt and brm-1 seedlings. Examination of global RNA expression in 14-day-old wt, brm-1, ref6-1 and brm-1 ref6-1 seedlings. Examination of H3K27me3 profiles in 14-day-old wt, brm-1, ref6-1, and brm-1 ref6-1 seedlings.
Project description:BRAHMA (BRM) is a conserved SWI/SNF-type chromatin remodeling ATPase implicated in many key nuclear events. Histone H3 Lysine 27 (H3K27) demethylases specifically remove the repressive histone mark, trimethylation of H3K27 (H3K27me3). Both proteins are thought to play active roles in regulating gene activities at the chromatin level, but their genome-wide coordination remains to be determined. In Arabidopsis thaliana, RELATIVE OF EARLY FLOWERING 6 (REF6, also known as JMJ12) is the first identified plant H3K27 demethylase. Here, genome-wide analyses revealed that REF6 targets to thousands of genes across the Arabidopsis genome and co-localizes with BRM at more than 1,000 genes, many of which are genes involved in response to various stimuli, especially plant hormones. Loss of REF6 activity results in decreased BRM occupancy at hundreds of BRM-REF6 co-targets, indicating that REF6 is required for the recruitment of BRM to chromatin. Further, REF6 targets to genomic loci that contains the CTCTGTTT motif in vivo through its C2H2 zinger finger domains. Consistently, the two proteins activate the expression of a set of common genes in plant cells. Thus, this work demonstrates a genome-wide coordination between an H3K27me3 demethylase and a chromatin remodelling protein. Examination of global RNA expression in 14-day-old wt, brm-1, ref6-1 and brm-1 ref6-1 seedlings. Three biological replicates for each one.
Project description:By using ChIP-seq, we found that loss of BRM activity in developing seedlings leads to ectopic and increased H3K27me3 deposition at several hundred genes, indicating the critical role of BRM in preventing the inappropriate deposition of this histone mark. Removal of CLF in brm mutant could partcially suppress the increased H3K27me3. Examination of H3K27me3 in 14-day-old wt, brm, clf, and brm clf seedlings. Two biological replicates for each one.
Project description:By using ChIP-seq, we found that loss of BRM activity in developing seedlings leads to ectopic and increased H3K27me3 deposition at several hundred genes, indicating the critical role of BRM in preventing the inappropriate deposition of this histone mark. Unexpectedly, BRM also facilitates PcG function at some PcG targets, suggesting the requirement for BRM in the deposition of this mark at certain PcG target genes. Examination of H3K27me3 in 14-day-old wt and brm seedlings. Two biological replicates for each one.
Project description:BRAHMA (BRM) is a conserved SWI/SNF-type chromatin remodeling ATPase implicated in many key nuclear events. Histone H3 Lysine 27 (H3K27) demethylases specifically remove the repressive histone mark, trimethylation of H3K27 (H3K27me3). Both proteins are thought to play active roles in regulating gene activities at the chromatin level, but their genome-wide coordination remains to be determined. In Arabidopsis thaliana, RELATIVE OF EARLY FLOWERING 6 (REF6, also known as JMJ12) is the first identified plant H3K27 demethylase. Here, genome-wide analyses revealed that REF6 targets to thousands of genes across the Arabidopsis genome and co-localizes with BRM at more than 1,000 genes, many of which are genes involved in response to various stimuli, especially plant hormones. Loss of REF6 activity results in decreased BRM occupancy at hundreds of BRM-REF6 co-targets, indicating that REF6 is required for the recruitment of BRM to chromatin. Further, REF6 targets to genomic loci that contains the CTCTGTTT motif in vivo
Project description:BRAHMA (BRM) is a conserved SWI/SNF-type chromatin remodeling ATPase implicated in many key nuclear events. Histone H3 Lysine 27 (H3K27) demethylases specifically remove the repressive histone mark, trimethylation of H3K27 (H3K27me3). Both proteins are thought to play active roles in regulating gene activities at the chromatin level, but their genome-wide coordination remains to be determined. In Arabidopsis thaliana, RELATIVE OF EARLY FLOWERING 6 (REF6, also known as JMJ12) is the first identified plant H3K27 demethylase. Here, genome-wide analyses revealed that REF6 targets to thousands of genes across the Arabidopsis genome and co-localizes with BRM at more than 1,000 genes, many of which are genes involved in response to various stimuli, especially plant hormones. Loss of REF6 activity results in decreased BRM occupancy at hundreds of BRM-REF6 co-targets, indicating that REF6 is required for the recruitment of BRM to chromatin. Further, REF6 targets to genomic loci that contains the CTCTGTTT motif in vivo
Project description:BRAHMA (BRM) is a conserved SWI/SNF-type chromatin remodeling ATPase implicated in many key nuclear events. Histone H3 Lysine 27 (H3K27) demethylases specifically remove the repressive histone mark, trimethylation of H3K27 (H3K27me3). Both proteins are thought to play active roles in regulating gene activities at the chromatin level, but their genome-wide coordination remains to be determined. In Arabidopsis thaliana, RELATIVE OF EARLY FLOWERING 6 (REF6, also known as JMJ12) is the first identified plant H3K27 demethylase. Here, genome-wide analyses revealed that REF6 targets to thousands of genes across the Arabidopsis genome and co-localizes with BRM at more than 1,000 genes, many of which are genes involved in response to various stimuli, especially plant hormones. Loss of REF6 activity results in decreased BRM occupancy at hundreds of BRM-REF6 co-targets, indicating that REF6 is required for the recruitment of BRM to chromatin. Further, REF6 targets to genomic loci that contains the CTCTGTTT motif in vivo through its C2H2 zinger finger domains. Consistently, the two proteins activate the expression of a set of common genes in plant cells. Thus, this work demonstrates a genome-wide coordination between an H3K27me3 demethylase and a chromatin remodelling protein.
Project description:RELATIVE OF EARLY FLOWERING 6 (REF6, also known as JMJ12) counteracts Polycomb mediated gene silencing through demethylating histone H3 lysine 27 trimethylation (H3K27me3) in Arabidopsis. Genome-wide analysis has demonstrated that REF6 dependent H3K37me3 demethylation occurs on hundreds of genes. However, how these genes are selectively subjected to H3K27me3 demethylation remains elusive. Here we show that a tandem array of four Cys2-His2 zinc finger domains (C2H2-ZF) at REF6 C-terminus are essential for REF6 function. Mechanistically, we find that C2H2-ZF cluster can directly recognize a specific DNA sequence motif, and is essential for binding of REF6 to its targets. In addition, we demonstrate that CUP-SHAPED COTYLEDON 1 (CUC1) and CUC3 harbor such sequence motif and are direct targets of REF6; while their close homolog, CUC2, without such binding motif is not bound by REF6. Furthermore, REF6 is essential for proper H3K27me3 level at CUC1 locus, CUC1 activation and cotyledon separation. Collectively, our study reveals not only a novel mechanism of H3K27me3 demethylase genome targeting to counteract Polycomb silencing, but also a new function of H3K27me3 demethylation in organ boundary formation. All seeds, except for H3K9me2 ChIP-seq, were grown on 1/2MS plate at 23°C under long day condition. 12 day after germination (12DAG), whole seedling were harvested for ChIP-seq. anti-HA ChIP-seq were performed with three samples: Col (WT,Negtive control), REF6-HA(REF6p::REF6-HA ref6-1),REF6-ZnF-HA(REF6p::REF6-ZnF-HA ref6-1). anti-H3K27me3 ChIP-seq were performed with four samples: Col (WT), ref6-1 (mutant), REF6-HA(REF6p::REF6-HA ref6-1),REF6-ZnF-HA(REF6p::REF6-ZnF-HA ref6-1).For H3K9me2 ChIP-seq, plants were grown in soil at 23°C under long day condition. Aerial part of plants were harvested for ChIP-seq 28d after germination.
Project description:We report transcriptome changes and genome-wide dynamics of H3K27me3 during seed germination in Arabidopdsis, and investigate the impact of REF6-mediated H3K27 demethylation on germination. Compared with transcriptome changes, we discover delayed H3K27me3 reprogramming closely associated with the embryo to vegetative cell fate switch. REF6-mediated H3K27 demethylation promotes germination but does not significantly contribute to H3K27me3 dynamics during germination, but rather stably establishes an H3K27me3-depleted state permissive to transcription. By analyzing REF6 genomic binding, we show that it is absent from mature embryo chromatin and gradually establishes occupancy during the course of germination to counteract increased PRC2 activity.
Project description:We report transcriptome changes and genome-wide dynamics of H3K27me3 during seed germination in Arabidopdsis, and investigate the impact of REF6-mediated H3K27 demethylation on germination. Compared with transcriptome changes, we discover delayed H3K27me3 reprogramming closely associated with the embryo to vegetative cell fate switch. REF6-mediated H3K27 demethylation promotes germination but does not significantly contribute to H3K27me3 dynamics during germination, but rather stably establishes an H3K27me3-depleted state permissive to transcription. By analyzing REF6 genomic binding, we show that it is absent from mature embryo chromatin and gradually establishes occupancy during the course of germination to counteract increased PRC2 activity.
Project description:We report transcriptome changes and genome-wide dynamics of H3K27me3 during seed germination in Arabidopdsis, and investigate the impact of REF6-mediated H3K27 demethylation on germination. Compared with transcriptome changes, we discover delayed H3K27me3 reprogramming closely associated with the embryo to vegetative cell fate switch. REF6-mediated H3K27 demethylation promotes germination but does not significantly contribute to H3K27me3 dynamics during germination, but rather stably establishes an H3K27me3-depleted state permissive to transcription. By analyzing REF6 genomic binding, we show that it is absent from mature embryo chromatin and gradually establishes occupancy during the course of germination to counteract increased PRC2 activity.