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SRX1526953: GSM2036545: H3K9me2_Col; Arabidopsis thaliana; ChIP-Seq
1 ILLUMINA (Illumina HiSeq 2000) run: 7M spots, 667.2M bases, 463.2Mb downloads

Submitted by: NCBI (GEO)
Study: REF6 recognizes specific DNA sequence to regulate H3K27me3 demethylation and organ boundary formation
show Abstracthide Abstract
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. Overall design: 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.
Sample: H3K9me2_Col
SAMN04395679 • SRS1244775 • All experiments • All runs
Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Selection: ChIP
Layout: SINGLE
Construction protocol: Chromatin Immunoprecipitation (ChIP) assays were performed using 1 g 12-day-old seedlings as previously described with minor modifications (Lu F. et al., 2010, Cell research 20(3):387-390).Antibodies used in ChIP were anti-H3K27me3 (Millipore, 07-449) and anti-HA (Sigma, H6908). The ChIP-Seq libraries were constructed using NEBNext® DNA Library Prep Master Mix Set for Illumina® (NEB, E6040S). ChIPed DNA was ligated with Illumina single-end genome sequencing adaptors, size fractionated to obtain 300-bp fragments
Experiment attributes:
GEO Accession: GSM2036545
Runs: 1 run, 7M spots, 667.2M bases, 463.2Mb
Run# of Spots# of BasesSizePublished


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