H3K27me3-rich genomic regions can function as silencers to repress gene expression via chromatin interactions

The mechanisms underlying gene repression and silencers are poorly understood. Here we investigate the hypothesis that H3K27me3-rich regions of the genome, defined from clusters of H3K27me3 peaks, may be used to identify silencers that can regulate gene expression via proximity or looping. We find that H3K27me3-rich regions are associated with chromatin interactions and interact preferentially with each other. H3K27me3-rich regions component removal at interaction anchors by CRISPR leads to upregulation of interacting target genes, altered H3K27me3 and H3K27ac levels at interacting regions, and altered chromatin interactions. Chromatin interactions did not change at regions with high H3K27me3, but regions with low H3K27me3 and high H3K27ac levels showed changes in chromatin interactions. Cells with H3K27me3-rich regions knockout also show changes in phenotype associated with cell identity, and altered xenograft tumor growth. Finally, we observe that H3K27me3-rich regions-associated genes and long-range chromatin interactions are susceptible to H3K27me3 depletion. Our results characterize H3K27me3-rich regions and their mechanisms of functioning via looping.


Statistics
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Software and code
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Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.
Melissa Jane Fullwood and Greg Tucker-Kellogg Dec 8, 2020 No software was used.
RNA-Seq, ChIP-Seq & 4Cdata analysis. For reads of RNA-seq and ChIP-seq, adptors are trimmed off by trimmomatic (0.38) with option 'TruSeq3-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36' and retained only those properly-paired reads after trimming. RNA-Seq reads of HAP1 EZH2KO/WT and K562 GSK343/DMSO were analysis with kallisto (0.44.0) with option '-b 100'. Differentially expressed genes were called using sleuth (0.29.0) with gene-level aggregation and wald test. ChIP-Seq reads of H3K2me3, H3K27ac and H3K4me3 were mapped by BOWTIE2 (v2.2.5) using default parameters in pair-end mode and filter out alignment with a mapq score smaller than 30. The two replicates were combined and peaks and bigWig files were generated by MACS2 (2.1.0.20150731) using option '-q 0.01' for H3K27ac and H3K4me3 and '--broad --broad-cutoff 0.1 -q 0.05' for H3K27me3. 4C reads were trimmed off HindIII digestion site using tagdust (2.33) and only those remained paired were mapped by BOWTIE2 (v2.2.5) with option '--end-to-end' in single-end mode. R3Cseq (1.24.0) was used to call significant interactions against Hind III digested genome background with a cut-off p value of 0.05. Significant interactions of two replicates were pooled and only one of the duplicated interacting regions was retained. Definition of H3K27me3-rich regions(MRRs). H3K27me3 ChIP-Seq signal and peaks were obtained from ENCODE, and used as inputs of an in-house customized script (https://bitbucket.org/YichaoCai/rose_strict_share/src/master/ ) that mimic the signal calculation of the ROSE (0.1) package. Missing H3K27me3 peaks from ENCODE were called using MACS2 (2.1.0.20150731)1with pooled replicates using option "--broad -q 0.05". First, ChIP-Seq peaks of H3K27me3 were stitched using a window size of 4 kb. After stitching, the treatment and control signal of the stitched peaks were calculated and used in the ranking in MRR calling. Super-enhancers were called in a similar manner except that a stitching window of 12.5 kb was used. Hi-C and 4C interactions were drawn in arc style using Sushi (1.16.0) from Bioconductor. Tracks of ChIP-Seq signal and peaks were generated by Gviz (1.22.3). Genomic feature enrichment analysis was performed using R package annotatr (1.8.0). Gene ontology, pathway enrichment analysis (REACTOME & KEGG) and map-view representation of enriched pathways were performed using R package clusterProfiler (3.10.1) and ReactomeRA (1.26.0).

October 2018
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

No data was excluded
For the RNA-Seq, ChIP-Seq and Hi-C data that was used, we performed RT-qPCR, ChIP-qPCR and 4C respectively in order to verify the data. In addition, a minimum of 3 replicates was used for the Reverse Transcriptase-quantitative PCR and Western Blot data, and a minimum The samples were prepared from experimental treatments where a cell line was treated with a drug or subjected to CRISPR. Covariates therefore were kept the same because the control cells came from the same cell line.
Blinding was not possible here because we performed experimental treatments, and one investigator prepared, collected and analyzed the experimental treatments. Dilution protocol of the antibodies can be found in the Methods section.
H3K4me3 (#ab8580, Abcam) (https://www.abcam.com/histone-h3-tri-methyl-k4-antibody-chip-grade-ab8580.html) Validation: Chromatin was prepared from U-2 OS (Human bone osteosarcoma epithelial cell line) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25 µg of chromatin, 2 µg of ab8580 (blue), and 20 µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.
H3K27ac (#ab4729, Abcam) (https://www.abcam.com/histone-h3-acetyl-k27-antibody-chip-grade-ab4729.html? productWallTab=ShowAll#top-653) Validation: Chromatin was prepared from HeLa (Human epithelial cell line from cervix adenocarcinoma) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25 µg of chromatin, 2 µg of ab4729 (blue), and 20 µl of Protein A/G sepharose beads.No antibody was added to the beads control (yellow).The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.
total H3 (abcam ab1791) (https://www.abcam.com/histone-h3-antibody-nuclear-loading-control-and-chip-grade-ab1791.html) Validation: All lanes : Anti-Histone H3 antibody -Nuclear Loading Control and ChIP Grade (ab1791)  Note that full information on the approval of the study protocol must also be provided in the manuscript.

ChIP-seq Data deposition
Confirm that both raw and final processed data have been deposited in a public database such as GEO.
Confirm that you have deposited or provided access to graph files (e.g. BED files) for the called peaks.

Data access links
May remain private before publication. Female CB17 SCID mice (6-8 weeks old) were used in this study. Mice were purchased from InVivos, Singapore and fed with standard laboratory diet and distilled water ad libitum. The animals were kept on a 12 h light/dark cycle at 22 ± 2°C in individually ventilated caging system with 50-65% humidity in the Biological Resource Centre, A-Star, Singapore.