H3k27me3-Rich Genomic Regions Can Function As Silencers to Repress Gene Expression Via Chromatin Interactions
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ARTICLE https://doi.org/10.1038/s41467-021-20940-y OPEN H3K27me3-rich genomic regions can function as silencers to repress gene expression via chromatin interactions Yichao Cai 1,2,10, Ying Zhang1,10, Yan Ping Loh1, Jia Qi Tng1, Mei Chee Lim 1,3, Zhendong Cao1,4, Anandhkumar Raju5, Erez Lieberman Aiden6, Shang Li3,7, Lakshmanan Manikandan5, Vinay Tergaonkar5, ✉ ✉ Greg Tucker-Kellogg 2,8 & Melissa Jane Fullwood 1,5,9 1234567890():,; 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 inter- actions. 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 mechan- isms of functioning via looping. 1 Cancer Science Institute of Singapore, National University of Singapore, Singapore, Singapore. 2 Department of Biological Sciences, National University of Singapore, Singapore, Singapore. 3 Cancer and Stem Cell Biology Programme, Duke-NUS Medical School, Singapore, Singapore. 4 Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 5 Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), Proteos, Singapore. 6 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA. 7 Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. 8 Computational Biology Programme, National University of Singapore, Singapore, Singapore. 9 School of Biological Sciences, Nanyang Technological University, Singapore, Singapore. 10These ✉ authors contributed equally: Yichao Cai, Ying Zhang. email: [email protected]; [email protected] NATURE COMMUNICATIONS | (2021) 12:719 | https://doi.org/10.1038/s41467-021-20940-y | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-20940-y he 3-dimensional organization of our genomes is impor- PRC2 Chromatin Interaction Analysis with Paired-End Tag Ttant for gene regulation1–3. The genome is organized into sequencing (ChIA-PET) in mouse embryonic stem cells. They large Topologically-Associated Domains (TADs) and concluded that PRC2-bound looping anchors function as tran- chromatin interactions. Gene transcription is controlled by scriptional silencers suggesting that we can identify silencers transcription factors (TFs) that bind to enhancers and promoters through investigating chromatin interactions13. to regulate genes4. TFs can bind to proximal enhancers in the However, there is no consensus yet in terms of how to identify genome, and enhancers distal to genes can loop to gene pro- silencers. Notably, each of these methods identify different moters via chromatin interactions to activate gene expression3. genomic regions as silencers, raising the question of whether Cancer cells show altered chromatin interactions2,3 including there may be different classes of silencers. Moreover, current altered chromatin loops to key oncogenes such as TERT5. methods for identifying silencers are laborious and require By contrast, mechanisms for gene repression are much less well complicated bioinformatics analyses and/or genome-wide understood. Silencers are regions of the genome that are capable screening (Supplementary Table 2, “comparison of different of silencing gene expression. Silencers have been shown to exist in human silencer identification methods”). A simple, easy to per- the human genome, but are less well characterized than enhan- form method to identify silencers in the genome in a high- cers. Until now, there are only a few known experimentally throughput manner would be ideal. Further investigation is validated silencers that have been demonstrated to repress target needed to understand whether there are different classes of genes in vitro, such as the human synapsin I gene6, the human silencers and to characterize the roles of silencers in the genome. BDNF gene7 and the human CD4 gene8,9 (experimentally vali- The term “super-enhancer”30 has been used to describe clusters dated silencer examples are discussed in Supplementary Table 1). of H3K27ac peaks which show very high levels of H3K27ac or The reason for the paucity of known silencers in the literature is other transcription-associated factors such as mediators as that methods to identify human silencer elements in a genome- determined from ChIP-seq data. Super-enhancers have high wide manner are only starting to be developed now. Moreover, levels of chromatin interactions to target genes31, and are asso- the mechanism by which silencers can regulate distant genes is ciated with oncogenes in cancer cells32 and cell fate-associated still uncharacterized. Distant silencers are thought to loop over to genes in embryonic stem cells30. While more research needs to be target genes to silence them10,11, and this mechanism has been done to determine if super-enhancers are a distinctly different demonstrated in studies of polycomb-mediated chromatin loops entity from enhancers, super-enhancers are thought as strong in Drosophila12 and in mice13. but no such examples have been enhancers, and the definition has been useful in identifying genes characterized to date in humans. important for cell-type specification33. Polycomb Group (PcG) proteins including Polycomb Repres- We reason that “super-silencers” or “H3K27me3-rich regions sive Complexes, PRC1 and PRC2 are widely recognized to (MRRs)” from clusters of H3K27me3 peaks in the genome can be mediate gene silencing of developmental genes14. During the identified through H3K27me3 ChIP-seq, as how super-enhancers development process, PRC1 and PRC2 have the ability to are defined. We hypothesize that H3K27me3-rich regions may be orchestrate genome architecture and repress gene expression15. a useful concept in identifying genomic regions that contain There are two different types of genomic domains: active domains silencers which can repress target genes either in proximity or via and repressive domains, which regulate gene expression and long-range chromatin interactions. The target genes may be establish cellular identity. Genes involved in cell self-renewal are tumor suppressors in cancer cells, and also cell fate-associated contained within the active domains which are governed by genes that need to be turned off for differentiation to occur. super-enhancers, while genes specifying repressed lineage are Here, we show that MRRs can be identified using H3K27me3 organized within chromatin structures known as PcG domains16. ChIP-seq data. MRRs show dense chromatin interactions con- Moreover, intact PcG domains have been shown to be necessary necting to target genes and to other MRRs. CRISPR excision of to maintain the chromatin interaction landscape17,18. However, two examples of looping silencers leads to gene up-regulation, the mechanisms of PcG domain formation and PcG proteins indicating they are indeed bona fide silencers. CRISPR excision recruitment are not fully characterized yet19, which makes finding leads to changes in chromatin loops, histone modifications, and silencers more difficult. cell phenotype including cell adhesion, growth and differentia- PcG domains are marked by H3K27me3, which is deposited by tion. Initial histone modification states predict changes in chro- the catalytic component of PRC2 complex, mainly Enhancer of matin loops. Finally, EZH2 inhibition leads to changes in Zeste Homolog 2 (EZH2) and sometimes EZH120. H3K27me3 chromatin interactions and histone modifications at MRRs, and marks are associated with gene repression for cell type-specific MRR-associated gene up-regulation. Taken together, silencers genes. Unlike H3K9me3 which remains silenced all the time and identified from clustering H3K27me3 peaks regulate key epige- prevents multiple TFs from binding21, H3K27me3 still allows nomic, transcriptomic and phenotypic events in cells. these genes to be activated through TF binding in a different cell state22. H3K27me3 is known to be a characteristic of silencers18,23. Although large blocks of H3K27me3-marked loci Results have been observed in previous studies24–26, their roles in chro- Identification and characterization of H3K27me3-rich regions matin loops and consequent regulatory actions were not explored (MRRs) in the human genome. We identified highly H3K27me3- in these manuscripts. rich regions (MRRs) from cell lines using H3K27me3 ChIP-seq Recently, several studies have proposed methods to identity data34 in the following manner: we first identified H3K27me3 silencer elements in a genome-wide manner. Huang et al. defined peaks, then clustered nearby peaks, and ranked the