Lineage-specific gene expression is modulated by a balance between transcriptional activation and repression during animal development. Knowledge about enhancer-centered transcriptional activation has advanced considerably, but silencers and their roles in normal development remain poorly understood. Here, we performed chromatin interaction analyses of Polycomb repressive complex 2 (PRC2), a key inducer of transcriptional gene silencing, to uncover silencers, their molecular identity and associated chromatin connectivity. Systematic analysis of cis-regulatory silencer elements reveals their chromatin features and gene-targeting specificity. Deletion of certain PRC2-bound silencers in mice results in transcriptional derepression of their interacting genes and pleiotropic developmental phenotypes, including embryonic lethality. While some PRC2-bound elements function as silencers in pluripotent cells, they can transition into active tissue-specific enhancers during development, highlighting their regulatory versatility. Our study characterizes the molecular profile of silencers and their associated chromatin architectures, and suggests the possibility of targeted reactivation of epigenetically silenced genes.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Young, R. A. Control of the embryonic stem cell state. Cell 144, 940–954 (2011).
Cook, P. R. A model for all genomes: the role of transcription factories. J. Mol. Biol. 395, 1–10 (2010).
Saurin, A. J. et al. The human Polycomb group complex associates with pericentromeric heterochromatin to form a novel nuclear domain. J. Cell Biol. 142, 887–898 (1998).
Zhang, Y. et al. Chromatin connectivity maps reveal dynamic promoter-enhancer long-range associations. Nature 504, 306–310 (2013).
Chakalova, L. & Fraser, P. Organization of transcription. Cold Spring Harb. Perspect. Biol. 2, a000729 (2010).
Maston, G. A., Evans, S. K. & Green, M. R. Transcriptional regulatory elements in the human genome. Annu. Rev. Genomics Hum. Genet. 7, 29–59 (2006).
Ogbourne, S. & Antalis, T. M. Transcriptional control and the role of silencers in transcriptional regulation in eukaryotes. Biochem. J. 331, 1–14 (1998).
Feuerborn, A. & Cook, P. R. Why the activity of a gene depends on its neighbors. Trends Genet. 31, 483–490 (2015).
Boyer, L. A. et al. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441, 349–353 (2006).
Bracken, A. P., Dietrich, N., Pasini, D., Hansen, K. H. & Helin, K. Genome-wide mapping of Polycomb target genes unravels their roles in cell fate transitions. Genes Dev. 20, 1123–1136 (2006).
Chamberlain, S. J., Yee, D. & Magnuson, T. Polycomb repressive complex 2 is dispensable for maintenance of embryonic stem cell pluripotency. Stem Cells 26, 1496–1505 (2008).
Pasini, D., Bracken, A. P., Hansen, J. B., Capillo, M. & Helin, K. The Polycomb group protein Suz12 is required for embryonic stem cell differentiation. Mol. Cell Biol. 27, 3769–3779 (2007).
Faust, C., Lawson, K. A., Schork, N. J., Thiel, B. & Magnuson, T. The Polycomb-group gene eed is required for normal morphogenetic movements during gastrulation in the mouse embryo. Development 125, 4495–4506 (1998).
O’Carroll, D. et al. The Polycomb-group gene Ezh2 is required for early mouse development. Mol. Cell Biol. 21, 4330–4336 (2001).
Pasini, D., Bracken, A. P., Jensen, M. R., Lazzerini Denchi, E. & Helin, K. Suz12 is essential for mouse development and for EZH2 histone methyltransferase activity. EMBO J. 23, 4061–4071 (2004).
Tiwari, V. K. et al. PcG proteins, DNA methylation, and gene repression by chromatin looping. PLoS Biol. 6, 2911–2927 (2008).
Ogiyama, Y., Schuettengruber, B., Papadopoulos, G. L., Chang, J. M. & Cavalli, G. Polycomb-dependent chromatin looping contributes to gene silencing during Drosophila development. Mol. Cell 71, 73–88 e5 (2018).
Bantignies, F. et al. Polycomb-dependent regulatory contacts between distant Hox loci in Drosophila. Cell 144, 214–226 (2011).
Tiwari, V. K., Cope, L., McGarvey, K. M., Ohm, J. E. & Baylin, S. B. A novel 6C assay uncovers Polycomb-mediated higher order chromatin conformations. Genome Res. 18, 1171–1179 (2008).
Schoenfelder, S. et al. Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome. Nat. Genet. 47, 1179–1186 (2015).
Denholtz, M. et al. Long-range chromatin contacts in embryonic stem cells reveal a role for pluripotency factors and Polycomb proteins in genome organization. Cell Stem Cell 13, 602–616 (2013).
Vieux-Rochas, M., Fabre, P. J., Leleu, M., Duboule, D. & Noordermeer, D. Clustering of mammalian Hox genes with other H3K27me3 targets within an active nuclear domain. Proc. Natl Acad. Sci. USA 112, 4672–4677 (2015).
Wani, A. H. et al. Chromatin topology is coupled to Polycomb group protein subnuclear organization. Nat. Commun. 7, 10291 (2016).
Kundu, S. et al. Polycomb repressive complex 1 generates discrete compacted domains that change during differentiation. Mol. Cell 65, 432–446 e5 (2017).
Bonev, B. et al. Multiscale 3D genome rewiring during mouse neural development. Cell 171, 557–572 e24 (2017).
Tolhuis, B. et al. Interactions among Polycomb domains are guided by chromosome architecture. PLoS Genet. 7, e1001343 (2011).
Li, L. et al. Widespread rearrangement of 3D chromatin organization underlies Polycomb-mediated stress-induced silencing. Mol. Cell 58, 216–231 (2015).
Fullwood, M. J. et al. An oestrogen-receptor-alpha-bound human chromatin interactome. Nature 462, 58–64 (2009).
Margueron, R. et al. Role of the Polycomb protein EED in the propagation of repressive histone marks. Nature 461, 762–767 (2009).
Oksuz, O. et al. Capturing the onset of PRC2-mediated repressive domain formation. Mol. Cell 70, 1149–1162 e5 (2018).
Cruz-Molina, S. et al. PRC2 facilitates the regulatory topology required for poised enhancer function during pluripotent stem cell differentiation. Cell Stem Cell 20, 689–705 e9 (2017).
Rada-Iglesias, A. et al. A unique chromatin signature uncovers early developmental enhancers in humans. Nature 470, 279–283 (2011).
Zentner, G. E., Tesar, P. J. & Scacheri, P. C. Epigenetic signatures distinguish multiple classes of enhancers with distinct cellular functions. Genome Res. 21, 1273–1283 (2011).
Shin, H. et al. TopDom: an efficient and deterministic method for identifying topological domains in genomes. Nucleic Acids Res. 44, e70 (2016).
Dixon, J. R. et al. Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 485, 376–380 (2012).
Shen, X. et al. EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol. Cell 32, 491–502 (2008).
Brand, A. H., Breeden, L., Abraham, J., Sternglanz, R. & Nasmyth, K. Characterization of a “silencer” in yeast: a DNA sequence with properties opposite to those of a transcriptional enhancer. Cell 41, 41–48 (1985).
Gray, S. & Levine, M. Transcriptional repression in development. Curr. Opin. Cell Biol. 8, 358–364 (1996).
Imakaev, M. et al. Iterative correction of Hi-C data reveals hallmarks of chromosome organization. Nat. Methods 9, 999–1003 (2012).
Dickinson, M. E. et al. High-throughput discovery of novel developmental phenotypes. Nature 537, 508–514 (2016).
Meehan, T. F. et al. Disease model discovery from 3,328 gene knockouts by the international mouse phenotyping consortium. Nat. Genet. 49, 1231–1238 (2017).
Zerbino, D. R., Wilder, S. P., Johnson, N., Juettemann, T. & Flicek, P. R. The ensembl regulatory build. Genome Biol. 16, 56 (2015).
Chen, X. et al. Integration of external signaling pathways with the core transcriptional network in embryonic stem cells. Cell 133, 1106–1117 (2008).
Gardiner-Garden, M. & Frommer, M. CpG islands in vertebrate genomes. J. Mol. Biol. 196, 261–282 (1987).
Deaton, A. M. & Bird, A. CpG islands and the regulation of transcription. Genes Dev. 25, 1010–1022 (2011).
Ku, M. et al. Genomewide analysis of PRC1 and PRC2 occupancy identifies two classes of bivalent domains. PLoS Genet. 4, e1000242 (2008).
Visel, A., Minovitsky, S., Dubchak, I. & Pennacchio, L. A. VISTA Enhancer Browser–a database of tissue-specific human enhancers. Nucleic Acids Res. 35, D88–D92 (2007).
Arner, E. et al. Transcribed enhancers lead waves of coordinated transcription in transitioning mammalian cells. Science 347, 1010–1014 (2015).
Consortium, E. P. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).
Guan, C., Ye, C., Yang, X. & Gao, J. A review of current large-scale mouse knockout efforts. Genesis 48, 73–85 (2010).
Lloyd, K. C. A knockout mouse resource for the biomedical research community. Ann. N. Y. Acad. Sci. 1245, 24–26 (2011).
Osterwalder, M. et al. Enhancer redundancy provides phenotypic robustness in mammalian development. Nature 554, 239–243 (2018).
Shim, S., Kwan, K. Y., Li, M., Lefebvre, V. & Sestan, N. Cis-regulatory control of corticospinal system development and evolution. Nature 486, 74–79 (2012).
Sur, I. K. et al. Mice lacking a Myc enhancer that includes human SNP rs6983267 are resistant to intestinal tumors. Science 338, 1360–1363 (2012).
Kazanets, A., Shorstova, T., Hilmi, K., Marques, M. & Witcher, M. Epigenetic silencing of tumor suppressor genes: paradigms, puzzles, and potential. Biochim. Biophys. Acta 1865, 275–288 (2016).
Crea, F., Paolicchi, E., Marquez, V. E. & Danesi, R. Polycomb genes and cancer: time for clinical application? Crit. Rev. Oncol. Hematol. 83, 184–193 (2012).
Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).
Tang, Z. et al. CTCF-Mediated human 3D genome architecture reveals chromatin topology for transcription. Cell 163, 1611–1627 (2015).
Wang, W., Zhang, Y. & Wang, H. Generating mouse models using zygote electroporation of nucleases (ZEN) technology with high efficiency and throughput. Methods Mol. Biol. 1605, 219–230 (2017).
Tunster, S. J. Genetic sex determination of mice by simplex PCR. Biol. Sex Differ. 8, 31 (2017).
Kurbatova, N., Mason, J. C., Morgan, H., Meehan, T. F. & Karp, N. A. PhenStat: a tool kit for standardized analysis of high throughput phenotypic data. PLoS ONE 10, e0131274 (2015).
Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Series B Stat. Methodol. 57, 289–300 (1995).
Li, G. et al. ChIA–PET tool for comprehensive chromatin interaction analysis with paired-end tag sequencing. Genome Biol. 11, R22 (2010).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).
Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at https://arxiv.org/abs/1303.3997 (2013).
Paulsen, J., Rodland, E. A., Holden, L., Holden, M. & Hovig, E. A statistical model of ChIA–PET data for accurate detection of chromatin 3D interactions. Nucleic Acids Res. 42, e143 (2014).
Liu, T. Use model-based analysis of ChIP-Seq (MACS) to analyze short reads generated by sequencing protein–DNA interactions in embryonic stem cells. Methods Mol. Biol. 1150, 81–95 (2014).
Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360 (2015).
Anders, S., Pyl, P. T. & Huber, W. HTSeq–a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Durand, N. C. et al. Juicebox provides a visualization system for Hi-C contact maps with unlimited zoom. Cell Syst. 3, 99–101 (2016).
Servant, N. et al. HiTC: exploration of high-throughput ‘C’ experiments. Bioinformatics 28, 2843–2844 (2012).
We thank R. Tewhey and C. Robinett for their feedback and comments on the manuscript, J. Denegre for coordinating mouse KO model generation and A. Lau for assistance with art images. Research reported in this publication was partially supported by the 4DN (grant no. U54 DK107967) and ENCODE (grant no. UM1 HG009409) consortia. C.-L.W. is supported by NIGMS (grant no. R01 GM127531-01A1). C.-L.W. and C.Y.N. are supported by NCI under award no. P30CA034196. A portion of this work was conducted with support from the US Department of Energy Joint Genome Institute by the Office of Science of the US Department of Energy under contract no. DE-AC02-05CH11231.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Pearson correlation coefficient, r, between individual ChIA-PET replicates for EED (n=6), EZH2 (n=7), SUZ12 (n=11) and the combined PRC2 libraries between three subunits. See Supplementary Table 1 for sample details. b, PRC2 chromatin interactions and binding profile across chr4:139,536,779-140,286,920. Tracks from the top: BA interaction, PRC2 binding profiles and SA interactions. Y-axis: interaction frequency represented by PET counts. c. Distribution of interaction frequency among BA and SA interactions. Each box represents first quartile (bottom) and third quartile (top) with median in the middle. Whiskers represent data range defined as 1.5 times interquartile from median (Q2 ± 1.5*(Q3-Q1)).
a, Examples of the multiple co-occurred chromatin looping patterns (P-P, P-G, P-I and intra-G interactions) in the Wnt6-Ihh (chr1:74,751,523-74,968,999) and Hoxb (chr11:96,161,617-96,425,610) regions are shown from EED (red), EZH2 (purple), SUZ12 (blue) and PRC2 (black) ChIA-PET libraries, respectively. b, Percentages of genes exhibit single, 2-type, 3-type and all 4-type of interactions. For example, among the 4,372 genes with P-P interactions, 14% of them have all 4-type of interactions (P-P, P-I, P-G and intra-G looping). c, Proposed model on how DREs can connect to their target genes and function as either enhancers or silencers by binding to RNAPII or PRC2.
a, Schematic overview of generating heterozygous founder mice strains and ES clones carrying deletion in the intergenic anchors by CRISPR/Cas9. b, Schematic description of genotype strategy and primer design used in screening of KO mice and derived ES clones.
PRC2 interactions and binding profiles from 5 of the 6 KO regions (si-Δchr9 is shown in Fig. 3a). Selective genes connected by the KO regions through the PRC2 loops are labelled. Chromosome location (from top to bottom) are as follow; chr11:118,861,894-119,194,521, chr5:28,100,320-28,484,061, chr3:107,423,514-107,782,737, chr7:143,061,554-143,537,289 and chr2:18,568,747-19,024,016.
a, Genotype confirmation by Sanger sequencing of the PCR products for all six successfully generated KO clones. b, PCR genotyping of KO derived mES clones to confirm deletion (deleted region on chromosome 9) in si-Δchr9 derived F1 and G9 clones, in triplicate (only representative results are shown here) in two independent experiments.. Additional primer R26 was designed to confirm heteroallelic deletion. Panel on the right determination of the gender of the KO clones are XY while wild type ES line is XX (refers to Methods). c, Genotyping by PCR to confirm deletion (deleted region on chromosome 7) in si-Δchr7 derived mES D4 and F4 clones.
a, Heatmap showing connectivity in previous study using Hi-C and current study using ChIA-PET. Example shown is chr1:36,282,810-192,258,731. b, Topological-associated domain analysis showed no difference in si-Δchr9, si-Δchr7 compared to wildtype. c, Loss of connecting loops in si-Δchr7 clones D4 and F4. Shown are chr7:142,557,623-14,3646,256 and zoom in region chr7:143,127,114-14,3550,277. d, Genes expression of connected of si-Δchr7 and non-connected genes from flanking 500kb and 1Mb regions. Only clone D4 is shown. n indicates number of genes in each category. See details in Supplementary Table 8B.
PRC2 interaction and binding profiles of the 1 Mb Igf2/Kcnq1 imprinting region. The si-Δchr7 (chr7:143,440,438-143,450,716) is marked in red. Three of the 10 genes with P-I interactions to this KO region located 15.5 Mb upstream. b, Normalized RNA-seq counts of the connected genes in wild type (+/+) (n=3) and 2 independent homozygous KO (-/-) ES clones D4 (n=3) and F4 (n=3). Gm44732 has no expression. N indicates number of biologically independent samples.
a, Venn diagram of differentially upregulated genes in si-Δchr9 clones F1 and G9. Differentially expressed genes in homozygous KO (-/-) ES clones G9 (n=3) compared with wild type (+/+) ESC (n=3) shown in volcano plot (p-value vs. fold change). Dysregulated genes found in both F1 and G9 (red), F1 only (orange) and G9 only (blue) are color labelled. Selected genes with the most striking upregulation are labelled. b, Circos plot shows the inter-chromosomal connectivity (iPET counts > 10) between the KO allele with the 29 upregulated gene loci. c, The distribution of interaction frequencies between the si-Δchr9 KO silencer locus and random background #1 (Left) or #2 (Right). TIFs between si-Δchr9 and the dysregulated genes are shown as red lines.
a, Enrichment fold of four histone modifications, RNAPII and CTCF binding over input across ±10Kb of promoter (P) and Gene (G)- anchor regions. b, Enrichment of H3K4me3 and ATAC-seq profile across ± 10 Kb of the promoter (P), gene (G) and intergenic (I) interaction anchors.
a, Heat maps H3K27me3, H3K27ac, H3K9me3 normalized signals of the 1,800 I-anchors through progressive developmental stages of kidney, limbs, hindbrain and liver. The color scales represented the fold enrichment of the ChIP vs input at log2 scale. b, Expression of eRNA in distal regulatory elements (DREs) and those overlapped with PRC2-bound silencers. Each box represents first quartile (bottom) and third quartile (top) with median in the middle. Whiskers represent data range defined as 1.5 times interquartile from median (Q2 ± 1.5*(Q3-Q1)). Points above whiskers represent outliers.
About this article
Cite this article
Ngan, C.Y., Wong, C.H., Tjong, H. et al. Chromatin interaction analyses elucidate the roles of PRC2-bound silencers in mouse development. Nat Genet 52, 264–272 (2020). https://doi.org/10.1038/s41588-020-0581-x
Nature Methods (2021)
Nature Reviews Genetics (2021)
Nucleic Acids Research (2021)
H3K27me3-rich genomic regions can function as silencers to repress gene expression via chromatin interactions
Nature Communications (2021)
The corepressors GPS2 and SMRT control enhancer and silencer remodeling via eRNA transcription during inflammatory activation of macrophages
Molecular Cell (2021)