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Chromatin interaction analyses elucidate the roles of PRC2-bound silencers in mouse development

Nature Genetics volume 52, pages 264–272 (2020)Cite this article

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Abstract

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.

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Fig. 1: ChIA–PET analysis defines PRC2 interactome in mESCs.
Fig. 2: PRC2 mediates extensive chromatin looping in genes with low transcriptional activity.
Fig. 3: Intergenic anchors function as transcriptional silencers.
Fig. 4: Mice with PRC2-bound silencer deletion display pleiotropic developmental defects.
Fig. 5: Intergenic anchors exhibit a poised chromatin state and acquire enhancer signature during differentiation.

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Data availability

All data described in this study have been deposited in NCBI’s Gene Expression Omnibus under accession GSE120393. Source data for Figs. 2–4 and Extended Data Fig. 8 are provided with the paper.

Code availability

For ChIA–PET Utilities, the code is available at https://github.com/cheehongsg/CPU). For ChiaSigScaled, the code is available at https://github.com/cheehongsg/ChiaSigScaled.

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Acknowledgements

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.

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Author notes

  1. Wenbo Wang

    Present address: The National Research Center for Translational Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China

  2. These authors contributed equally: Chew Yee Ngan, Chee Hong Wong, Harianto Tjong.

Authors and Affiliations

  1. The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA

    Chew Yee Ngan, Chee Hong Wong, Harianto Tjong, Rachel L. Goldfeder, Hao He, Liang Gong, Meihong Li & Chia-Lin Wei

  2. The Jackson Laboratory, Bar Harbor, ME, USA

    Wenbo Wang, Barbara Urban, Vivek Philip & Stephen A. Murray

  3. US Department of Energy Joint Genome Institute, Walnut Creek, CA, USA

    Cindy Choi, Junyan Lin, Julianna Chow & Joanne Lim

  4. State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, People’s Republic of China

    Haoyi Wang

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Contributions

C.Y.N., C.H.W., H.T. and C.-L.W. designed the studies and wrote the manuscript. C.Y.N., C.C., J. Lin, J.C., J. Lim and M.L. performed wet lab experiments. C.H.W., H.T. and R.L.G. performed informatic analysis. C.H.W. led the development of the ChIA–PET data processing pipeline. W.W., B.U., H.H., V.P., S.A.M. and H.W. performed CRISPR–Cas9 knockout, phenotyping experiments and analysis. L.G. supported the validation experiments. All co-authors read and approved the manuscript.

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Correspondence to Chia-Lin Wei.

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Extended data

Extended Data Fig. 1 Reproducibility of PRC2 ChIA-PET analysis.

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)).

Extended Data Fig. 2 Extensive chromatin interactions between DREs and PRC2 bound genes.

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.

Extended Data Fig. 3 Experimental validation of intergenic silencers in vivo.

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.

Extended Data Fig. 4 Intergenic anchors deleted in the mouse KO strains by CRISPR-Cas9.

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.

Extended Data Fig. 5 Validation of KO.

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.

Extended Data Fig. 6 The loss of connectivity triggers genes reactivation.

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.

Extended Data Fig. 7 Upregulation of genes associated with si-Δchr7.

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.

Extended Data Fig. 8 Upregulation of genes associated with si-Δchr9.

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.

Source data

Extended Data Fig. 9 Histone profiles of PRC2 interaction anchors.

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.

Extended Data Fig. 10 Features of intergenic anchors in developmental stages.

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.

Supplementary information

Supplementary Information

Supplementary Tables 2, 6 and 11

Reporting Summary

Supplementary Tables

Supplementary Tables 1, 3–5, 7–10 and 12–14

Source data

Source Data Fig. 2

Statistical source data for Fig. 2c,d.

Source Data Fig. 3

Statistical source data for Fig. 3f.

Source Data Fig. 4

Statistical source data for Fig. 4f.

Source Data Extended Data Fig. 8

Statistical source data for Extended Data Fig. 8b.

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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

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  • DOI: https://doi.org/10.1038/s41588-020-0581-x

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