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CTCF mediates dosage- and sequence-context-dependent transcriptional insulation by forming local chromatin domains

Nature Genetics volume 53, pages 1064–1074 (2021)Cite this article

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Abstract

Insulators play a critical role in spatiotemporal gene regulation in animals. The evolutionarily conserved CCCTC-binding factor (CTCF) is required for insulator function in mammals, but not all of its binding sites act as insulators. Here we explore the sequence requirements of CTCF-mediated transcriptional insulation using a sensitive insulator reporter in mouse embryonic stem cells. We find that insulation potency depends on the number of CTCF-binding sites in tandem. Furthermore, CTCF-mediated insulation is dependent on upstream flanking sequences at its binding sites. CTCF-binding sites at topologically associating domain boundaries are more likely to function as insulators than those outside topologically associating domain boundaries, independently of binding strength. We demonstrate that insulators form local chromatin domain boundaries and weaken enhancer–promoter contacts. Taken together, our results provide genetic, molecular and structural evidence connecting chromatin topology to the action of insulators in the mammalian genome.

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Fig. 1: A sensitive insulator reporter assay measures the insulation activity of different CBSs at the Sox2 locus in mESCs.
Fig. 2: Multiple CTCF sites in tandem enable strong transcriptional insulation.
Fig. 3: Synthetic insulators reveal sequence requirements for CTCF-mediated enhancer-blocking activity.
Fig. 4: Enhancer-blocking insulator forms local chromatin domains and reduces Sox2 enhancer–promoter chromatin contacts.
Fig. 5: Effects of an enhancer-blocking insulator on chromatin topology and transcription revealed by multiplexed FISH.

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Data availability

All next-generation sequencing data are available under GEO accession GSE153403. Raw images of multiplexed FISH experiments and raw FACS data for specific mESC colonies are available on request. Source data are provided with this paper.

Code availability

Code for analyses of multiplexed FISH data can be found on Github at https://github.com/epigen-UCSD/huang-natgen2021.

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Acknowledgements

We are grateful for comments from members of the Ren laboratory. This study was supported by funding from the Ludwig Institute for Cancer Research and NIH (U54 DK107977 and UM1HG011585, to B.R., X.Z., M.H. and M.N., and 3U54DK107977-05S1 to B.R.). Y.Z. was supported by 1K99CA252020-01. X.Z. is a Howard Hughes Medical Institute Investigator.

Author information

Authors and Affiliations

  1. Ludwig Institute for Cancer Research, La Jolla, CA, USA

    Hui Huang, Adam Jussila, Yanxiao Zhang, Miao Yu, Rong Hu & Bing Ren

  2. Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, CA, USA

    Hui Huang

  3. Center for Epigenomics, Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA

    Quan Zhu, Yuanyuan Han, Colin Kern & Bing Ren

  4. Bioinformatics and Systems Biology Graduate Program, University of California San Diego, La Jolla, CA, USA

    Adam Jussila

  5. Howard Hughes Medical Institute, Department of Chemistry and Chemical Biology and Department of Physics, Harvard University, Cambridge, MA, USA

    Bogdan Bintu & Xiaowei Zhuang

  6. Dipartimento di Fisica, Università di Napoli Federico II, and INFN Napoli, Complesso di Monte Sant’Angelo, Naples, Italy

    Mattia Conte, Simona Bianco, Andrea M. Chiariello & Mario Nicodemi

  7. Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, CA, USA

    Melodi Tastemel & Bing Ren

  8. Department of Quantitative Health Sciences, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, USA

    Ivan Juric & Ming Hu

  9. Berlin Institute for Medical Systems Biology, Max Delbrück Centre (MDC) for Molecular Medicine, Berlin, Germany

    Mario Nicodemi

  10. Berlin Institute of Health (BIH), Berlin, Germany

    Mario Nicodemi

  11. Institute of Genomic Medicine and Moores Cancer Center, School of Medicine, University of California, San Diego, La Jolla, CA, USA

    Bing Ren

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Contributions

B.R. and H.H. conceived the study. B.R. supervised the study. H.H. performed insulator assays and related analysis. R.H. and M.Y. performed PLAC-seq and Hi-C experiments. I.J. and M.H. analyzed PLAC-seq data. M.T. performed western blot experiments. Y.Z. performed Hi-C analysis. Q.Z. and Y.H. performed chromatin tracing experiments with help from B.B. and X.Z. A.J., B.B., C.K., M.C., S.B., A.M.C. and M.N. analyzed chromatin tracing data. The manuscript was written by H.H. and B.R. with input from all coauthors.

Corresponding author

Correspondence to Bing Ren.

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Competing interests

B.R. is a cofounder and consultant for Arima Genomics, Inc. and cofounder of Epigenome Technologies. X.Z. is a cofounder and consultant for Vizgen, Inc. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Genetics thanks Qiang Wu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Genotyping mESC reporter cell lines.

a, Genotyping egfp and mcherry labeled Sox2 gene. Left, Sanger sequencing results for allele-specific PCR products. Allele-specific SNP is highlighted. Right, the construct of the clone and the SNP information used to distinguish the two alleles. The reverse primer was common, while the forward primer was allele-specific, matching with egfp and mcherry sequence, respectively. b-c, Genotyping the Insulator reporter and control cell lines. Left, Sanger sequencing and SNP information. Right, Construct of the clone and positions of PCR primers. The forward primer is specific to the inserted HyTK gene. b, insulator reporter cell line. c, Insulator control cell line.

Extended Data Fig. 2 Insulation features of CBSs from the Sox9-Kcnj2 TAD boundary.

a, Hi-C contact map of the Sox9-Kcnj2 locus in mouse ES cells. Zoom in view shows the four CTCF binding sites cloned for insulator activity test. b, ChIP-seq of CTCF in the no insertion clone and the clone with an extra copy of the four Sox9-Kcnj2 TAD boundary CBS inserted inside the Sox2 domain. c, Reduction in Sox2-eGFP expression by one additional CBS. The comparison was between the clones presented in Fig. 2b. (0 CBS, n = 8; 1 CBS inside, n = 23; 2 CBS inside, n = 18; 3 CBS inside, n = 13; 4 CBS inside, n = 5; Data are mean ± sd). d, Allele-specific Sox2 expression in the no insertion clone (n = 2), the 4CBS clone (n = 3), and the 4CBS downstream clone (n = 2) as measured by RNA-seq. Sox2 expression from the CAST and 129 allele was represented by normalized read counts (rpkm) of the tagged egfp and mcherry gene, respectively. e, Relative Sox2 expression in the 4CBS and the 4CBS downstream clone in d measured by RNA-seq and FACS. The Sox2 expression from the egfp allele was first normalized to the mcherry allele, then compared to the no insertion clone. f, FACS profiling of the no insertion clone and the 4CBS clone. g, FACS profiling of GFPlow, GFPhigh sub-populations, and the unsort total population of the 4CBS insertion clone in f after extended culturing for 8 days. Left, GFP signal, right, mCherry signal from the same cells. h, ChIP-seq of H3K4me3 and H3K27ac in the no insertion clone and the 4CBS clone (n = 2). i, Allelic quantification of H3K27ac signal on the Sox2 super-enhancer of clones in h. H3K27ac ChIP-seq reads on the Sox2 super-enhancer were normalized by the total reads mapped to chromosome 3 for each allele.

Extended Data Fig. 3 Insulation effects of synthetic CTCF binding sites.

a, Additive insulation by synthetic CBS from boundary regions. Left top, compositions of one 139bp-CBS that was synthesized; Left bottom, tandemly arrayed 139bp-CBSs tested for insulator activity. Right, normalized Sox2-eGFP expression of clones with the tandemly arrayed 139bp-CBSs inserted between the Sox2 gene and its super-enhancer. Blue, CBS core motifs were in forward orientation; Red, CBS core motifs were in reverse orientation. Insertions were on the CAST allele only. n = 3, unpaired t-test, two-tailed. Data are mean ± sd. b, Insulation effects of PCR cloned large size CBSs (1–4 kb) and the synthesized 139bp-CBSs that contain the same CTCF motifs. (n = 12, paired t-test, two-tailed, ***P = 0.0007.). c, CTCF binding strength at selected boundary sites and non-boundary sites in mouse ES cells. ChIP-seq signals of CTCF are shown in 2-kb window. d, ChIP-seq of CTCF and Rad21 in clones with the insertion of six (nBd-syn6) or fifteen (nBd-syn15) 139-bp CBSs obtained from non-boundary regions. ChIP-seq reads were mapped to a customized mm10 genome that included the inserted sequence at the target site. Insertion position is highlighted in the red box.

Extended Data Fig. 4 ChIP-seq analysis of CTCF and cohesin binding at the synthetic insulators in various insulator reporter clones.

a, ChIP-seq signal tracks of CTCF in clones with the insertion of different synthetic CBS variants (n = 2). Each insertion consists of six CBSs that were tandemly arrayed in forward orientation (Supplementary Table 4). The insertion location is highlighted in the yellow box. b, CTCF binding strength (counts per million uniquely mapped reads) at the insertion location in the clones in (a). For each clone, ChIP-seq reads were mapped to a specific customized genome that contains the corresponding insertion in the Sox2 locus (n = 2; for bd core with bd adj, n = 4; for nbd core with bd adj n = 3.). c, ChIP-seq signal tracks of Rad21 in the same clones in (a) (n = 2). The insertion location is highlighted in the yellow box. d, Rad21 binding strength (counts per million uniquely mapped reads) at the insertion location in the clones in (c). For each clone, ChIP-seq reads were mapped to a specific customized genome that contains the corresponding insertion in the Sox2 locus (n = 2). e, Sequence alignment of the six boundary CBSs. Each CBS consists of 19-bp core motif plus 20-bp adjacent sequences on both sides. The color indicates the base frequency at each position. The CTCF motifs are highlighted in the red box.

Extended Data Fig. 5 Impact of CTCF ZF-9-11 deletion on transcriptional insulation by a synthetic insulator.

a, A schematic shows the experimental design to delete ZF9-11 of CTCF in mESCs. The exon 10, 11, partial of exon12, and an SV40 polyA signal were inserted into exon 9, resulting in the skip of exon9 in mRNA of the CTCF gene. b, PCR genotyping CTCF ΔZF9-11 mutant colonies. Genotyping primers spanned the insertion in exon 9. Highlighted in red boxes were homozygous mutant colonies evidenced by a single large-sized PCR fragment. PCR products from the homozygous mutant clones were further confirmed by Sanger sequencing. Genotyping of the homozygous mutants was repeated once with similar results. c, Western blot of CTCF in wild type and an exemplary CTCF ΔZF9-11 mutant clone. The primary antibody was the same one used for ChIP-seq (catalog: ab70303, lot GR3281212-7). Bottom, TBP loading control (primary antibody: sc-421, lot #B0304). Western blot was repeated once with similar results. d, Impact of CTCF zinc fingers 9–11 deletion on insulation effects of boundary CBSs with sixty-base-pair adjacent sequences. Left, eGFP profile of exemplary clones expressing wild-type and mutant CTCF protein; middle, mCherry profile of the same cells; right, normalized eGFP signal (eGFP/mCherry) of the wild-type and mutant clones. e, Impact of CTCF zinc fingers 9–11 deletion on insulation effects of boundary CBSs with ten-base-pair adjacent sequences. Left, eGFP profile of exemplary clones expressing wild-type and mutant CTCF protein; middle, mCherry profile of the same cells; right, normalized eGFP signal (eGFP/mCherry) of the wild-type and mutant clones.

Source data

Extended Data Fig. 6 Chromatin contacts at inserted CBSs.

a, K-R normalized Hi-C matrix (Observed/Expected) in the clone with the two forward Sox9-Kcnj2 TAD boundary CBSs inserted between the Sox2 promoter and super-enhancer (n = 2, replicates were merged). Hi-C reads were mapped to a customized chromosome 3 containing the insertion of the two forward CBSs. ChIP-seq signal of CTCF and orientations of the inserted CBSs and CBSs around the Sox2 promoter and super-enhancer were shown. The black arrow indicates the interactions between the inserted CBSs and the CBS on the Sox2 super-enhancer. b, Virtual 4C derived from Hi-C contacts in (a) at the viewpoint of the two inserted CBSs. Contacts were counted in each 5kb-bin. Contacts between the inserted CBSs and the Sox2 promoter or super-enhancer were compared to expected values, two-sided Poisson test. c, K-R normalized Hi-C matrix (Observed/Expected) in the clone with the four Sox9-Kcnj2 TAD boundary CBSs inserted between the Sox2 promoter and super-enhancer (n = 2, replicates were merged). Hi-C reads were mapped to a customized chromosome 3 containing the insertion of the four CBSs. ChIP-seq signal of CTCF and orientations of the inserted CBSs and CBSs around the Sox2 promoter and super-enhancer were shown. The black arrows indicate the interactions between the inserted CBSs and the CBS on the Sox2 promoter and super-enhancer. d, Virtual 4C derived from Hi-C contacts in (c) at the viewpoint of the two reverse-orientated CBSs inserted between the Sox2 promoter and super-enhancer. Contacts were counted in each 5kb-bin. Contacts between the two reverse CBSs and the Sox2 promoter or super-enhancer were compared to expected values, two-sided Poisson test.

Extended Data Fig. 7 Allele classification by multiplexed DNA FISH.

a-c, Bar plots showing detect efficiency of the 42 segments of chromatin tracing experiments in the ‘4CBS’ clone (a), the ‘4CBS mutant’ clone (b), and the ‘4CBS downstream’ clone (c). Detect efficiency of each segment was calculated as the fraction of chromosomes that showed a positive fluorescence signal at the specific imaging round. d, Exemplary images of allele classification. Left, nuclei segmentation and the positions of CAST and 129 allele in the nucleus. Right, images of the forty-two 5-kb segments (chr3:34,601,078-34,811,078) of the CAST and 129 allele. The hybridization probes of the 26th segment (highlighted in the red box) specifically targeted the 4CBS sequence. The chromosome positive for the 26th segment (inserted 4CBS) was classified as CAST allele, the negative chromosome in the same cell was classified as 129 allele. Cells with both chromosomes positive or both chromosomes negative for the 26th segment were discarded.

Extended Data Fig. 8 Spatial organization of the Sox2 locus in engineered mESCs.

a, Bulk Hi-C contact matrix (K-R normalized) of the Sox2 locus in the 4CBS clone. b, Median pairwise distance of the same Sox2 region measured by chromatin tracing experiment in the same clone in a, CAST and 129 chromosomes were combined. c, Correlation between the Hi-C contact frequency matrix (a) and median distance matrix(b). d, Normalized Sox2-eGFP expression in the no insertion clone(n = 8), the ‘4CBS’ clone (same cells in a-b, n = 2), and two insertion controls, ‘4CBS mutant’ (n = 3) and ‘4CBS downstream’ (n = 3). One-way analysis of variance with Bonferroni’s multiple comparisons test. Data are mean ± sd. e-f, Median spatial-distance matrix for the 210 kb Sox2 region (chr3: 34601078-34811078) of 129 (left) and CAST (right) chromosomes of the ‘4CBS mutant’ clone(e) and the ‘4CBS downstream clone’(f). The 26th segment was imaged by 4CBS specific probes in e. Similarly, the 38th segment was imaged by 4CBS specific probes in f. g-h, The probability of forming single-chromosome domain boundaries at each segment for the two alleles of the ‘4CBS mutant’ clone (g), and the ‘4CBS downstream’ clone (h). i, The distribution of single-chromosome insulation scores for each of the alleles between Sox2 promoter – 4CBSΔ insertion (segments 10–25) and 4CBSΔ insertion – Sox2 super-enhancer (segments 26–33), respectively. Two-sided Wilcoxon rank-sum test was performed. j, The distribution of single-chromosome insulation scores for each of the alleles between the same two domains (segment 10–25 and segment 26–33) in (i) for the ‘4CBS downstream’ clone. Insulation score was calculated in the same way as in (i). Two-sided Wilcoxon rank-sum test was performed.

Extended Data Fig. 9 Allele differences in median spatial distance.

a-c, Difference of the median distance matrices between the CAST and 129 allele of the ‘4CBS’ clone (a), the ‘4CBS mutant’ clone (b) and the ‘4CBS downstream’ clone(c).

Extended Data Fig. 10 Imaging of both nascent transcripts and chromatin structure at the Sox2 locus.

a, Example images of RNA FISH(left) and chromatin tracing(right) in the same cells. Circles indicate individual nuclei. RNA probes targeting Sox2, efgp, and mcherry are color-coded. Transcripts from the CAST allele are indicated by dots in purple pseudo color. Transcripts from the 129 allele are indicated by dots in cyan pseudo color. Arrows highlight examples of bursting alleles. b, Bursting frequencies of the CAST and 129 allele in the 4CBS clone and the control clone with 4CBS inserted downstream of the Sox2 SE. The numbers of bursting chromosomes and total chromosomes are indicated on the top of each bar. c, Median spatial distance matrices of the CAST and 129 allele in the 4CBS and the control clone (4CBS inserted downstream of the Sox2 super-enhancer). Multiplexed DNA FISH experiments were performed in the same cells following the RNA FISH experiments. d, Enhancer-promoter distances of the bursting and resting chromosomes in the 4CBS and the 4CBS downstream clone. A two-sided KS test between the distributions and a two-sided Wilcoxon test were performed.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2 and unprocessed gel of Supplementary Fig. 1b

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Supplementary Tables 1–9

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Source Data Extended Data Fig. 5

Unprocessed gels and western blots.

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Huang, H., Zhu, Q., Jussila, A. et al. CTCF mediates dosage- and sequence-context-dependent transcriptional insulation by forming local chromatin domains. Nat Genet 53, 1064–1074 (2021). https://doi.org/10.1038/s41588-021-00863-6

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