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CTCF chromatin residence time controls three-dimensional genome organization, gene expression and DNA methylation in pluripotent cells

Abstract

The 11 zinc finger (ZF) protein CTCF regulates topologically associating domain formation and transcription through selective binding to thousands of genomic sites. Here, we replaced endogenous CTCF in mouse embryonic stem cells with green-fluorescent-protein-tagged wild-type or mutant proteins lacking individual ZFs to identify additional determinants of CTCF positioning and function. While ZF1 and ZF8–ZF11 are not essential for cell survival, ZF8 deletion strikingly increases the DNA binding off-rate of mutant CTCF, resulting in reduced CTCF chromatin residence time. Loss of ZF8 results in widespread weakening of topologically associating domains, aberrant gene expression and increased genome-wide DNA methylation. Thus, important chromatin-templated processes rely on accurate CTCF chromatin residence time, which we propose depends on local sequence and chromatin context as well as global CTCF protein concentration.

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Fig. 1: Generation of CTCF ZF-deletion ESC lines and genomic binding profile of mutant CTCF proteins.
Fig. 2: Interphase localization and dynamic behaviour of CTCF proteins.
Fig. 3: Mitotic localization and dynamic behaviour of CTCF proteins.
Fig. 4: A/B compartmentalization and long-range SE clustering in ESCs expressing WT or d8 CTCF.
Fig. 5: ZF8 deletion destabilizes TAD organization in ESCs.
Fig. 6: CTCF ZF8 deletion results in transcriptional defects.
Fig. 7: CTCF ZF8 deletion results in aberrant global DNA methylation.

Data availability

All sequencing the datasets generated for this study are available in the GEO data repository under accession number GSE154009. Selected RNA-seq and Hi-C results can be found in Supplementary Tables 1 and 2. Previously published genomics data that were re-analysed here are available under the following accession codes: GSE123636 (RNA-seq of RBRi CTCF-expressing ESCs); GSE98671 (RNA-seq and Hi-C of auxin-inducible degron-mediated CTCF-depleted ESCs); GSE62380 (H3K27Ac, H3K4me3, H3K27me3, H3K36me3 and H3K79me2 ChIP-seq of ESCs); GSE96611 (H3K4Me2 ChIP-seq and ATAC-seq of ESCs); GSE44286 (Oct4 ChIP-seq of ESCs); GSE40910 (MNAse-seq of ESCs); GSE20485 (RNA polymerase II ChIP-seq of ESCs); and GSE136860 (PRO-seq of ESCs). Source data are provided with this paper. All other data that support the conclusions of this study are available from the authors on reasonable request.

Code availability

All codes used in this manuscript are publicly available except for Python scripts to analyse MeD-seq data, which are proprietary (Erasmus MC/Methylomics).

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Acknowledgements

We would like to thank I. Zampeta for performing the Dnmt3a western blots, and R. van der Linden, R. Janssens and M. de Bruijn for performing flow cytometry. This work was supported by grants from the Netherlands Organisation for Scientific Research (ALW 822.02.018) and the Dutch Cancer Society (KWF EMCR 2008-4109). G.S. was supported by a Marie Skłodowska-Curie fellowship (H2020-MSCA-IF-2016, miRStem) and by the ‘Fundación Científica de la Asociación Española Contra el Cáncer’. R.S. is supported by the Netherlands Organization for Scientific Research (VENI 91617114) and an Erasmus MC Fellowship.

Author information

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Authors

Contributions

W.S., F.S. and M.v.d.R. generated and characterized the GFP-tagged ESC lines. W.S., F.S., M.v.d.H., Z.O., W.F.J.v.IJ., S.B., S.C.H., R.S., N.G. and M.B. performed and analysed the RNA-seq and ChIP-seq experiments. S.C.H., S.B., B.G., W.A.v.C., A.H. and N.G. performed and analysed the imaging experiments. S.B., G.S., E.V. and R.S. performed and analysed the Hi-C experiments. S.B., R.B., J.B., J.G., N.G. and R.S. performed and analysed the DNA methylation experiments. S.B., S.M., J.G. and N.G. performed and analysed the FISH experiments. R.R., T.G., A.H., F.G., R.S. and N.G. conceived many of the experiments presented in this work and analysed results. All authors contributed to the writing of this manuscript.

Corresponding authors

Correspondence to Frank Grosveld, Ralph Stadhouders or Niels Galjart.

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Competing interests

R.B., J.B., W.F.V.v.IJ. and J.G. report being shareholders in Methylomics B.V., a commercial company that applies MeD-seq to develop methylation markers for cancer staging. The other authors declare no conflicts of interest or financial interests.

Additional information

Peer review information Nature Cell Biology thanks Bing Ren and the other, anonymous, reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

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

Extended data

Extended Data Fig. 1 Characterization of ESC lines expressing GFP-CTCF or mutant CTCF proteins.

a) CTCF zinc finger position and sequence. ZFs are based on ref. 14. Cysteines (C) and histidines (H) important for zinc coordination are boxed. b) Designed amino acid deletions in the various CTCF ZF mutant proteins. c) Results of the ES cell rescue experiment. For each construct that was transfected, the number of colonies analyzed (left column) and of colonies with full Cre-mediated Ctcfdelneo allele deletion (middle column) are shown. Colonies were analyzed by various methods to verify the intended deletion. The final conclusion regarding functional CTCF substitution is depicted in the right hand column. Of note, we only obtained one ES cell line expressing del1. Sequencing revealed that it contained a deletion of one extra amino acid (K) at the N-terminus of the originally intended del1 stretch (which begins with TFQ), and that the D residue situated at the C-terminus of the intended deletion stretch (which ends with HTD) was not deleted but was instead mutated into an E. Both changes are indicated in Fig. 1a. d) Log2 fold change in CTCF ChIP-Seq signal in del8- versus GFP-CTCF-expressing ES cells. e) Motif analysis of DNA binding by CTCF ZF mutants compared to GFP-CTCF (top row). Sites with more than 2-fold increased binding were included (number of sites are between brackets). Core (C), upstream (U), and downstream (D) motifs are indicated. Arrows point to U motif nucleotides. These are shifted towards the core in the del8 mutant. Asterisks point to GC residues appearing in all mutant motifs.

Source data

Extended Data Fig. 2 Interphase localization and behaviour of GFP-tagged CTCF proteins.

a) Fluorescence intensities in the indicated ES cell lines, measured by FACS, which reveals mean fluorescence intensity (MFI) per cell. WT: wild type ES cells, GC: GFP-CTCF-expressing ES cells, 1: del1-, 8: del8-, 9: del9-, 10: del10-, 11: del11-expressing ES cells (n = 2 independent experiments for each line, 10,000 cells measured per experiment). b) Fluorescence intensity comparison of GFP-CTCF, del8, and EB3-GFP. To estimate the average number of expressed GFP-CTCF or del8 molecules in ESC nuclei (see right hand panel for still image of an ES cell colony expressing GFP-CTCF; scale bar: 12 micron), we determined the fluorescence intensity of various dilutions of a purified GFP-tagged protein (EB3-GFP) of known concentration, and generated a calibration curve (lower graph, regression line (red) and function are shown). We next measured GFP-CTCF (GC) and del8 (d8) fluorescence intensity (FI) per nuclear area in arbitrary units (a.u., left hand graph, GC: n = 44 nuclei from 6 independent ESC colonies, d8: n = 70 nuclei from 6 independent ESC colonies, median values with 75% percentile indicated). We then compared intensities of EB3-GFP to that of GFP-CTCF or del8. Based on this analysis we calculated that GFP-CTCF has an average nuclear concentration of 290 nM and del8 of 190 nM. c) Affinity of GFP-CTCF and del8 for chromatin. Taking into account the concentrations of diffusing GFP-CTCF and del 8 (95 nM and 264 nM, respectively, see Fig. 2f), we calculated experimental kon (= apparent kon/[Free CTCF]) and affinity (Kd = koff/ Experimental kon) of GFP-CTCF and del8 for chromatin.

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Extended Data Fig. 3 Behaviour of GFP-CTCF- and del8-expressing cell lines.

a) Proliferation assay. GFP-CTCF-expressing ESCs (GC), and three ES cell lines expressing del8 (d8.1, d8.2, d8.3) were seeded at 100000 cells/dish at day 0. We counted cells after 1, 2, or 3 days (n = 3 experiments per cell line per time point, averages ± SD). b) Intracellular distribution of GFP-CTCF (GC) and of del8 in three independently isolated ES cell lines expressing (d8.1, d8.2, d8.3). Asterisk depicts a nuclear area of low fluorescence in the GC line, representing the nucleolus. This area is not visible in the del8 lines. Scale bar: 8 micron. c) Fluorescence intensity (FI) in arbitrary units (a.u.) in the indicated ES cell lines, measured by confocal microscopy in two independent experiments. GC, GFP-CTCF-expressing ES cells (n = 183 nuclei measured), d8: del8-expressing ES cells (d8.1: n = 322 nuclei measured, d8.2: n = 159 nuclei measured, d8.3: n = 167 nuclei measured). Data are presented as whisker plots with median and IQR. Mean values are also indicated (black stripes inside whisker plots, for d8.2 and d8.3 median and mean values overlap). d) Fluorescence recovery after photobleaching (FRAP) experiments in ES cells expressing GFP-CTCF (GC) or del8 (d8.1, d8.2, d8.3). Average values of two FRAP experiments are shown (GC: 22 cells measured, d8.1: 32 cells measured, d8.2: 24 cells measured, d8.3: 27 cells measured). For clarity we did not indicate SEMs. Note that d8.2 is the line used in many of the other experiments presented in this manuscript whereas the GC line is a second independently isolated line expressing GFP-CTCF. Recovery of the del8 mutant proteins is very similar in the three lines, in part because the nuclear concentrations (as examined in panel (c)) are similar. Recovery of GFP-CTCF in the second GC line is also very similar to that of GFP-CTCF in the first GC line shown in Fig. 2.

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Extended Data Fig. 4 Analysis of 3D genome topology in ES cells expressing GFP-CTCF or the del8 mutant.

a) Principal component analysis (PCA) of the top 1000 genes with highest standard deviation in ES cells expressing del8 (d8) or GFP-CTCF (GC). We performed RNA-Seq on three biological replicate cultures of three independently isolated d8 lines (d8.1, d8.2, d8.3) and two GC lines (GC1, GC2). One RNA-Seq sample of clone d8.3 showed poor quality metrics and was therefore excluded. b) Reproducibility scores (based on spectral decomposition; on a 0 to 1 scale, see Methods for details) across in-situ Hi-C matrices between the indicated pairwise comparisons of conditions. Note high reproducibility scores (>0.7) amongst biological replicates, which significantly drop (<0.4) when correlating in-situ Hi-C datasets from GC and d8-expressing cells. c) Distance decay curves of chromosome-wide interactions for wildtype and del8 expressing ES cells. d) Absolute PC1 values of genome-wide A and B compartment regions (100 kb bins) that switch compartment when comparing GC and d8-expressing cells. Data shown are merged values from three independent biological replicates. Center lines denote median values; box limits indicate 25th-75th percentiles with whiskers extending 1.5 times the interquartile range. e) Change in mRNA expression levels as measured by RNA-Seq for genes located in genomic bins that switch compartment when comparing GC- and del8-expressing cells. Data shown are values from 2-3 independent biological replicates obtained from 2-3 independently generated ESC clones. Center lines denote median values; box limits indicate 25th-75th percentiles with whiskers extending 1.5 times the interquartile range. f) Absolute PC1 values of genome-wide A and B compartment regions (100 kb bins) for untreated and CTCF-depleted cells. Data were obtained from ref. 29. Data shown are merged values from two independent biological replicates. Center lines denote median values; box limits indicate 25th-75th percentiles with whiskers extending 1.5 times the interquartile range. Pie chart indicated genomic regions (100 kb bin size) that switch compartment (4.1% of the genome) due to CTCF depletion. ***P < 0.001; A: P = 0.00039, B: P = 0.00081 (two-sided Mann Whitney U test). g) 2D meta-plot quantifying inter-TAD interaction frequencies between superenhancers in untreated and CTCF-depleted cells (ref. 29).

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Extended Data Fig. 5 Aspects of TAD organization in ES cells expressing GFP-CTCF or del8.

a) Percentage of conserved TAD borders between GC and d8 ESCs (1 bin = 50 kb). b) TAD size distribution in GC and d8 ESCs. Data shown are merged values from three independent biological replicates (presented as whisker plots with IQR and median). c) Log2 fold change (FC) in IS at TAD borders in GC ESCs. Borders were categorized based on >20% increase in IS (‘gain’), >20% decrease in IS (‘loss’) or <20% change in IS (‘stable’) in d8 compared to GC ESCs. d) Overlap between A/B compartment junctions and three TAD border categories. Outward or inward extension indicates positive or negative enrichment, respectively. Dashed circles indicate relative enrichment of 1. Table shows border and overlap statistics. e) IS TAD border correlation plot, comparing IS change in GC vs d8 to 48 h of acute CTCF depletion (data obtained from ref. 29). f) Differential IS at TAD borders in GC vs d8 (left) or after CTCF depletion (ref. 29) (right). Data shown are merged values from two independent biological replicates (presented as whisker plots with IQR and median). g) IS changes (GC-d8 differential values) for TAD borders bound or not bound by CTCF in GC cells. Data shown are merged values from three independent biological replicates (presented as whisker plots with IQR and median; ***P = 0.00094 (two-sided Mann Whitney U test)). h) Overlap between SMC1 loop anchors (top) or published ES cell loops (bottom) and three TAD border categories. Outward or inward extension indicates positive or negative enrichment, respectively. Dashed circles indicates a relative enrichment of 1. i) Meta-plot of Hi-C interaction signals for validated ES cell loops (ref. 50) in GC- and d8-expressing ES cells. j) Sex chromosome analysis of GC- and d8-expressing ESCs. Left upper panel: sex chromosome-specific PCR (m, f: DNA from male or female mouse, respectively). Right hand panels: representative fluorescent x-chromosome images (red). DAPI (green); scale bar: 5 micron. Bar plot (bottom left): x-chromosome number per ESC (n = 5 independent counting experiments, averages ± SD). k) Differential Hi-C interaction map of chromosome X. CTCF peak calls in GC- and d8-expressing cells are indicated on the left. l) Gene expression output (sum of all RPKM values from genes within a border) of three TAD border categories. n.s. = non significant; P > 0.05 (two-sided Mann Whitney U test). m) Precision nuclear run-on sequencing (PRO-Seq) at CTCF sites within ‘stable’ and ‘loss’ category TAD borders. PRO-Seq signals were divided into regions of 2 kb upstream or downstream of CTCF border sites; signal was also compared to a randomly shuffled set of all CTCF peaks (P < 0.0001 for all ‘loss’ versus ‘stable’ comparisons; two-sided Mann Whitney U Test). Data show values from two independent biological replicates (presented as whisker plots with IQR and median). n) CTCF binding site distribution with respect to distance to nearest TSS. Upper graph: all CTCF binding sites, lower graph: UC-containing sites. Inset: detailed distribution in a 5 kb window surrounding the TSS.

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Extended Data Fig. 6 Gene expression and DNA methylation changes in ES cells expressing the del8 CTCF mutant.

a) Dnmt3a levels in GC or d8-expressing ESCs. Left: immunofluorescence staining with anti-Dnmt3a antibodies (red); DAPI (blue). We enhanced brightness and contrast to visualize the low Dnmt3a signal in GC cells. This leads to an overexposed image in the d8 panel. Right: quantification of Dnmt3a signal (data are presented as whisker plots with IQR and median, GC, n = 52 nuclei measured, d8, 60 nuclei measured; >5 ESC colonies measured per line, data are from one representative experiment; P = 4.89 E-12, two-sided t-test). b) Western blot analysis of cell lysates from two GC-expressing ESC lines (GC1, GC2) and three d8-expressing ESC lines (d8.1, d8.2, d8.3). Western blots were probed with Dnmt3a (upper) or tubulin (lower) antibodies. ‘L’ and ‘S’ depict long and short Dnmt3a isoforms, respectively. Quantification of Dnmt3a levels (relative to GC2 by measuring Dnmt3a intensity over tubulin. Data are shown below the lanes. The western blot was done three times. c) Dnmt3b levels in GC- and d8-expressing ESCs. Data were taken from the RNA-Seq in Supplementary Table 2 (GC, n = 6 RNA-Seq experiments, d8, n = 8 experiments). ***P < 0.0052 (two-sided Wald test corrected for multiple testing). d) 5MC levels in GC or d8-expressing ESCs. Left: immunofluorescence staining with anti-5mC antibodies (red). Right: quantification of fluorescent 5mC signal (data are presented as whisker plots with IQR and median, GC, n = 49 nuclei, d8, 61 nuclei; >5 ESC colonies measured per line, data are from one representative experiment; P = 1.61 E-12, two-sided t-test). e) Principal component analysis (PCA) of differentially methylated regions (DMRs) showing >2 fold change in methylation in d8- or GC-expressing ESCs. We performed MedSeq in duplicate for three independent d8 lines (d8.1, d8.2, d8.3) and two GC lines (GC1, GC2). f) Genomic localization of differentially methylated regions (DMRs) divided in hyper-methylated (top, n = 290) or hypo-methylated (bottom, n = 82) regions in del8-expressing cells as compared to wildtype ES cells. g) Correlation scatter plot comparing changes in DNA methylation (log2 d8-GC, y-axis) and changes in CTCF ChIP-Seq signal (log2 d8-GC, x-axis) for 87 DMRs (>3 fold change in DNA methylation) that contain a CTCF binding site.

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Supplementary information

Supplementary Information

Supplementary Notes 1 and 2 and Supplementary Fig. 1

Reporting Summary

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Supplementary Tables

Supplementary Table 1: Hi-C data statistics. Supplementary Table 2: Differential gene expression analysis using RNA-seq data from GFP–CTCF and del8-expressing ES cells. (A) RPKM. (B) DEG. Supplementary Table 3: Primers used for ZF constructs and sex chromosome analysis.

Supplementary Video 1

GFP–CTCF behaviour during mitosis.

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Soochit, W., Sleutels, F., Stik, G. et al. CTCF chromatin residence time controls three-dimensional genome organization, gene expression and DNA methylation in pluripotent cells. Nat Cell Biol 23, 881–893 (2021). https://doi.org/10.1038/s41556-021-00722-w

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