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DamC reveals principles of chromatin folding in vivo without crosslinking and ligation

Nature Structural & Molecular Biology volume 26, pages 471–480 (2019)Cite this article

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Abstract

Current understanding of chromosome folding is largely reliant on chromosome conformation capture (3C)-based experiments, where chromosomal interactions are detected as ligation products after chromatin crosslinking. To measure chromosome structure in vivo, quantitatively and without crosslinking and ligation, we implemented a modified version of DNA adenine methyltransferase identification (DamID) named DamC, which combines DNA methylation-based detection of chromosomal interactions with next-generation sequencing and biophysical modeling of methylation kinetics. DamC performed in mouse embryonic stem cells provides the first in vivo validation of the existence of topologically associating domains (TADs), CTCF loops and confirms 3C-based measurements of the scaling of contact probabilities. Combining DamC with transposon-mediated genomic engineering shows that new loops can be formed between ectopic and endogenous CTCF sites, which redistributes physical interactions within TADs. DamC provides the first crosslinking- and ligation-free demonstration of the existence of key structural features of chromosomes and provides novel insights into how chromosome structure within TADs can be manipulated.

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Fig. 1: DamC: methylation-based measurement of chromosomal interactions.
Fig. 2: Physical model of methylation dynamics.
Fig. 3: An inducible mESC line used to perform DamC and test the model predictions.
Fig. 4: DamC confirms the existence of TAD boundaries and quantitatively correlates with 4C and Hi-C.
Fig. 5: DamC-based detection of CTCF loops.
Fig. 6: Ectopic CTCF insertion leads to the formation of new loops and stripes.
Fig. 7: Scaling analysis of contact probabilities in vivo.

Data availability

The sequencing data from this study, including bedgraph files for the visualization of DamC and 4C profiles from all samples described in the manuscript, are available at the NCBI Gene Expression Omnibus with accession code GEO GSE128017. A University of California, Santa Cruz session containing all the DamC and 4C tracks used can be found at https://genome.ucsc.edu/s/zhan/DamC_publication_2019. The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium via the PRIDE72 partner repository with the dataset identifier PXD013507. Source data for Figs. 1 and 3–7 and Supplementary Figs. 1–3, 5 and 6 are available online.

Code availability

The custom-made codes used to analyze the data are available at https://github.com/zhanyinx/NMSB_2019_redolfi_et_al.

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Acknowledgements

This work is dedicated to the memory of M. Dahan. Research in the Giorgetti laboratory is funded by the Novartis Foundation and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation (grant agreement no. 759366, ‘BioMeTre’). The Kind laboratory was funded by the ERC (grant agreement no. 678423, ‘EpiID’) and EMBO (no. LTF 1214-2016 to I.G.). R.S.G. acknowledges support from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 705354 and an EMBO Long-Term fellowship (no. ALTF 1086-2015). We would like to thank P. Cron for cloning TetO-piggyBac plasmids; S. Aluri and S. Thiry for assistance with high-throughput sequencing; M. Stadler for help with bioinformatics analysis; S. Grzybek and H.-R. Hotz for server supports; and E. Heard and R. Galupa (Institut Curie, PSL Research University) for kindly providing PGK cells. We are grateful to D. Schuebeler and R. Galupa for critically reading the manuscript, and to G. Fudenberg for useful comments on scaling behavior. We acknowledge The ENCODE Project Consortium and, in particular, the Ren and Hardison laboratories for ChIP-Seq datasets in ESC.

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Author notes

  1. These authors contributed equally: J. Redolfi, Y. Zhan, C. Valdes-Quezada.

Authors and Affiliations

  1. Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

    Josef Redolfi, Yinxiu Zhan, Mariya Kryzhanovska, Vytautas Iesmantavicius, Ralph S. Grand, Sebastien A. Smallwood & Luca Giorgetti

  2. University of Basel, Basel, Switzerland

    Josef Redolfi & Yinxiu Zhan

  3. Oncode Institute, Hubrecht Institute–KNAW, Utrecht, the Netherlands

    Christian Valdes-Quezada, Isabel Guerreiro, Jop Kind & Wouter de Laat

  4. University Medical Center Utrecht, Utrecht, the Netherlands

    Christian Valdes-Quezada, Isabel Guerreiro, Jop Kind & Wouter de Laat

  5. EMBL, Heidelberg, Germany

    Tim Pollex

  6. Department of Cell Biology, Erasmus MC, Rotterdam, the Netherlands

    Eskeatnaf Mulugeta

  7. Università degli Studi di Milano and INFN, Milan, Italy

    Guido Tiana

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Contributions

J.R. generated cell lines and performed DamC experiments. Y.Z. wrote the model with assistance from G.T. and analyzed the data. C.V.-Q. performed 4C in W.dL.’s laboratory. M.K. assisted with cell culture and DamC library preparation and performed Hi-C experiments. I.G. and J.K. helped with experimental design and data analysis. V.I. performed mass spectrometry experiments and analysis. T.P. provided constructs for initial experiments and discussed the data. R.S.G. provided CTCF site sequences and tested CTCF binding in preliminary experiments. E.M. contributed to design of the initial experiments. S.A.S. developed the DamC library preparation protocol and performed piggyBac insertion mapping experiments. L.G. designed the study and wrote the paper with J.R. and Y.Z. and input from all the authors.

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Correspondence to Luca Giorgetti.

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Integrated supplementary information

Supplementary Fig. 1 Parameter study of model predictions.

a) Left: DamC enrichment is plotted as a function of the concentrations of rTetR-Dam and TetO viewpoints, imposing specific and non-specific dissociation constants to 1 nM and 80 nM respectively. Right: the rTetR-Dam concentration where the DamC enrichment is maximal is linearly correlated with the concentration of TetO viewpoint. b) DamC enrichment shows a maximum irrespective of the choice of the numerical parameters. This is exemplified by plots of DamC enrichment as a function of rTetR-Dam concentration when varying the TetO specific affinity and keeping the nonspecific affinity fixed (left panel) and vice versa (right panel).

Source data

Supplementary Fig. 2 Experimental system and optimized DamC protocol.

a) rTetR-Dam-EGFP-ERT2 becomes increasingly localized to the nucleus upon increasing 4-OHT concentration in the culture medium, as shown by the increasingly nuclear accumulation of EGFP. Maximum intensity projections of 10 wide-field Z planes are shown. Bright spots indicate binding of rTetR-Dam-EGFP-ERT2 to the 256x TetO array on chromosome X (see Fig. 1c). b) Schematics of the strategy for measuring rTetR-Dam-EGFP-ERT2 nuclear concentrations as a function of 4-OHT concentration. After exposing the cells to different concentrations of 4-OHT, nuclei were extracted and prepared for mass spectrometry. The relative abundance of nuclear rTetR-EGFP-Dam-ERT2 was measured using parallel reaction monitoring (PRM) using two replicate samples from all 4-OHT concentrations. Absolute quantification was performed in triplicate uniquely in the 500 nM 4-OHT sample using proteomic-ruler based mass spectrometry measurements (Wiśniewski et al. Mol. Cell. Proteomics 13, 3497–3506, 2014). We then extrapolated absolute nuclear rTetR-Dam copy numbers at all concentrations of 4-OHT based on the absolute quantification at 500 nM 4-OHT and the relative PRM quantification. Finally, the nuclear concentration of Dam-fusion Protein was calculated based on the average nuclear volume determined based on DAPI staining. Contamination from cytoplasmic proteins was estimated by comparing protein copy numbers of nuclear and whole-cell extracts, and subtracted from nuclear copy numbers. c) Protein copy numbers determined in nuclear extracts at 500 nM 4-OHT using the proteomic ruler strategy (Wiśniewski et al. Mol. Cell. Proteomics 13, 3497–3506, 2014). Data from three biological replicates are plotted before correction for cytoplasmic contamination. d) Schematics of the DamC library preparation. Genomic DNA is extracted from cells expressing the Dam-fusion protein. To avoid nonspecific ligation events in step 2, DNA is treated with shrimp alkaline phosphatase prior to DpnI digestion. After digestion with DpnI, a non-templated adenine is added to the 3’ blunt end of double-stranded DNA followed by ligation of the UMI-Adapter. Next, double-stranded DNA is denatured before random annealing of the second single stranded Adapter. In step 4, a T4-DNA-Polymerase is used for removal of 3’ overhangs and synthesis in the 5´→ 3´ direction. Finally, libraries are amplified by PCR and prepared for next generation sequencing. UMI: Unique Molecular Identifier. e) The DamC sequencing library preparation protocol includes UMIs allowing to filter ~40% of duplicated reads, and increases by roughly 30% the coverage of methylated GATC sites genome-wide compared to classical DamID (Peric-Hupkes et al. Mol. Cell 38, 603–613, 2010). at the same sequencing depth. f) Median DamC enrichment at the same viewpoints used for Fig. 3d as a function of 4-OHT concentration. Significant amounts of DamC enrichment in our experimental system can be observed in a range of rTetR-Dam nuclear concentrations corresponding to 5–10 and 0.1-1 nM 4-OHT for the lines carrying 890 and 135 viewpoints, respectively.

Source data

Supplementary Fig. 3 Characterization of the TetO-piggyBac clonal cell line and saturation analysis.

a) DamC enrichment from single DpnI fragments within +/− 100 kb from individual TetO viewpoints is plotted for two biological replicates performed with 0.1-1 nM 4-OHT. The Spearman correlation coefficient between the two replicates is indicated. b) The percentage of TetO viewpoints inserted in close proximity (<1 kb) from an active promoter or enhancer, or from a CTCF site that is bound in ChIP-seq (Nora et al. Cell 169, 930-944.e22, 2017). c) 4C interaction profiles obtained using a TetO viewpoint within 2 kb from an endogenous CTCF site and the partner CTCF locus as a reverse viewpoint. d) DamC and 4C interaction profiles measured from a TetO viewpoint inserted at the 3’UTR of the Chic1 gene within the Tsix TAD in the X inactivation center. Dashed lines indicate the interactions of Chic1 with the Linx and Xite loci. e) Definition of a deviation score measuring local differences between DamC and 4C. The deviation score is defined as the average quadratic difference between the DamC and the 4C signal in a 20-restriction fragment interval, normalized by the mean of the signal in the same interval. Two intervals are shown on the right to illustrate the differences between deviation scores of ~1 and ~3. f) Left: the 10% most dissimilar 20-fragment intervals are enriched in active chromatin, based on the dominant ChromHMM state (Ernst & Kellis. Nat. Methods 9, 215–216, 2012) in the interval using four chromatin states (ChromHMM emissions) (Chi-Square Test: pvalue < 10−9). ‘Inert’ corresponds to chromatin that is not enriched in H3K9me3, H3K27m3, H3K36me3, H3K9ac, nor H3K27ac. See the Methods section for more details. Right: The distributions of deviation scores in 20-fragment intervals where the dominant ChromHMM state is either inert, repressive, polycomb-associated or active, showing that active chromatin tends to show higher local dissimilarity between 4C and DamC (p-values from Wilcoxon test, two-sided). Cf. panel f for an example of a deviation score of ~3, corresponding to the average dissimilarity at active chromatin regions. g) Left: correlation between DamC signal in the -Dox sample and DNase-seq in mESC from ENCODE datasets. Each point in the scatter plot represents the aggregated signal in 20 kb; all 20 kb intervals genome-wide are shown along with their Spearman correlation. Right: One representative megabase on Chr1 showing the high correlation between the two signals. DamC and DNase-seq data were normalized to have equal average signal over the genomic interval shown here. h) Left: Removing DNase hypersensitive GATCs (see Methods) does not lead to increased local similarity between DamC and 4C. Distributions of local deviation scores are calculated over all 130 valid profiles and deviation scores between two DamC biological replicates is shown for comparison (p-values from Wilcoxon test, one-sided).

Source data

Supplementary Fig. 4 Additional DamC and 4C profiles from TetO viewpoints.

DamC (red) and 4C (black) profiles from forty TetO viewpoints in the pure clone with 135 TetO insertions.

Supplementary Fig. 5 TetO-piggyBac insertions do not perturb chromosome structure.

a) Insertion of TetO arrays does not perturb genome structure. Hi-C heatmaps of three different genomic locations harboring an array of 50xTetO sites and the corresponding wild-type locus are shown. Hi-C data are binned at 10 kb resolution. b) In windows of +/− 50 or +/− 200 kb surrounding the TetO integration sites, no significant changes can be detected in Hi-C at 5 and 10 kb resolution, respectively. Indeed, deviation scores between wild-type and TetO cells obtained at TetO insertion sites (green violin plot) are similar to those obtained at random wild-type genomic viewpoints (pink violin plot), and significantly smaller than those obtained by comparing virtual 4C profiles from pairs of different random genomic viewpoints (blue) (p-values are from Wilcoxon test, one-sided). c) Left: scheme of viewpoints used for the 4C experiment shown on the right. In cells harboring the TetO insertions, the ‘forward’ 4C viewpoint is within the TetO array as in main Fig. 3; in wild-type cells, the viewpoint is adjacent to the insertion genomic coordinate. The reciprocal viewpoint is the same in the two cases. Right: 4C profiles at the locus shown in panel c using the viewpoints shown on the left are indistinguishable.

Source data

Supplementary Fig. 6 Analysis of TetO-CTCF insertions.

a) Percentage of TetO-CTCF viewpoints occurring in close proximity (<1 kb) from an active promoter or enhancer, or a CTCF site that is bound in ChIP-seq (Nora et al. Cell 169, 930-944.e22, 2017). b) Distribution of peaks detected by peakC per viewpoint in TetO-CTCF (left) and TetO line (right) c) Examples of interaction profiles from TetO-CTCF viewpoints occurring in regions that are either devoid of (left) or densely bound by CTCF (right). d) Two further examples of ectopic structures formed as a consequence of the insertion of TetO-CTCF viewpoints. Hi-C data are binned at 10 kb resolution. e) Scheme of Cre-mediated excision of the ectopic CTCF cassette and genotyping. f) Genotyping PCR showing Cre-mediated excision of the CTCF cassette from the two integration sites shown in Fig. 6 in the same mESC clone (A4).

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Supplementary Fig. 7 Additional DamC and 4C profiles from TetO-CTCF viewpoints.

DamC (red) and 4C (black) profiles from forty TetO-CTCF viewpoints in the pure clone with 91 TetO insertions.

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Supplementary Information

Supplementary Figs. 1–7, Supplementary Note 1

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Redolfi, J., Zhan, Y., Valdes-Quezada, C. et al. DamC reveals principles of chromatin folding in vivo without crosslinking and ligation. Nat Struct Mol Biol 26, 471–480 (2019). https://doi.org/10.1038/s41594-019-0231-0

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