Recent efforts have succeeded in surveying open chromatin at the single-cell level, but high-throughput, single-cell assessment of heterochromatin and its underlying genomic determinants remains challenging. We engineered a hybrid transposase including the chromodomain (CD) of the heterochromatin protein-1α (HP-1α), which is involved in heterochromatin assembly and maintenance through its binding to trimethylation of the lysine 9 on histone 3 (H3K9me3), and developed a single-cell method, single-cell genome and epigenome by transposases sequencing (scGET-seq), that, unlike single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq), comprehensively probes both open and closed chromatin and concomitantly records the underlying genomic sequences. We tested scGET-seq in cancer-derived organoids and human-derived xenograft (PDX) models and identified genetic events and plasticity-driven mechanisms contributing to cancer drug resistance. Next, building upon the differential enrichment of closed and open chromatin, we devised a method, Chromatin Velocity, that identifies the trajectories of epigenetic modifications at the single-cell level. Chromatin Velocity uncovered paths of epigenetic reorganization during stem cell reprogramming and identified key transcription factors driving these developmental processes. scGET-seq reveals the dynamics of genomic and epigenetic landscapes underlying any cellular processes.
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We thank all the members of the COSR and Tonon laboratory for discussions, support and critical reading of the manuscript. We are grateful to E. Brambilla and F. Ruffini for preparation of the iPSCs and NPCs and A. Mira for assistance in the preparation of the organoids. We would like to thank S. de Pretis for the thoughtful discussions about chromatin velocity. We are grateful to G. Bucci for providing raw exome sequencing data and P. Dellabona for the coordination of the metastatic colon cancer sample collection and analysis. We also thank D. Gabellini, M. E. Bianchi, A. Agresti and S. Biffo for helpful discussions and for reviewing the manuscript. A.B. and L.T. are members of the EurOPDX Consortium. This work was partially supported by the Italian Ministry of Health with Ricerca Corrente and 5 × 1000 funds (S.M. and S.P.), by Associazione Italiana per la Ricerca sul Cancro (AIRC) investigator grants 20697 (to A.B.) and 22802 (to L.T.), AIRC 5 × 1000 grant 21091 (to A.B. and L.T.), AIRC/CRUK/FC AECC Accelerator Award 22795 (to L.T.), European Research Council Consolidator Grant 724748 BEAT (to A.B.), H2020 grant agreement 754923 COLOSSUS (to L.T.), H2020 INFRAIA grant agreement 731105 EDIReX (to A.B.), Fondazione Piemontese per la Ricerca sul Cancro-ONLUS, 5 × 1000 Ministero della Salute 2014, 2015 and 2016 (to L.T.), AIRC investigator grants (to G.T.) and by the Italian Ministry of Health with 5 × 1000 funds, Fiscal Year 2014 (to G.T.), AIRC 5 × 1000 ID 22737 (to G.T.) and the AIRC/CRUK/FC AECC Accelerator Award ‘Single Cell Cancer Evolution in the Clinic’ A26815 (AIRC number program 2279) (to G.T.).
M.T., F.G., D.L., S.P., D.C. and G.T. have submitted a patent application, pending, covering TnH.
Peer review information Nature Biotechnology thanks Kun Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work
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a, General scheme of TAM-ChIP technique (created with BioRender.com). A primary antibody (ChIP-validated antibody, dark grey) binds to a specific histone modification (light grey) over the genome (blue-red). A secondary antibody (TAM-ChIP conjugate, blue) is linked to the Tn5 transposon, which is made of Tn5 transposase (yellow) and the respective barcoded adapters (green). Upon the binding of the secondary antibody to the primary antibody, the linked Tn5 transposase targets and cuts the genomic regions flanking the histone modification, adding the barcoded adapters. TAM-ChIP was performed on two biological replicates for each condition (H3K4me3, H3K9me3 and NoAb). b, H3K4me3 (green) and H3K9me3 (red) enrichment profiles obtained either by ChIP-seq or TAM-ChIP-seq, compared with Input ChIP control (violet). c, Enrichment profile of heterochromatic genes FAM5B, NTF3, CACNA1E obtained from TAM-ChIP libraries assessed by Real Time-qPCR confirms Tn5 is able to target heterochromatic loci when redirected by H3K9me3 antibody. For each biological replicate three technical replicates were analyzed by Real-Time qPCR; one of the two H3K4me3 biological replicate was excluded because no appreciable signal was detected for any condition. Whiskers represent standard deviations (n = 3 technical replicates). Data shown in b and c refer to experiments performed on Caki-1 cell line.
a, Two different lengths of HP1α polypeptide (spanning amino acids 1-93 and 1-112) were linked to Tn5, using either a 3 or 5 poly-tyrosine–glycine–serine (TGS) linker, resulting in four hybrid construct, TnH#1-4. TnH#1 made of 1-93aa (HP1α) - 3x(TGS) - Tn5; TnH#2 made of 1-93aa (HP1α) - 5x(TGS) - Tn5; TnH#3 made of 1-112aa (HP1α) - 3x(TGS) - Tn5; TnH#4 made of 1-112aa (HP1α) - 5x(TGS) - Tn5. The 1-93 or 1-112aa spanning regions of HP1α include 1-75aa of CD followed by 18 or 37aa of natural linker, respectively (Created with BioRender.com). b-c, Tagmentation profiles relative to the four hybrid constructs (TnH#1-4) showing no difference in tagmentation efficiency relative to the native Tn5 enzyme (Nextera and Tn5 in-house produced) when targeting either genomic DNA, panel b, or native chromatin on permeabilized nuclei, panel c. d, Enrichment profiles relative to ATAC-seq performed with the four hybrid constructs (TnH#1-4, red) compared with native Tn5 enzyme (Nextera and Tn5 in-house produced) and with H3K4me3 and H3K9me3 ChIP-seq signals (green). e, Distribution of the enrichment of four TnH hybrid constructs (TnH#1-4) relative to genomic background in regions enriched for H3K4me3 (orange) or H3K9me3 (blue) expressed as log2(ratio) of the signal over the genomic Input. Enrichment over the same regions for native Tn5 enzyme (Nextera and Tn5 in-house produced), H3K4me3 and H3K9me3 ChIP-seq are reported as reference. Ec: global enrichment over H3K9me3-marked regions; Eo: global enrichment over H3K4me3-marked regions; Mc: modal enrichment over H3K9me3-marked regions; Mo: modal enrichment over H3K4me3-marked regions. Data shown in b, c and d refer to experiments performed on Caki-1 cell line.
Extended Data Fig. 3 Optimization of ATAC-seq protocol introducing a combination of Tn5 and TnH transposases.
a, Effect of altering Tn5 (green) to TnH (red) ratio on tagmentation profiles when adding both enzymes simultaneously at the beginning of the 60 minutes of the transposition reaction. b, Sequential addition of the same quantity of Tn5 and then TnH enzyme after 30 minutes of the transposition reaction results in a balanced distribution of enrichment signals between the two enzymes. Experiments performed on Caki-1 cell line.
a Abundance of unique cell barcodes retrieved by scATAC-seq performed on Caki-1 cells using the provided ATAC transposition enzyme (10X Tn5; 10X Genomics) (blue) compared to cell barcodes countable by TnH (orange) or Tn5 (green) alone. scGET-seq performance (Tn5 + TnH) is represented in red. The curves are largely overlapping, indicating no evident bias in single cell identification; b Distribution of per-cell normalized coverage over fixed-size genomic bins (5 kb) is reported for 10X Tn5 (blue) and for signal obtained by TnH (orange) and Tn5 (green). While Tn5 is comparable to 10X Tn5, TnH returns higher and less overdispersed per-bin coverages. White dot in boxplots reprents the median, boxes span between the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range. n = 3363, 1281 and 1537 cells in one experiment; c Saturation analysis for selected libraries. Dotted lines show the fitted incomplete Gamma functions on subsampled data; red solid lines show subsampling data from the same libraries; d Tn5 (green) and TnH (red) enrichment profiles obtained from scGET-seq (pseudo-bulk) or from ATAC-seq performed by using the two enzymes separately, compared with H3K4me3 (green) and H3K9me3 (red) ChIP-seq data. Data shown refer to experiments performed on Caki-1 cells.
a, Segmentation profiles in individual cells profiled by 10X Tn5 (scATAC-seq) (left panel) or TnH scGET-seq (right panel) at 500 kb. b, Segmentation profiles in individual cells profiled by 10X Tn5 (scATAC-seq) (left panel) or TnH scGET-seq (right panel) at 1 Mb. c, Segmentation profiles in individual cells profiled by 10X Tn5 (scATAC-seq) (left panel) or TnH scGET-seq (right panel) at 10 Mb. On top of each heatmap the genome-wide coverage of bulk sequencing of corresponding cell lines is represented. Centromeric regions and gaps (in white) have been excluded from the analysis.
a, evaluation of clonal structure of two PDO (CRC6 and CRC17) by exome sequencing; the histogram show the distribution of the cancer cell fraction estimated from the analysis of somatic mutations; in both organoids we observe a monoclonal structure b, 5X (left panel) and 10X (right panel) magnification contrast phase images of PDO #CRC17 obtained from a liver metastasis of a CRC patient (n>5); c absolute copy number of CRC17 and CRC6 as revealed by whole exome sequencing; data in panel c are equivalent to barplots over heatmaps in Fig. 3a.
a, UMAP embedding of individual cells as in Fig. 3, colored by the time PDX were harvested. b, Segmentation profiles in individual cells profiled by scGET-seq at 1 Mb resolution expressed as log2(ratio) over the median signal. Cells are clustered according to genetic clones. Red: positive values; Blue: negative values. Centromeric regions (white) have been excluded from the analysis because they correspond to low mapping and not fully characterized regions.
a, Distribution of early-to-late ratio of 2-stage Repli-seq data for NIH-3T3 cells. Violin plots represent the value of log2(E/L) values over DHS regions which are differential in the high-vs-low coverage cells in Fig. 4a (Mann-Whitney U = 36169.5, p = 1.403e-84). White dot in boxplots represents the median, boxes span between the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range. n = 35438 regions. b, Distribution of lamin-B1 DamID scores for NIH-3T3 cells. Violin plots represent the value of DamID scores over DHS regions which are differential in the high-vs-low coverage cells in Fig. 4a (Mann-Whitney U = 723.0, p = 4.621e-6). White dot in boxplots represents the median, boxes span between the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range. n = 35438 regions. c, UMAP embedding of individual cells coloured by cell groups, identified by Leiden algorithm with resolution parameter set to 0.2. d, Results of the linear model calculating the group-wise differences between TnH and Tn5 enrichment. For each group we reported the coefficient of the model, the p-value and the Benjamini-Hochberg corrected p-value. Values are reported for the two genomic regions including the Major primers (see text). Barplot indicates the proportion of shScr-treated for each cell group.
a, UMAP embedding of individual cells colored by the probability of being included in a trajectory branch estimated by Palantir. Three major branches have been identified, roughly corresponding to the three cell types profiled in this study. b, Schematic representation of the phase portraits underlying Chromatin Velocity. In RNA-velocity, the time derivative of the unspliced/spliced RNA is used to estimate synthesis or degradation of RNA; in Chromatin Velocity, the same procedure is applied on Tn5/TnH data to estimate chromatin relaxation or compaction. d, UMAP embedding of individual cells colored by cell clusters. e, Heatmap shows average expression profiles of TF with the top 10 most negative on PLS2 during the early brain development. Darker color indicates higher expression. w.p.c.: weeks post conception.
Counts of cells from organoid CRC6 or CRC7 found in different clones identified using TnH (above) or Tn5 (below).
Enrichment analysis over KEGG pathways and Reactome pathways of genes associated with DHS sites that are found to be differentially enriched in epigenetic clones. Enrichment was performed using the Enrichr platform.
Mutations: list of somatic mutations of the primary tumor as a result of exome sequencing data. scGET-seq mutations: list of mutations profiled by scGET-seq. Only variants that have an impact on protein sequence have been reported.
Analysis of differential Tn5 signal enrichment according to different cell types. For each cell type, we report log fold change, P value and adjusted P value as a result of a t-test over each region. For each region, we report the closest genes (GENCODE v36) and the distance. We also report the log fold change, P value and adjusted P value of differential expression of the associated genes in each cell type
Analysis of differential Tn5 signal enrichment with respect to the cell entropy as estimated by Palantir. Regions are sorted for decreasing coefficient of the generalized linear model. Genes associated with regions by proximity are also reported.
Enrichment analysis of genes associated with top DHS regions with the dynamical profile. Analysis was performed using gProfiler.
Analysis of global transcription factor activity. HOCOMOCO v11 ID, PWM identification code; Gene Symbol, associated gene symbol; PLS1 and PLS2, loading of the TF after PLS regression, corresponds to the horizontal/vertical displacement of the TF arrows in Fig. 6e.
Sequencing statistics for all scGET-seq experiments presented in the manuscript. n_reads, number of sequencing fragments; n_reads_in_cell, number of fragments associated to a cell; n_duplicated, number of PCR duplicates; target cells, number of target cells in the experiment; PF cells, number of cells passing the initial processing filters (coverage by cell and by region); Compound Coverage, coverage estimate as number of mapped reads in cells (without duplicates) by read length divided by genome size; Per cell Coverage, average per cell coverage as Compound Coverage divided by the number of PF cells.
Amino acid sequences of TnH constructs (TGS residues underlined; H stands for histidine residue that is an artifact introduced as a consequence of the cloning strategy); Modified Tn5ME-A and TnHMe-A sequences with Tn- or TnH-associated barcode are underlined.
Representative code snippets to postprocess scGET-seq data.
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Tedesco, M., Giannese, F., Lazarević, D. et al. Chromatin Velocity reveals epigenetic dynamics by single-cell profiling of heterochromatin and euchromatin. Nat Biotechnol (2021). https://doi.org/10.1038/s41587-021-01031-1