Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion, termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility, and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control.
At a glance
- Genome architecture: domain organization of interphase chromosomes. Cell 152, 1270–1284 (2013). &
- Self-organization in the genome. Proc. Natl. Acad. Sci. USA 106, 6885–6886 (2009).
- Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013). , , , &
- An oestrogen-receptor-alpha-bound human chromatin interactome. Nature 462, 58–64 (2009). et al.
- High-resolution profiling of histone methylations in the human genome. Cell 129, 823–837 (2007). et al.
- Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293 (2009). et al.
- The potential of 3D-FISH and super-resolution structured illumination microscopy for studies of 3D nuclear architecture: 3D structured illumination microscopy of defined chromosomal structures visualized by 3D (immuno)-FISH opens new perspectives for studies of nuclear architecture. BioEssays 34, 412–426 (2012). et al.
- CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells. Proc. Natl. Acad. Sci. USA 112, 11870–11875 (2015). , , , &
- Haplotype-resolved whole genome sequencing by contiguity preserving transposition and combinatorial indexing. Nat. Genet. 46, 1343–1349 (2014). et al.
- Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523, 486–490 (2015). et al.
- Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing. Science 348, 910–914 (2015). et al.
- The hierarchy of the 3D genome. Mol. Cell 49, 773–782 (2013). &
- A comparison of constitutive heterochromatin staining methods in two cases of familial heterochromatin deficiencies. Hum. Genet. 52, 133–138 (1979). , , , &
- Dynamic genome architecture in the nuclear space: regulation of gene expression in three dimensions. Nat. Rev. Genet. 8, 104–115 (2007). , , , &
- Neutrophil recruitment and function in health and inflammation. Nat. Rev. Immunol. 13, 159–175 (2013). &
- Individuality and variation of personal regulomes in primary human T cells. Cell Syst. 1, 51–61 (2015). et al.
- Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions. Nature 453, 948–951 (2008). et al.
- Nuclear lamins are not required for lamina-associated domain organization in mouse embryonic stem cells. EMBO Rep. 16, 610–617 (2015). &
- Nuclear architecture of rod photoreceptor cells adapts to vision in mammalian evolution. Cell 137, 356–368 (2009). et al.
- Neutrophil extracellular traps kill bacteria. Science 303, 1532–1535 (2004). et al.
- PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. J. Exp. Med. 207, 1853–1862 (2010). et al.
- Inhibition of PAD4 activity is sufficient to disrupt mouse and human NET formation. Nat. Chem. Biol. 11, 189–191 (2015). et al.
- Citrullination regulates pluripotency and histone H1 binding to chromatin. Nature 507, 104–108 (2014). et al.
- Changes in chromatin compaction during the cell cycle revealed by micrometer-scale measurement of molecular flow in the nucleus. Biophys. J. 102, 691–697 (2012). , , &
- Cdc2-cyclin E complexes regulate the G1/S phase transition. Nat. Cell Biol. 7, 831–836 (2005). , &
- Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. Genome Res. 24, 2033–2040 (2014). et al.
- Chromatin in situ proximity (ChrISP): single-cell analysis of chromatin proximities at a high resolution. Biotechniques 56, 117–124 (2014). et al.
- Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009). , , &
- Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008). et al.
- GREAT improves functional interpretation of cis-regulatory regions. Nat. Biotechnol. 28, 495–501 (2010). et al.
- edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010). , &
- ChromHMM: automating chromatin-state discovery and characterization. Nat. Methods 9, 215–216 (2012). &
- Supplementary Figure 1: Validation of ATAC-seq library from Atto-Tn5 (128 KB)
a, genome-wide comparisons of ATAC-seq reproducibility with Atto-Tn5 in GM12878 cells (GM). b, genome-wide comparisons of ATAC-seq reproducibility with Nextera Tn5 in GM12878 cells (GM). c, Insert size distribution of GM12878 ATAC-seq libraries transposed by Nextera Tn5. d, Insert size distribution of GM12878 cells ATAC-seq libraries from Atto-Tn5. e, Calculation of regulatory element enrichment from ChromHMM in previously published GM12878 ATAC-seq libraries and new ATAC-seq libraries using either Atto-Tn5 or Illumina Nextera Tn5. f: density plot of differential ATAC-seq peaks fold change (FC)(log2 value) in GM12878 from Atto-Tn5 and Nextera Tn5 among technical replicates and different enzyme.
- Supplementary Figure 2: Validation of ATAC-seq library in fixed cells (102 KB)
a, Insert size distribution of HT1080 cells ATAC-seq with standard protocol. b, Insert size distribution of fixed HT1080 cells ATAC-seq without reverse crosslinking step. c, Genomic tracks of ATAC-seq data from HT1080 cells among different conditions: No fixation, with fixation. X-axis is genomic coordinates; Y-axis is normalized ATAC-seq read counts. d, Insert size distribution of fixed HT1080 cells ATAC-seq with reverse crosslinking step. e, genome-wide comparisons of ATAC-seq reproducibility with Nextera Tn5 in non-fixed HT1080 cells (HT). f, genome-wide comparisons of ATAC-seq reproducibility with Nextera Tn5 in fixed HT1080 cells (HT) with reverse crosslinking.
- Supplementary Figure 3: Confirmation of ATAC-see principle (236 KB)
a, Left: representative whole frame images of ATAC-see from normal reaction and with 50 mM EDTA treated in HT1080 cells. The white squares in the DAPI channel indicate the cropped cells in Figure 2a. Scale bar=2 μm. Right: Signal intensity of ATAC-see was quantified in both normal reaction and 50 mM EDTA control samples. 20 cells were counted in each condition with independent replication, ** (p<0.005, student t-test), error bar is standard deviation. b, Representative whole frame images of ATAC-see co-staining with Lamin B1 and mitochondria protein marker (Mito) in HT1080 cells. The white square in the DAPI channel indicates the cropped cells in Figure 2b. Scale bar=2 μm. c, Correlation coefficient of ATAC-see with different epigenetic markers and active form of RNAP II in HT1080 cells (n= cell number); RNAPII Ser-2 P = RNA polymerase II phosphorylation ser-2, and RNAPII Ser-5 P= RNA polymerase II phosphorylation ser-5. d, Representative images of ATAC-see co-staining with different epigenetic markers and active form of RNAP II in HT1080 cells. e, ATAC-see and XIST RNA-FISH in mouse Neural Progenitor Cells. Upper panel: representative images (the white arrows in the ATAC-see panel indicate the location of XIST RNA FISH signal); lower panel: signal intensity quantification of ATAC-see within and outside of XIST area. 30 cells were counted in each condition with independent replicate, ** (p<0.005, student t-test), error bar is standard deviation.
- Supplementary Figure 4: Validation of ATAC-seq library after ATAC-see imaging (168 KB)
a, Schematic workflow of ATAC-seq library preparation after ATAC-see imaging. b, genome-wide comparisons of ATAC-seq reproducibility with Atto-Tn5 on slides from HT1080 cells (HT). c, density plot of differential ATAC-seq peaks fold change (FC)(log2 value) in HT1080(HT) from Atto-Tn5 (on slide) and Nextera Tn5 (in solution) among technical replicates and different enzyme. d, Sensitivity assay (with different input) of global DNA accessibility of ATAC-seq after imaging (on slides) from HT1080 cells.
- Supplementary Figure 5: ATAC-see in different cell types (143 KB)
a, Representative whole frame images of ATAC-see from different cell types. Scale bar=2 μm. Multi images (n>5) were taken in each cell type with independent replicates. b, Cell type specific accessible chromatin organization in the intact nucleus. The violin plot represents of the correlation coefficients between ATAC-see signal and DAPI signal per nucleus in different cell types. Each cell type has a unique profile, and neutrophils stand out as an outlier. B-cell = B-lymphoblastoid GM12878 cells.
- Supplementary Figure 6: Systematic imaging processing of ATAC-see (148 KB)
a, Schematic of image processing workflow of making mitochondria masks from ATAC-see and define the bright area of ATAC-see signal in the nucleus. b, Analysis of two different ATAC-see patterns in CD4+ T cells. The two plots in the upper panel represent the radial distribution of ATAC-see signal intensity and DAPI signal intensity in two different groups of CD4+ T cells. The blue line shows the ATAC-see signal intensity is low at the nucleus periphery, the green line illustrates the ATAC-see signal form a rim structure at the nucleus periphery (“Cap pattern”) and the red line indicates the means of all signal intensity. The line plot in the middle panel shows the ratio of ATAC-see signal intensity and DAPI signal intensity. The scatter plot in the lower panel represents the correlation of ATAC-see signal intensity and DAPI signal intensity among the population cells. The red lines in the plot represent the average values of single intensity from all groups.
- Supplementary Figure 7: Cell type specific accessible chromatin organization in the intact nucleus. (168 KB)
For each cell type (organized in rows), we display from left to right columns: (i) A representative ATAC-see image (red color is ATAC-see and blue is DAPI, scale bar = 2 μm). (ii) Signal intensity of ATAC-see and DAPI as a function of distance from nuclear periphery. Each trace is one nucleus; n= number of nuclei analyzed. (iii) Correlation of ATAC-see and DAPI signal intensity; Pearson correlation (r) is indicated. (iv) ATAC-see clusters, quantified as the ratio of ATAC-see bright areas vs total nucleus area.
- Supplementary Figure 8: Unique pattern of ATAC-see in the human neutrophil (134 KB)
a, Representative whole frame images show ATAC-see co-staining with Lamin B1 and mitochondria protein marker (Mito) in human neutrophils. The dotted lines in the Lamin B1 panel show the location of the nucleus periphery based on DAPI staining. Multi images (n>5) were taken with independent replicates. Scale bar=2 μm. b, The similarity of ATAC-seq libraries after imaging (on slide) with ATTO-590 from different donors. c, The boxplot represents the ATAC-seq peaks enrichment within NKI LADs and outside of NKI LADs, 0= outside of LADs, 1= within LADs. d, the black squares show the location of BAC clones chosen for DNA FISH according to the genomic tracks of ATAC-seq data in human neutrophil; BAC clones were chosen from both LAD region (RP11-626N18, RP11-832P24) and none LAD regions (RP11-63J14, RP11-637D5, RP11-368K11, RP11-116A9). e, Representative DNA FISH image in human neutrophils: the left panel: extended focus, right four panels: side view of 3D images. X-Y= X dimension and Y dimension, X-Z= X dimension and Z dimension. Scale bar=2 μm.
- Supplementary Figure 9: Immunostaining in human neutrophils and NETosis (175 KB)
a, Immunostaining of epigenetic markers in human neutrophil. RNAPII Ser-5 P= RNA polymerase II phosphorylation ser-5. Multi images (n>5) were taken with independent replicates in each staining. b, The representative images show DAPI staining of control, PMA and PAD4 inhibitor (PAD4i) treated human neutrophils. Scale bar=2 μm. c, The bar graph presents the quantification of NETosis in PMA and PAD4 inhibitor treated human neutrophils based on DAPI staining, Error bar= standard deviation; n=60x2 for each condition. d, Representative whole frame images of H3 citrullination staining in control, 5 h PMA treated and 5 h PAD4 inhibitor (PAD4i) treated human neutrophils. Multi images (n=5x2) were taken in each condition. Scale bar=2 μm.
- Supplementary Figure 10: ATAC-see and –seq in the human NETosis. (57 KB)
a, Representative images ATAC-see in 3h PMA stimulation human neutrophils. Scale bar=2 μm.
b, Epigenomic landscape of 3h PMA stimulation human neutrophils. Left column: Genomic tracks of ATAC-seq data. Locations of NKI Lamin associated domains (LADs) are indicated. X-axis is genomic coordinates; Y-axis is ATAC-seq normalized read counts. Middle: Metagene plot of ATAC-seq signal centered on the boundary between NKI LADs and neighboring sequences. Right: ATAC-seq insert size distribution for the corresponding samples. Diagnostic insert sizes for accessible DNA, mononucleosome, and di-nucleosome are labeled.
- Supplementary Figure 11: FACS analysis of ATAC-see and DAPI dual staining in GM12878 cells. (191 KB)
a, Left panel: cell cycle histogram from DAPI staining. Right panel: ATAC-see signal intensity histogram. b, violin plots show the ATAC-see signal intensity of the sorted cells measured by confocal microscopy. n= cell number measured under the confocal microscopy. *** (p<0.001, ANOVA test). c, Heatmap of the correlation coefficient of different FACS sorted groups. Each group contains independent replicate. d, Heatmap represents the cluster of accessible regions (FD>2, FDR<0.05) in FACS sorted groups: G1 low, G1 high, S phase and G2. Each group contains independent replicate. e, genome-wide comparison of accessible regions between different ATAC-see sorted groups: G1 low vs. G1 high; S vs. G1 high; G2 vs. S; G1 low vs. G2.
- Supplementary Figure 12: Classification of accessible regions in G1 (80 KB)
a, Genomic tracks of ATAC-seq data from ATAC-see G1 low and G1 high group after FACS sorting. X-axis is genomic coordinates; Y-axis is normalized ATAC-seq read counts. b, ChromHMM classification of accessible regions in asynchronized GM12878 cells (left), more accessible G1 high (middle) and more accessible G1 low regions (right). Each chromatin state was color-coded.
- Supplementary Figure 13: FACS analysis of ATAC-see and cell surface marker staining in mouse bone marrow progenitor cells. (99 KB)
a: FACS plots was gated (see Methods) as common myeloid progenitors (CMP), granulocyte-macrophage progenitors (GMP), and band neutrophils; b: Representative images of sorted populations shown in (a) by confocal microscopy, and multi images (n>5) were taken with independent replicates in each group.
- Supplementary Text and Figures (5,383 KB)
Supplementary Figures 1–13 and Supplementary Tables 1 and 2.