Trac-looping measures genome structure and chromatin accessibility

Abstract

Long-range chromatin interactions play critical roles in genome organization and regulation of transcription. We now report transposase-mediated analysis of chromatin looping (Trac-looping) for simultaneous detection of multiscale genome-wide chromatin interactions among regulatory elements and chromatin accessibility. With this technique, a bivalent oligonucleotide linker is inserted between two interacting regions such that the chromatin interactions are captured without prior chromatin fragmentation and proximity-based ligation. Application of Trac-looping to human CD4⁺ T cells revealed substantial reorganization of enhancer-promoter interactions associated with changes in gene expression after T cell receptor stimulation.

Figure 1.. Scheme of Trac-looping

a.Top panel shows scheme of Trac-looping: (1) transposition of bivalent linker “in trans” into two interacting chromatin regions; (2) fragment DNA with a 4bp cutter and enrich biotinylated DNA by Strepavidin beads; (3) circulization of DNA; (4) Amplification of circular DNA; and (5) paired-end sequencing of the Trac-looping libraries. Bottom panel shows three classes of Trac-looping PETs: (1) both ends locate at the same open region (<150 bp); (2) two ends passes several nucleosomes (150 bp to 1,000 bp); (3) two ends form a loop by chromatin interaction (>1,000 bp). b.Plot of chromatin contact probability as a function of distance (>1,000 bp) measured by Trac-looping, in situ Hi-C, and H3K4me2 ChIA-PET. c.Percentage of intra-TAD PETs (>1,000 bp) for in situ Hi-C, Trac-looping, and H3K4me2 ChIA-PET for naïve CD4+ T cells. Data are shown as mean ± s.d. of repeated experiments for each method (HiC: n=2; TrAC-looping: n=4; ChIA-PET: n=2).

Figure 2.. Short-distance Trac-looping PETs detect accessible chromatin regions and capture short-range nucleosome interactions

a.Genome Browser display showing that Trac-looping and ATAC-seq detect similar chromatin accessibility profiles in fixed and unfixed cells. b.Plots of contact probability as a function of genomic distance (150–1,500 bp) defined by Trac-looping PETs within accessible, inaccessible, H3K9me3-marked or H3K27me3-marked regions. c.Heat maps show contact matrix at a resolution of 10 bps around TSSs of repressed and active genes, centers of CTCF binding sites, and centers of CTCF-binding-free non-promoter DHS defined by Trac-looping (upper panels). For side-by-side comparison, heat maps generated from in situ Hi-C data were also shown in the lower panels.

Figure 3.. Trac-looping efficiently detects interactions between accessible chromatin regions

a.Trac-looping PETs (>1,000 bp, magenta) are highly enriched in H3K4me3 and H3K27ac peaks. H3K4me2 ChIA-PET (green) and in situ Hi-C (blue) PETs are also plotted surrounding the H3K4me3 and H3K27ac peaks. b.WashU genome browser showing the read density of PETs with distance less than 150 bps (for accessibility) and interaction matrices generated from Trac-looping (red) and from in-situ Hi-C (blue) for a genomic region enclosing vav1 gene locus in chromosome 19. The interaction matrices were visualized at a resolution of 2Kb. Interacting PETs longer than 200K bps were not shown. Also included for comparison is an interaction matrix from deeply sequenced in situ Hi-C data generated for GM12878 (Rao et al., 2014). Circles: interacting regions previously confirmed by H3K4me2 CHIA-PETs in resting CD4+ T cells (Chepelev et al., 2012). The predicted significant interactions for Trac-looping were also presented in the bottom panel.

Figure 4.. Trac-looping efficiently detects interactions between accessible chromatin regions

a.Genome browser displays the Trac-looping detected interactions around IL2RA gene locus. Accessible regions, raw contact matrix (resolution = 2kb), all significant interactions (color indicates PET count) and the interactions linked to IL2RA promoter were shown. The (NG) Capture-C data using IL2 promoter (negative control) as the bait and the IL2RA promoter as the bait were also included. The IL2RA promoter region was highlighted in red bar. Three dCAS9-KRAB repression targets were indicated by red arrows. The PPRV region was also indicated in the accessibility panel. b.Promoters tend to interact with nearby accessible enhancers. The interaction between the top 1,000 most interactive promoters and the 10 nearest DHSs on each side of the promoter was examined as indicated by the cartoon on the top. +1 and −1 DHSs are closest to the promoters; +10 and −10 DHSs are most distant to the promoters. Red color indicates interactions.

Figure 5.. Reorganization of enhancer-promoter interaction upon TCR stimulation of CD4+ T cells

a.Volcano plot shows increased (red) and decreased (blue) interactions (FDR<1e-3, accessibility-normalized FC>2). b.Plots of accessibility-normalized fold change of interacting PETs vs. accessibility change at anchors for all the identified interactions. Significant increased (FDR<1e-3, FC>2, red) or decreased (FDR<1e-3, FC>2, blue) interactions were highlighted. a-b: Data shown represents total 63,427 merged interactions identified from resting and activated CD4 T cells. Details of identifying significantly changed interactions are presented in the online Methods. c.Heat maps show the contact intensity defined by Trac-looping PETs (red, bin size=2kb) and in situ Hi-C PETs (blue, bin size=10kb because of low sequence depth) in resting and stimulated T cells. Tracks for the accessibility were shown at the top. Black circles: examples of interaction increase with no accessibility increase observed at anchor regions. Orange circle: example of interaction increase with also accessibility increase at anchor regions. The bottom two panels show the (NG) Capture-C data using IL2 promoter as the bait. The anchors (including IL2 promoter) of the highlighted interaction examples in the matrix were also highlighted for the Capture-C data.