Nanoscale 3D DNA tracing reveals the mechanism of self-organization of mitotic chromosomes

Abstract

How genomic DNA is folded during cell division to form the characteristic rod-shaped mitotic chromosomes essential for faithful genome inheritance is a long-standing open question in biology. Here, we use nanoscale DNA-tracing in single dividing cells to directly visualize how the 3D fold of genomic DNA changes during mitosis, at scales from single loops to entire chromosomes. Our structural analysis reveals a characteristic genome scaling minimum at 6-8 Mbp in mitosis. Combined with data-driven modeling and molecular perturbations, we can show that very large and strongly overlapping loops formed by Condensins are the fundamental structuring principle of mitotic chromosomes. These loops compact chromosomes locally and globally to the limit set by chromatin self-repulsion. The characteristic length, density and increasingly overlapping structure of mitotic loops we observe in 3D, fully explain how the rod-shaped mitotic chromosome structure emerges by self-organization during cell division.

Figure 1.. Multiscale DNA tracing from interphase to metaphase

A Schematic of multiscale probe libraries targeting chromosome 2, which was decoded using a combination of 15 Mb segment barcode and 1 Mb resolution spot barcodes. Full chromosome libraries were supplemented with intermediate scale (30 kb spots every 200 kb for 10-12 Mb) and high resolution (12 kb spots contiguously tiled for 1.2 Mb) libraries targeting the same and different (Chr5, Chr14, Chr18) chromosomes. B Schematic of multiscale chromosome trace acquisition. Whole chromosomes were segmented based on the full library signal, while sequential images of 15 Mb segments and 100 kb spots were used to decode and uniquely identify and 3D localize all detected spots. Intermediate and high-resolution libraries were segmented by regional barcodes and traced by sequential spot fitting in 3D. C Exemplary maximum-intensity projected micrographs of cell nuclei (DAPI, grey) with full chromosome traces (color-coded sequential 15 Mb segments). Arrowheads indicate fiducial beads used for drift correction. D Reconstructed 3D traces (multi-coloured) from libraries targeting whole chromosome 2 and intermediate- and high-resolution regions. Data representative of in total 8879 traces in 1396 cells in 5 independent experiments.

Figure 2.. Mitosis-specific chromatin loop and distance scaling signatures

A Illustration of single-trace metrics. Euclidean distance-based filtering of close contacts (<100 nm) with a genomic distance over 30 kb defines “loops”. Based on their overlap with other loops or loop bases, contacts are classified into base loops, nested loops (when 2 or more base loops coincide to form a second, larger loop) or z-loops (2 loops partially overlap). Radius of gyration is a measure of overall compaction of the trace. B Trace metrics calculated for the high-resolution (12 kb) 1.2 Mb region on chromosome 5 (149,500,723-150,699,962). Trace metrics were calculated from traces that were more than 80% complete. Data from n=278 (510), n=22 (44), n=124 (297) and n=152 (398) inter-, pro-, prometa- and metaphase cells (traces) from 3 independent experiments. Median, quartiles and whiskers are shown in the plots. C, D, E Traces of a high-resolution region (chromosome 5, C), intermediate scale (chromosome 2, B) and whole chromosome 2 (E) and corresponding pairwise distance scaling plots. Exemplary pairwise distances (black) for a single spot (red) within the DNA trace are highlighted. Inset with arrow highlights the characteristic scaling dip at 6-8 Mb. Plots show median ± standard error of the mean. Data from (C) n=400 (824), n=39 (109), n=241 (609) and n=244 (644), 3 experiments; (D) n=165 (237), n=27 (44), n=175 (325) and n=187 (346); 2 experiments; and (E) n=331 (803), n=28 (83), n=241 (736) and n=212 (686), 3 experiments; inter-, pro-, prometa- and metaphase cells (traces).

Figure 3.. Mitotic chromosome structural features depend on Condensins

A Wild type (WT) HeLa Kyto cells and HeLa Kyoto cells with SMC4-mAID-Halo acutely depleted for 3-4 hours with 5-Ph-IAA before mitotic entry (ΔSMC4). Exemplary maximum-intensity projected micrograph of metaphase chromatin (DAPI, grey) with full chromosome 2 traces (DNA-FISH, multi-coloured). Data representative of 212 WT metaphase traces from 3 independent experiments and 84 metaphase ΔSMC4 traces from 2 independent experiments. B Trace metrics from chr5:149500723-150699962 (1.2 Mb, 12 kb resolution) for WT and ΔSMC4 cells. Data from 152 (398) WT cells (traces), 3 independent experiments, and 153 (265) ΔSMC4 cells (traces), 2 independent experiments. C 3D DNA traces for chromosome 5, 1 Mb scale (12 kb resolution), chromosome 2, 10 Mb scale (200 kb resolution) and whole chromosome 2 (1 Mb resolution) from WT and ΔSMC4 cells at metaphase. Data representative of Chr5, 1 Mb: 152 (398) WT cells (traces), 3 independent experiments, and 153 (265) ΔSMC4 cells, 2 independent experiment; Chr 2, 10 Mb: 159 (286) WT cells (traces), 2 independent experiments and 106 (153) ΔSMC4 cells (traces), 2 independent experiments; Chr2 whole: 130 (212) WT cells (traces), 3 independent experiments and 51 (62) ΔSMC4 cells (traces), one experiment. D Distance scaling plots for chromosome 5, 1 Mb scale, chromosome 2, 10 Mb scale and whole chromosome 2 at metaphase. Data from Chr5, 1 Mb: 244 (644) WT cells (traces), 3 independent experiments and 212 (608) ΔSMC4 cells (traces), 2 independent experiments; Chr2, 10 Mb: 187 (346) WT cells (traces), 3 independent experiments and 124 (193) ΔSMC4 cells (traces), 2 independent experiments; Chr2, whole: 212 (686) WT cells (traces), 3 independent experiments and 206 (503) ΔSMC4 cells (traces), 2 independent experiments.

Figure 4.. Loop extrusion and self-repulsion correctly predict mitotic chromosome structure

A Polymer simulation of mitotic progression for a 100 Mb chromosome sampled at the indicated timepoints, displayed at 2 kb resolution (multi-colored, thin line) and as a 1 Mb rolling average (grey, thick line). Data representative of 20 dynamically simulated chromosomes. B Condensin I (magenta) and Condensin II (green) loop lengths shown for a 10 Mb stretch of the simulation example shown in A. Loop height scales with length for clarity. C Reconstructed traces from simulated regions from 100 Mb chromosomes at metaphase (40 minutes) and corresponding experimental data (Chr5 1 Mb scale, Chr2 10 Mb scale, Chr2 100 Mb from q-arm). Simulated traces were sampled as experimental data (12 kb tiled probes, 30 kb probes with 200 kb resolution and 100 kb probes with 1 Mb resolution). Simulated data representative of 20 simulated 100 Mb chromosomes, experimental data representative of Chr 5, 1 Mb: 152 (398) cells (traces); Chr 2, 10 Mb: 159 (286) cells (traces); Chr2, 100 Mb: 130 (212), 3 independent experiments. D Distance scaling plots for simulated metaphase chromosomes and corresponding experimental data. Simulated chromosomes were sampled as in C either 10 times (10 and 100 Mb scales) or 60 times (1 Mb scale) with different starting positions in each of twenty 100 Mb simulated chromosomes for representative sampling. Experimental data from Chr5, 1 Mb: n=244 (644) cell (traces), 3 independent experiments; Chr2, 10 Mb: n=187 (346) cell (traces), 2 independent experiments; Chr2q, 100 Mb: n=212 (686) cell (traces), 3 independent experiments.

Figure 5.. Global compaction is influenced by self-repulsion

A Wild type (WT) HeLa Kyto cells and HeLa Kyoto cells treated with TSA before mitotic entry. Exemplary maximum-intensity projected micrograph of metaphase chromatin (DAPI, grey) with full chromosome 2 traces (DNA-FISH, multi-coloured). Data representative of 212 WT metaphase cells from 3 independent experiments and 205 prometa-/metaphase TSA-treated cells from 2 independent experiments. B Trace metrics from chr5:149500723-150699962 (1.2 Mb, 12 kb resolution) for WT and TSA-treated cells. Data from 152 (398) WT cells (traces), 3 independent experiments, and 67 (168) TSA-treated cells (traces), 2 independent experiments. C Reconstructed traces for chromosome 5, 1 Mb scale (12 kb resolution), chromosome 2, 10 Mb scale (200 kb resolution) and whole chromosome 2 (1 Mb resolution) from WT and TSA-treated cells at metaphase. Data representative of Chr5, 1 Mb: 152 (398) WT cells (traces), 3 independent experiments, and 67 (168) TSA-treated cells, 2 independent experiment; Chr 2, 10 Mb: 159 (286) WT cells (traces), 2 independent experiments and 69 (126) TSA-treated cells (traces), 2 independent experiments; Chr2 whole: 130 (212) WT cells (traces), 3 independent experiments and 19 (31) TSA-treated cells (traces), two independent experiments. D Distance scaling plots for chromosome 5, 1 Mb scale, chromosome 2, 10 Mb scale and whole chromosome 2 at metaphase of TSA-treated cells together with corresponding WT and ΔSMC4 data. Data from Chr5, 1 Mb: 71 (198) TSA-treated cells (traces), 2 independent experiments; Chr2, 10 Mb: 73 (135) TSA-treated cells (traces), 2 independent experiments; Chr2, whole: 24 (69) TSA-treated cells (traces), 2 independent experiments. See Fig. 4 for details on WT and ΔSMC4 data. E Distance scaling plots from simulated 100 Mb WT and TSA-treated chromosomes, sampled as the corresponding experimental data (see D for details). TSA-treatment was simulated by increasing the repulsive potential between monomers. Simulated data from 20 dynamically simulated chromosomes per condition, sampled at metaphase (40 minutes).