Helical coiling of metaphase chromatids

Abstract

Chromatids of mitotic chromosomes were suggested to coil into a helix in early cytological studies and this assumption was recently supported by chromosome conformation capture (3C) sequencing. Still, direct differential visualization of a condensed chromatin fibre confirming the helical model was lacking. Here, we combined Hi-C analysis of purified metaphase chromosomes, biopolymer modelling and spatial structured illumination microscopy of large fluorescently labeled chromosome segments to reveal the chromonema - a helically-wound, 400 nm thick chromatin thread forming barley mitotic chromatids. Chromatin from adjacent turns of the helix intermingles due to the stochastic positioning of chromatin loops inside the chromonema. Helical turn size varies along chromosome length, correlating with chromatin density. Constraints on the observable dimensions of sister chromatid exchanges further supports the helical chromonema model.

Figure 1.

Hi-C data analysis and polymer model of barley metaphase chromosomes. (A) Hi-C contact probability (P_C) determined for barley chromosome 5H at metaphase (blue) and interphase (orange). The statistics of all Hi-C datasets are shown in Supplementary Table S1. (B) Hi-C contact matrix of metaphase chromosome 5H. (C) Derivative of the log-transformed contact probability in metaphase (blue) and interphase (orange). The two dashed lines mark: 500 kb, where the derivative is close to null and the probability decay brakes, and 30 Mb, where the derivative is null, and the probability reaches a peak. (D) Contact probability ratio between metaphase (P_CM) and interphase (P_CI). The dashed line marks high contact peaks that distinguish metaphase from interphase. (E) Turn length (T_l) along all chromosomes based on the local Hi-C contacts (Supplementary Table S2). The chromosome positions were scaled to a relative distance from the centromere. (F) A helical model illustrating the variation in turn length for chromosome 5H. (G) Bottle-brush polymer model (equilibrated after 100 000 steps) representing barley metaphase chromosomes. Five turns with intercalating magenta and green colours are shown on the left and the bases of the major loops are highlighted on the right. (H) Contact probability comparing Hi-C data (blue) and the polymer model (red).

Figure 2.

FISH confirms the helical organization of barley metaphase chromosomes. (A) Design of the oligo-FISH probes covering the 157 Mb-long region of chromosome 5HL (Supplementary Table S3). (B) FISH-labeled metaphase chromosomes. The enlarged oligo-painted region of 5HL (asterisks) shows the chromatin arrangement in both sister chromatids as predicted by the Hi-C-based helical chromatin arrangement model (Figure 1G). Due to chromosome tilting, Moa signals show, in a top-side view, a turn of the ∼400 nm thick chromatin fibre (marked with yellow lines). (C) Ortho-view (Supplementary Movie S3) and (D) surface rendering (Supplementary Movie S5) of the same 5H homologue. (E) Helical arrangement of the target region illustrating the changes of the turn lengths (T_l). (F) T_I calculated from the Hi-C data, across a 300 Mb region of 5HL encompassing the designed oligo-FISH probes. The regions covered by each probe are coloured according to the probe colour. (G) Relative heights (H) of the measured oligo-FISH probe signals as a percentage of the whole chromosome height. (H) Relative volume (V) of the measured oligo-FISH probe signals as a percentage of the whole chromosome volume. The black dots are the percentage of the DNA content of the probes relative to the entire chromosome. (I) Positions of telomeres (white) and subtelomeres (red) vary at both termini of different chromosomes. Subtelomeres form ring-like structures (right, arrows). Chromatin was counterstained with DAPI (blue). For (G) and (H), the total number of measured chromosomes per oligo probe are in parentheses.

Figure 3.

Sister chromatid exchanges (SCEs) confirm the helical organization of barley metaphase chromosomes. (A) Metaphase chromosomes labeled with EdU to detect SCEs creating a harlequin pattern. The exchanged chromatin regions appear as thin bands with a minimum height of ∼400 nm (enlarged regions I and II in dashed rectangles). (B) Example of the height and width measurements taken from the exchanged segments. (C) The colour bar indicates the ratio between the exchanged segments and the chromatid widths. (D) The height and width of 257 measured exchanged segments. The lack of values in the dashed region indicates that exchanged segments higher than ∼400 nm and not covering the complete chromatid width (left) never occurred. The black line indicates the median height (∼380 nm) of exchanged segments with incomplete width (less than chromatid width), and the grey area spans the lower and upper quartiles.

Figure 4.

A combination of oligo-FISH and EdU labeled SCEs endorse the helical chromatin organization. Metaphase chromosome spread (top) showing all barley chromosomes after EdU and oligo-FISH labeling. In both homologous oligo-FISH painted 5HL regions two SCEs occurred (bottom). The left exchanged segment does not span the entire chromatid width, but the right one does (insets in the merged images). The SCEs are present at the transition from the Rhea region to the not labeled Moa (left, red arrow) and Ostrich (right, white arrow) regions. The heights of the exchanged segments, oligo-FISH probe and EdU-free regions are similar (∼380–450 nm).

Figure 5.

Helical coiling of the chromonema. (A) Model of the chromatin organization in barley metaphase chromosomes. Bottom right: loops of the 80 nm lower-order chromatin fibre, formed of consecutive nucleosomes. Consecutive loops of simulated chromatin coloured as in our microscopic observation form the chromonema, which coils fill completely the chromatid without large cavities. Adjacent chromonema turns intermingle at their edges due to the flexibility of the smaller 80 nm fibres present within the chromonema. The helical order is interrupted at centromeres and secondary constrictions displaying mainly straight 80 nm chromatin fibers. The chromosomal termini contain less condensed, more flexible chromatin. Due to this flexibility, the telomeres may be embedded into the subtelomeric chromatin and not appear at the very end of the chromatid. The left long chromatid is shown as a stretched helix representing its higher-order chromatin folding based on oligo-FISH labeling. The different colours represent incomplete (white + green) and complete (magenta + green) chromonema turns, respectively. (B) Parameters of helically organized somatic metaphase chromosomes of barley compared to other species. Chromatid helix turn sizes and nested loop sizes are based on Hi-C data.