Condensin II drives large-scale folding and spatial partitioning of interphase chromosomes in <i>Drosophila</i> nuclei

<div><p>Metazoan chromosomes are folded into discrete sub-nuclear domains, referred to as chromosome territories (CTs). The molecular mechanisms that underlie the formation and maintenance of CTs during the cell cycle remain largely unknown. Here, we have developed high-resolution chromosome paints to investigate CT organization in <i>Drosophila</i> cycling cells. We show that large-scale chromosome folding patterns and levels of chromosome intermixing are remarkably stable across various cell types. Our data also suggest that the nucleus scales to accommodate fluctuations in chromosome size throughout the cell cycle, which limits the degree of intermixing between neighboring CTs. Finally, we show that the cohesin and condensin complexes are required for different scales of chromosome folding, with condensin II being especially important for the size, shape, and level of intermixing between CTs in interphase. These findings suggest that large-scale chromosome folding driven by condensin II influences the extent to which chromosomes interact, which may have direct consequences for cell-type specific genome stability.</p></div

Condensin II and cohesin drive different scales of chromatin folding during interphase.

<p>(A) Top: Schematic of three-color Oligopaints. Three 3–4 Mb sized probes were tiled along chromosome 2L. The centromere-proximal probe (cen) is shown in cyan, the telomere-proximal probe (tel) is shown in green, and the middle probe (mid) is shown in pink. Bottom: Representative nuclei with three-color FISH are shown from control (Brown), Cap-H2, or Rad21 depleted cells, or cells over-expressing Cap-H2 (OX). DNA (Hoechst) is shown in blue. Scale bars equal 5 μm. 3D rendering of structures are shown on the right. (B) Tukey box plot showing the volume of the mid signal probe after RNAi. The data shown are from a single biological replicate (n>500 cells). These results were confirmed by at two additional biological replicates. ***p < 0.0001; Mann-Whitney test. (C) Tukey box plot showing the minimal edge-to-edge distance between the cen and tel probes after RNAi. The data shown are from a single biological replicate (n>500 cells). These results were confirmed by at two additional biological replicates. ***p < 0.0001; Mann-Whitney test. (D) Schematic representation of eight possible chromosome configurations detected using this approach. Configurations were classified as either ‘closed’ (cen and tel-proximal probes touching), or open (cen and tel-proximal probes not touching). The percentage of untreated Kc167 cells with a particular 2L configuration is noted inside the corresponding circle. (E) Bar graph showing the fraction of cells with chromosome 2L in either an open or closed configuration after RNAi. The data shown are from a single biological replicate (n>500 cells). These results were confirmed by at two additional biological replicates. ***p < 0.0001; Fisher’s exact test. (F) Tukey box plot showing the minimal edge-to-edge distance between the cen and tel probes before in control cells and after Cap-H2 overexpression (OX). The data shown are from a single biological replicate (n>500 cells). These results were confirmed by at two additional technical replicates. ***p < 0.0001; Mann-Whitney test. (G) Bar graph showing the fraction of cells with chromosome 2L in either an open or closed configuration in control cells and after Cap-H2 overexpression (OX). The data shown are from a single technical replicate (n>500 cells). These results were confirmed by at two additional technical replicates. ***p < 0.0001; Fisher’s exact test.</p

Chromosome arms form independent territories.

<p>(A) Schematic of chromosome arm-specific Oligopaints. (B) Representative Kc167 cell nucleus with Oligopaints labeling chromosomes X (green), 2L (red), 2R (cyan), 3L (yellow), and 3R (magenta). Total DNA (Hoechst stain) is shown in blue. Scale bar equals 5 μm. (C) Tukey box plot showing CT volumes as a fraction of nuclear volume. n>500 cells. (D) Heatmap showing median CT overlap for a population of n>500 Kc167 cells, where overlap is shown as a percentage of CT volume. Values in the bottom half of the heatmap are normalized to the structures listed along the bottom, while the top half are normalized to the structures listed on the left. (E) Heatmap showing pairwise contact frequencies for a population of n>500 Kc167 cells. The diagonal boxes represent whole-chromosome pairing frequencies. (F) Radial position of chromosomes in the nucleus determined by shell analysis with five shells of equal volume, where shell 1 is the closest to the nuclear periphery and shell 5 is the nuclear center. n>500 cells. ***p<0.0001, Mann-Whitney test. (G) Left: plot of DNA content in Kc167 cells after FACS. Right: Representative nuclei after FACS with Oligopaints labeling chromosomes X (green), 2L (red), and 2R (cyan). Dashed lines represent nuclear edge. Scale bar equals 5 μm. (H) Left: Tukey box plot showing nuclear volumes of G1, S, and G2 phased cells after FACS sorting, determined by Hoechst stain. Right: Tukey box plot showing CT volumes after FACS sorting. The data shown represent one technical replicate (n = 220–350 cells per cell cycle phase). These data were confirmed by two additional technical replicates. (I) Top: heatmaps showing median CT overlap for a population of n>200 FACS sorted Kc167 cells, where overlap is shown as a percentage of the structures listed along the bottom. Bottom: heatmaps showing pairwise contact frequencies for a population of n>200 FACS sorted Kc167 cells. (J) Histogram showing X-2L CT overlap as a percent of X CT volume after FACS. Binned data from a single technical replicate are shown (n>200 cells). These results were confirmed by at two additional technical replicates. (K) Scatter plot of nuclear volume (X-axis) versus 2L CT volume or X-2L overlap volume (Y-axis). Chromosome 2L volume data are shown in blue, while X-2L overlap data are shown in gray. n = 835 cells.</p

Condensin II is sufficient to drive whole-chromosome separation.

<p>(A) Left: Oligopaints labeling chromosomes X (green), 2L (red), and 2R (cyan) on representative Kc167 cell nuclei depleted of Brown (control) or the condensin II regulator SLMB, or stably expressing a copper sulfate-inducible Cap-H2-GFP construct (OX). Dashed lines represent the nuclear edge. Scale bar equals 5 μm. n>500 cells per condition. Right: 3D renderings of segmented structures. (B) Dot plot showing nuclear volume normalized to control. Each dot represents the average of a biological (SLMB RNAi) or technical (Cap-H2 OX) replicate with n>500 cells. *p = 0.02; t-test. (C) Dot plot showing 2L Oligopaint volume normalized to control. Each dot represents the average of a biological (SLMB RNAi) or technical (Cap-H2 OX) replicate with n>500 cells. *p = 0.04; t-test. (D) Histogram showing the binned distribution of 2L shape from a single biological (SLMB RNAi) or technical (Cap-H2 OX) replicate with n>300 cells. Higher compacity values indicate a more spherical structure. These results were confirmed by at least two additional biological or technical replicates. ***p < 0.0001; Mann-Whitney test. (E) IF/FISH on representative Kc167 cell nuclei depleted of Brown (control) or SLMB, or stably expressing a copper sulfate-inducible Cap-H2-GFP construct (OX). Heterochromatin is labeled with anti-H3K9me2 antibody (green) and heterochromatin FISH probes (Het) labeling AATAT, AATAG, AACAC, 359, and dodeca in red. Dashed lines represent the nuclear edge. DNA (Hoechst stain) is shown in blue. Scale bar equals 5 μm. n>300 cells per condition. (F) Tukey box plot of the number of Het foci shown in (E), showing the mean (black line) and distribution (minus outliers). Data shown are from a single biological (SLMB RNAi) or technical (Cap-H2 OX) replicate (n>300 cells each). These results were confirmed by two additional biological or technical replicates, respectively. ***p < 0.0001; Mann-Whitney test. (G) Heatmap showing median CT overlap for a population of n>500 cells per condition, where overlap is shown as a percentage of the structures listed along the bottom. (H) Histogram showing X-2L CT overlap as a percent of X CT volume. Binned data from a single biological or technical replicate are shown (n>500 cells). These results were confirmed by at least two additional biological or technical replicates. ***p < 0.0001; Mann-Whitney test. (I) Heatmap showing pairwise contact frequencies for a population of n>500 cells per condition. The diagonal boxes are whole-chromosome pairing frequencies.</p

<i>Drosophila</i> Kc167 cells form robust CTs.

<p>(A) Schematic representation of <i>D</i>. <i>melanogaster</i> karyotype. The unlabeled heterochromatin is depicted in white, while euchromatin is depicted in green (X-chromosome), pink (chromosome 2), and gray (chromosome 3). (B) Representative Kc167 cell nucleus with Oligopaints labeling chromosomes X (green), 2 (pink), and 3 (gray). Total DNA (Hoechst stain) is shown in blue. Scale bar equals 5 μm. (C) Tukey box plot showing structure volume as a fraction of nuclear volume. X-axis denotes the structure being measured. (D) Top: Representative nucleus of IF/FISH in Kc167 cells with Oligopaints labeling chromosomes X, 2, and 3 all in red (all CTs), and anti-H3K9me2 IF in cyan. Bottom: IF to euchromatin (H3K4me3) in red and heterochromatin (H3K9me2) in cyan. Scale bar equals 5 μm. (E) Left: Heatmap of pairwise contact frequencies for a population of n>500 Kc167 cells. The diagonal boxes are whole-chromosome pairing frequencies. Right: Heatmap of median CT overlap for a population of n>500 Kc167 cells. Overlap is shown as a percentage of CT volume. Values in the bottom half of the heatmap are normalized to the structures listed along the bottom, while the top half are normalized to the structures listed on the left. (F) Top: Representative images of Kc167 cells with whole chromosome Oligopaints, showing the varying degrees of CT intermixing found in the population. Scale bar equals 5 μm. Bottom: histogram showing CT overlap as a percent of CT volume. Overlap between chromosomes X and 2, and X and 3, are shown as a percent of chromosome X CT volume. Overlap between chromosomes 2 and 3 is shown as a percent of chromosome 2 CT volume.</p

Heterochromatin clustering is dispensable for CT formation.

<p>(A) IF/FISH on representative Kc167 cell nuclei depleted of Brown (control), CAL1, or HP1a. Heterochromatin is labeled with anti-H3K9me2 antibody (green) and heterochromatin FISH probes (Het) labeling the AATAT, AATAG, AACAC, 359, and dodeca satellites in red. DNA (Hoechst stain) is shown in blue. Dashed lines represent the nuclear edge. Scale bar equals 5 μm. n>500 cells per condition. (B) Tukey box plot of the number of Het foci shown in (A). Data shown are from a single biological replicate (n>500 cells each). These results were confirmed by two additional biological replicates. ***p < 0.0001; Mann-Whitney test. (C) Left: Oligopaints labeling chromosomes X (green), 2L (red), and 2R (cyan) on representative Kc167 cell nuclei depleted of Brown (control), CAL1, or HP1a. Dashed lines represent the nuclear edge. Scale bar equals 5 μm. n>500 cells per condition. Right: 3D renderings of segmented structures. (D) Dot plot showing nuclear volume normalized to control. Each dot represents the average of a biological replicate with n>500 cells. The differences shown are not significant (t-test). (E) Dot plot showing 2L Oligopaint volume normalized to control. Each dot represents the average of a biological replicate with n>500 cells. The differences shown are not significant (t-test). (F) Histogram showing the binned distribution of 2L shape from a single biological replicate with n>500 cells. Higher compacity values indicate a more spherical structure. These results were confirmed by at least two additional biological replicates. The differences shown are not significant (Mann-Whitney test). (G) Heatmap showing median CT overlap for a population of n>500 cells per condition, where overlap is shown as a percentage of the structures listed along the bottom. (H) Histogram showing X-2L CT overlap as a percent of X CT volume. Binned data from a single biological replicate are shown (n>500 cells). These results were confirmed by at least two additional biological replicates. The differences shown are not significant (Mann-Whitney test). (I) Heatmap showing pairwise contact frequencies for a population of n>500 cells per condition. The diagonal boxes are whole-chromosome pairing frequencies.</p

Socio-Economic Practices of Resilience in Ireland

This working paper is a qualitative analysis of socio-economic practices of resilience in Irish households. It was produced as the Irish national report for Work Package 4 (D4.19) within the FP7 Project, ‘RESCuE: Patterns of resilience during socio-economic crisis among households in Europe.’ The paper includes an overview of the socio-economic characteristics of participating households in Ireland. It discusses the narratives of hardship provided by Irish participants and considers the varying practices adopted within households to cope with hardship. It concludes with an analysis of the degrees of hardship and forms of resilience experienced and displayed across different household types, and discusses some of the limitations of the concept of resilience

Pairing models involving both candidate pairing promoting and anti-pairing factors.

<p>a, Summary of candidate pairing factors identified in the screen. Green boxes denote candidate pairing promoters (hatched green were those identified only in the primary screen) and red boxes denote candidate anti-pairers. A representative sampling of pairing promoters were tested (italicized) and found (asterisk) to be important for euchromatic pairing. Proteins are grouped based on either a known function or localization pattern. Candidate pairing promoters found to elicit RNAi phenotypes dependent on Cap-H2 are presented as potential condensin II regulators (question marks). Note one dsRNA targets both CG42550 and CG14463 (separated by comma). b, Model for how compaction and intrachromosomal interactions compete with homolog pairing. Although all chromosomal regions may transiently unpair prior to or during S-phase, homolog pairing (red circles) of heterochromatic centromeric regions (grey lines) may be in competition with intrachromosomal interactions (black circles), causing pairing to occur less often, more slowly, or with less stability than homolog pairing of less compacted euchromatic regions (blue lines), where the paucity of repeated sequences reduces the likelihood of intrachromosomal interactions. This figure is not meant to imply a causal or dependent relationship between heterochromatic and euchromatic pairing, although such a relationship may exist. c, Model for pairing through the cell cycle. Proper spindle formation and chromosome segregation during anaphase/telophase of mitosis may bundle centromeric heterochromatic regions to spindle poles and directly facilitate or accelerate homolog recognition. Such interactions would then be maintained through G1. During S-phase, however, the pairing of regions is perhaps more dynamic, becoming antagonized and then re-paired subsequently. In this case, not all pairing interactions would be reestablished until the following mitosis. Euchromatic pairing is not depicted.</p

Homologous chromosomes enter the germline unpaired.

<p>A, Left: Schematic of a germarium showing pre-meiotic mitotic cell divisions as well as maturation of the meiotic cysts. The GSCs (purple nuclei) are positioned adjacent to the somatic niche (brown) and express high levels of SXL (green cytoplasm). Each GSC divides asymmetrically to produce a renewed stem cell and a differentiating cystoblast (CB, blue nucleus surrounded by green cytoplasm), which is positioned distal to the niche. The CB will undergo four more rounds of mitotic divisions to form a 16-cell cyst. Following these pre-meiotic stages, the 16-cell cyst will enter meiotic prophase, as defined by the initiation (zygotene) and complete formation (pachytene) of the synaptonemal complex (SC, red) between the paired homologs in two of the sixteen cells. Only a single cell will complete meiosis within each 16-cell cyst to form a mature egg (not shown) Arrow, direction of maturation. Right: Wild-type germarium stained for DNA (blue) and SXL (green). A GSC and CB are indicated by arrows and identified by SXL staining and relative position to somatic niche. Approximately 1–2 GSCs and 1–2 CBs are present in each germarium. Scale bar represents 10 µm. B, Drosophila chromosomes and targets of FISH probes (red). Heterochromatin is denoted in grey and rDNA cluster on the X-chromosome is in purple. C, Image of a GSC nucleus (dashed circle) at the tip of a germarium identified by DAPI (blue) surrounded by cytoplasmic SXL (green) staining and combined with FISH targeting AACAC (red) and dodeca (grey). Two signals for each FISH target represent separated homologous loci. Scale bar represents 5 µm. D, Percentage of nuclei exhibiting paired and unpaired loci in GSCs (left panel) and CBs (right panel). 15–30 ovaries were scored for each stage with a combined total of 242 GSC nuclei and 262 CB nuclei (approximately 30 nuclei for each locus at each stage). E, CB nuclei identified with SXL staining in combination with two-color FISH targeting AACAC (grey) and 24D (red) on Chromosome 2. Cartoon depicts hypothetical arrangement of homologous chromosomes as either unpaired or partially paired. Scale bars represents 5 µm.</p

RNAi of candidate pairing promoters disrupts pairing.

<p>a, Representative FISH images are shown for RNAi knockdown of candidate pairing promoters (<i>slmb</i>, <i>lin19</i>, <i>shtd</i>, <i>klp61f</i>, <i>dhc64C</i>, <i>mcph1</i>, <i>borr</i>), where the number of FISH signals per nucleus is increased compared to control. The percentage of single-signal nuclei is noted for both 359 and dodeca. n denotes number of nuclei scored. Scale bars equal 5 µm. b, Relative frequencies of interhomolog distances (unpaired = two signals >1.0 µm apart) based on dodeca FISH ± SD for three tests. dsRNA targets are either grouped based on known interactions (SCF, APC, CPC) or localization patterns of the proteins they encode (MTOC). All significantly reduced the percentage of paired nuclei compared to control (<i>P</i><0.05). c, Chromosomal targets of euchromatic FISH probes 16E and 28B and graph displaying the percentage of single-signal nuclei ± SD following RNAi. Asterisks denote a significant reduction from control (<i>P</i><0.05). A minimum number of 100 nuclei were scored for each dsRNA.</p