Establishment of Centromeric Chromatin by the CENP-A Assembly Factor CAL1 Requires FACT-Mediated Transcription

SummaryCentromeres are essential chromosomal structures that mediate accurate chromosome segregation during cell division. Centromeres are specified epigenetically by the heritable incorporation of the centromeric histone H3 variant CENP-A. While many of the primary factors that mediate centromeric deposition of CENP-A are known, the chromatin and DNA requirements of this process have remained elusive. Here, we uncover a role for transcription in Drosophila CENP-A deposition. Using an inducible ectopic centromere system that uncouples CENP-A deposition from endogenous centromere function and cell-cycle progression, we demonstrate that CENP-A assembly by its loading factor, CAL1, requires RNAPII-mediated transcription of the underlying DNA. This transcription depends on the CAL1 binding partner FACT, but not on CENP-A incorporation. Our work establishes RNAPII passage as a key step in chaperone-mediated CENP-A chromatin establishment and propagation

The Co-evolution of CAL1 and CENP-A Mediates Centromere Divergence in Drosophila

Centromeres mediate the conserved and essential process of chromosome segregation, yet centromeric DNA and the centromeric histone, CENP-A, are rapidly evolving. The rapid evolution of loop 1 (L1) of Drosophila CENP-A is thought to modulate the DNA-binding preferences of CENP-A to suppress centromere drive, the preferential transmission of chromosomes with expanded centromeric satellites during female meiosis. Consistent with this model, CENP-A from D. bipectinata (bip) fails to localize to D. melanogaster (mel) centromeres due to amino acid differences between mel and bip L1. Here, I show that this result is, in fact, due to the inability of the mel CENP-A chaperone, CAL1, to incorporate bip CENP-A into chromatin. Co-expression of bip CENP-A and bip CAL1 in mel cells restores centromeric localization, and similar findings apply to other Drosophila species. Furthermore, two co-evolving regions, CENP-A L1 and the CAL1 N-terminus, are identified as critical for lineage-specific CENP-A incorporation. Collectively, these data show that the rapid evolution of L1 regulates CAL1-mediated CENP-A assembly, suggesting an alternative mechanism for the suppression of centromere drive

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

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

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

Focus on Forestry ; Winter 2003

Magazine of the OSU College of Forestry