Fig 1.
RNAi screen to identify genes that promote dosage compensation.
(A) Male rescue assay. RNAi-mediated depletion of the indicated genes in the him-8 xol-1 sex-1 background led to rescue of the indicated percentage of males. Depleting DCC components DPY-21 and DPY-27 rescues a larger percentage of males than depletion of the other genes identified in this screen Asterisks indicate statistical significance based on Chi square test analysis of results, with expected rescue being equivalent to vector RNAi. * = p<0.05, ** = p<0.01, *** = p<0.001. (B) Raw data and expected table used in Chi square analysis. (C) Proposed mechanism of anchoring heterochromatic regions to the nuclear lamina. HMTs methylate H3K9. The chromodomain protein CEC-4 binds to this chromatin mark. Bound genomic regions are enriched for interactions with the nuclear lamina protein LEM-2.
Fig 2.
X chromosome decondensation in mutants.
(A) X chromosome paint FISH (red) in representative images of intestinal nuclei (DAPI, blue) of hermaphrodite adult worms in each genotype. The X chromosomes are compact and peripherally localized in wild type (N2), hpl-1, hpl-2 and met-1 mutant hermaphrodites, but are decondensed and more centrally located in the other mutants. Scale bar, 5 μm. (B) Quantification of X chromosome volumes normalized to nuclear size (n = 20 nuclei). Error bars indicate standard deviation. n.s = p>0.05 not significant; *** = p<0.001 by Student's t-test (N2 compared to appropriate mutant).
Fig 3.
The X chromosome relocates centrally in the nucleus.
(A) A diagram of the three-zone assay. An optical section from the middle of the nucleus was divided into three concentric rings of equal area. The proportion of the X chromosome paint signal in each zone (peripheral-intermediate-central) was quantified. (B) Results of quantification of the three-zone assay using whole X paint FISH probes in hermaphrodite intestinal nuclei (n = 10). In tethering mutants, a larger portion of the X chromosome is located in the central zone compared to wild type hermaphrodites. Relocation to a central region is less significant in DCC mutants or DCC-depleted hermaphrodites. Asterisks indicate statistical analysis (Student's t-test) of the centrally located portion of the X chromosome (shown in blue). n.s. = p>0.05, * = p<0.05, ** = p<0.01, *** = p<0.001. See S1 Table for statistical data.
Fig 4.
The middle region of the X chromosome is most affected in the absence of heterochromatic tethers.
(A) Autosomes are anchored to the nuclear lamina at both chromosome arms (anchors shown in green), while the X chromosome only has a significant anchored domain at the left end. Probes used in FISH analysis are indicated in red. Each probe covered an approximately 3–4 Mb genomic region. (B) Representative images of X-left, X-mid, X-right FISH analysis in each genotype. The mid-X region appears most decondensed and most centrally located in mutants. Scale bar, 5 μm. (C) Quantification of volumes occupied by the indicated FISH probes, normalized to nuclear size (n = 12 nuclei). Error bars indicate standard deviation. The greatest degree of decondensation in mutants is observed for the mid-X probe. (D) Three zone assay for each probe (n = 12 nuclei). The greatest degree of central relocation is observed for the mid-X probe. Asterisks indicate statistical analysis of mutant to wild type comparisons of volumes in (C) and centrally located portion of the X in (D) using Student's t-test. n.s. = p>0.05, * = p<0.05, ** = p<0.01, *** = p<0.001. See S1 Table for statistical data.
Fig 5.
The X chromosome is decondensed and centrally located in the absence of dosage compensation in XO animals.
(A) Chromosome paint FISH (red) in intestinal nuclei (DAPI, blue) of male adult worms, XO hermaphrodites and set-25 mutant males using whole X paint probe, and probes to the left, middle, and right domains of the X chromosome. The X chromosome, and the middle region of the X chromosome, appear large and diffuse and are located more toward the nuclear interior. set-25 mutations do not have additional effects on X chromosome morphology in males. Scale bar, 5 μm. (B) Quantification of volumes occupied by the X paint probe (n = 20 nuclei). The wild type hermaphrodite data point (wt herm) is repeated from Fig 2B and is marked by $ sign. (C) Three-zone assay for the whole chromosome X paint probe (n = 10). Wt herm data point is repeated from Fig 3B ($). (D) Quantification of volumes occupied by the X-left, X-mid, and X-right probes normalized to nuclear size (n = 20). Wt herm data points are repeated from Fig 4C ($). (E) Three zone assay for the mid-X probe (n = 10). Wt herm data point is repeated from Fig 4D ($). Error bars in (B) and (D) indicate standard deviation. Asterisks indicate statistical analysis compared to wild type hermaphrodites for volumes in (B) and (D) and centrally located portion of the X or mid-X (C) and (E), using Student's t-test. wt male and set-25 male comparisons are also shown as indicated. n.s. = p>0.05, * = p<0.05, ** = p<0.01, *** = p<0.001. See S1 Table for statistical data.
Fig 6.
Chromosome I structure and organization is not affected in tethering mutants.
(A) Chromosome I paint FISH (red) in representative images of intestinal nuclei (DAPI, blue) of hermaphrodite adult worms. Chromosome I appears comparably sized in each background. Scale bar, 5 μm. (B) Quantification of chromosome I volumes normalized to nuclear size (n = 12 nuclei). Error bars indicate standard deviation. (C) Three-zone assay for whole Chr I paint (n = 10 nuclei). The chromosome did not relocate to a more central position in any of the mutants (D) FISH analysis of the left, middle and right regions of Chr I in wild type (N2) and set-25(n5021) mutant hermaphrodites. Diagram (left) indicates locations of probes, representative images are shown on the right. The left and right ends of the chromosome are peripherally located, but the middle appears more centrally located in both backgrounds. (E) Quantification of volumes occupied by Chr I domains (n = 20 nuclei). Error bars indicate standard deviation. (F) Three-zone assay for the left, middle and right domains of Chr I (n = 10 nuclei). The middle domain is more centrally located than the left and right arms in both genotypes. Student's t-test did not reveal any statistically significant differences for volume measurements in (B) and (E), or for the portion of chromosome located in the central zone in (C) and (F), mutant compared to wild type. n.s. = p > 0.05. See S2 Table for statistical data.
Fig 7.
Immunofluorescence analysis with antibodies specific to H3K9me3 (green), combined with antibodies specific to DCC subunit CAPG-1 (red) to mark the location of the X chromosomes. To illustrate the spatial proximity of the bright H3K9me3 foci to the X territory, single focal planes are shown. Maximum intensity projections of whole nuclei are shown for reference (right, MIP). The H3K9me3 signal is distributed diffusely in the nucleus with some peripherally localized bright foci. H3K9me3 signal intensity is only affected in set-25 and set-32 mutants. Scale bar, 5 μm.
Fig 8.
DCC localization and H4K20me1 enrichment in tethering mutants.
(A) Combined X paint fluorescence in situ hybridization (red) and immunofluorescence with antibodies specific to DCC component DPY-27 (green). The DCC remains localized on the decondensed X chromosomes of tethering mutants. (B) Immunofluorescence images with antibodies specific to H4K20me1 (green) and DCC component CAPG-1 (red) to mark the location of the X chromosome. H4K20me1 remains enriched on DCC-bound X chromosomes. Scale bar, 5 μm.
Fig 9.
RNA-seq analysis of gene expression changes in tethering mutants.
(A-C) Boxplots show the distribution of log2 expression ratios on the X chromosomes and autosomes (A), as well as all individual autosome (I, II, III, IV, V) and the rest of the genome (G) between dpy-27(RNAi) and control (A), cec-4(ok3124) mutant and control (B), and met-2(n4256) set-25(n5021) mutant and control (C). The X chromosome was significantly derepressed compared to autosomes, but the only autosome that showed derepression compared to the rest of the genome is chromosome V. (D-F) Boxplots show log2 expression ratios on the X chromosome and on individual autosomes in dpy-27(RNAi) (D), cec-4(ok3124) mutants (E), and met-2(n4256) set-25(n5021) mutants (F). The X chromosome is more derepressed than any individual autosome in all three backgrounds. Differences in gene expression changes from the X was tested between the X and all autosomes, or the X and individual autosomes by one-sided Wilcoxon rank-sum test, and between a given autosome and the rest of the genome by two-sided Wilcoxon rank-sum test (n.s. = not significant, * = p < 0.05, ** = p<0.01, *** = p < 0.001).
Fig 10.
Comparison of gene expression changes in tethering mutants and partial DCC depletions.
The magnitude of log2 expression ratios of X-linked (dark) and autosomal linked genes (light) between cec-4(ok3124) mutant and control plotted against dpy-27(RNAi) and control RNAi (A), met-2(n4256) set-25(n5021) mutant and control plotted against dpy-27(RNAi) and control RNAi (B), and met-2(n4256) set-25(n5021) mutant and control plotted against cec-4(ok3124) mutant and control (C). Red circles indicate a group of genes that are repressed by MET-2 and SET-25 independent of dosage compensation or cec-4 function. Percent of X-linked (dark numbers) and autosomal genes (light numbers) with greater than 10% (log2 of 0.1) change in expression are indicated in each quadrant (D) The R-squared of each regression and the Pearson correlation values are shown for X-linked genes (X), for each individual autosome (I-V), and all autosomal genes (A) for each comparison. Values for a control analysis (dpy-27 versus lsm-1 mutants) are also indicated.
Fig 11.
Model showing the effects of tethering and DCC function on X chromosome compaction and nuclear localization.
Model 1: In differentiated cells, heterochromatin tethers (black anchors) and additional mechanisms (blue anchors) tether the left end of the X chromosome to the nuclear lamina. When heterochromatic tethers are lost, the left end of the X remains near the periphery, but the rest of the chromosome relocates to a more central position. Model 2: In wild type cells, the DCC organizes the X chromatin into topologically associating domains (TADs) and uses heterochromatic anchors to compact the X chromosome and bring it to the nuclear periphery. In the absence of the DCC, the left end of the X remains peripheral and compact due to the action of the tethering proteins, and its TAD structure is maintained. The rest of the X chromosome loses its TAD organization, decondenses and moves more internally. When heterochromatic anchors are lost, redundant tethers keep the left end of the X near the nuclear lamina, but the DCC is unable to compact the rest of the chromosome and bring it to the periphery.