Caenorhabditis elegans chromosome arms are anchored to the nuclear membrane via discontinuous association with LEM-2 - PubMed (original) (raw)

Caenorhabditis elegans chromosome arms are anchored to the nuclear membrane via discontinuous association with LEM-2

Kohta Ikegami et al. Genome Biol. 2010.

Abstract

Background: Although Caenorhabditis elegans was the first multicellular organism with a completely sequenced genome, how this genome is arranged within the nucleus is not known.

Results: We determined the genomic regions associated with the nuclear transmembrane protein LEM-2 in mixed-stage C. elegans embryos via chromatin immunoprecipitation. Large regions of several megabases on the arms of each autosome were associated with LEM-2. The center of each autosome was mostly free of such interactions, suggesting that they are largely looped out from the nuclear membrane. Only the left end of the X chromosome was associated with the nuclear membrane. At a finer scale, the large membrane-associated domains consisted of smaller subdomains of LEM-2 associations. These subdomains were characterized by high repeat density, low gene density, high levels of H3K27 trimethylation, and silent genes. The subdomains were punctuated by gaps harboring highly active genes. A chromosome arm translocated to a chromosome center retained its association with LEM-2, although there was a slight decrease in association near the fusion point.

Conclusions: Local DNA or chromatin properties are the main determinant of interaction with the nuclear membrane, with position along the chromosome making a minor contribution. Genes in small gaps between LEM-2 associated regions tend to be highly expressed, suggesting that these small gaps are especially amenable to highly efficient transcription. Although our data are derived from an amalgamation of cell types in mixed-stage embryos, the results suggest a model for the spatial arrangement of C. elegans chromosomes within the nucleus.

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Figures

Figure 1

Figure 1

Chromosome arms are associated with the nuclear membrane. (a) Immunofluorescence analysis of C. elegans embryos with anti-LEM-2 antibodies (green), and the mAb414 antibody, which labels nuclear pore complexes (red). In the merged image, DNA stained by DAPI is shown in blue. The top row is wild-type N2 embryos; the bottom row is the lem-2 null mutant embryos. The arrowhead indicates the nucleus shown more closely in (b). (b) Enlarged image of the nucleus indicated by arrowhead in (a). (c) LEM-2 or negative control ChIP-chip (Array) or ChIP-seq (Seq) profiles. LEM-2* and LEM-2† indicate antibody Q3891 and Q4051, respectively. Vertical bars in the tracks indicate average ChIP-chip signals (MA2C scores) or ChIP-seq signals (z-scores of (IP - input)) in 5-kb windows. The y-axis range is -2 to 2. (d,e) LEM-2 ChIP-chip signals (5-kb window MA2C scores), recombination rate (interpolated genetic position of genes in centimorgans (cM)), and coverage of repetitive sequences in 50-kb windows are shown on chromosomes III (d) and X (e). The other chromosomes are shown in Figure S2c in Additional file 1. Dashed lines indicate the edges of LEM-2 domains as judged by visual inspection.

Figure 2

Figure 2

Within large LEM-2 domains, a finer level of organization consists of LEM-2 subdomains and gaps. (a,b) Representative LEM-2 subdomains on chromosomes IV (a) and V (b). Top panels with box indicate the chromosomal positions of regions shown below. Vertical bars in the tracks indicate ChIP-chip MA2C scores (-2 to 2) or ChIP-seq z-scores (-2 to 2). (c) Size distribution of LEM-2 subdomains and gaps. Subdomains or gaps were binned according to their size (log10 scale), and the number of regions for each bin are plotted. (d) Relationship between chromosome size and LEM-2 occupancy (total base pairs of LEM-2 subdomains divided by chromosome size (bp)). The line indicates a linear regression for autosomes by the least squares fit (intercept, 31.4; slope, 1.48).

Figure 3

Figure 3

Repeats are associated with the nuclear membrane. (a) Coverage of repetitive sequences within LEM-2 subdomains or gaps. Percentages of bases covered by repetitive sequences are plotted. The bottom and top of boxes indicate the 25th and 75th percentiles, respectively, and bands in the boxes indicate medians. Whiskers indicate the lowest or the highest data points within 1.5 × interquartile range from the box. Wilcoxon rank sum test was used for the statistical analysis. (b) Average counts of repetitive sequences across LEM-2 subdomain-gap boundaries. The number of repeats were counted (according to each repeat's central coordinate) within sliding 1 kb windows (500 bp offset) for the 354 boundaries (Materials and methods). The average count in each window is plotted. Average LEM-2 ChIP-chip MA2C scores of sliding windows (100 bp window, 50 bp offsets) are also plotted.

Figure 4

Figure 4

Genes reside preferentially in the gaps between LEM-2 subdomains. (a) Coverage of genes within subdomains or gaps. Percentages of bases covered by transcribed regions are plotted. Box plot representation and the statistical analysis are according to Figure 3a. (b) Average counts of coding genes across LEM-2 subdomain-gap boundaries. The number of translation start sites within sliding 1-kb windows (500-bp offset) were counted for the 354 boundaries. The average gene count in each window is plotted. Average LEM-2 ChIP-chip MA2C scores of sliding windows (100-bp window, 50-bp offsets) are also plotted. (c) Same as (b) but genes with the indicated orientations are plotted separately.

Figure 5

Figure 5

Genes at the nuclear membrane are inactive. (a) Expression level of genes within subdomains or gaps in mixed-stage embryos. Genes were categorized based on the size of gaps where they reside: extra large gap (XL), >1 Mb; large gap (L), 100 kb to 1 Mb; medium gap (M), 10 to 100 kb; and small gap (S), <10 kb. Box plot representation and the statistical analysis are according to Figure 3a. (b) Expression status during development for transcripts undetectable in early embryos. Transcripts were categorized in LEM-2 subdomains (top), large/medium/small gaps (middle) or extra large gaps (bottom) based on their start coordinates. We defined transcripts that were undetectable in early embryos as those with RNA-seq dcpm (depth of coverage per base per million reads) equals 0 in early embryos. E Emb, early embryo; L Emb, late embryo; L, larva stage; Adult, young adult.

Figure 6

Figure 6

H3K27me3 widely decorates LEM-2 subdomains except at active genes. (a) A representative genomic region showing ChIP-chip signals for LEM-2, H3K9me2, H3K9me3 and H3K27me3. The top panel indicates the chromosomal position of the enlarged region. The y-axes represent MA2C scores (-2 to 2) for LEM-2 ChIP-chip or z-scores (-2 to 2) for LEM-2 ChIP-seq and histone modification ChIP-chip. (b) Average H3K9 and H3K27 methylation profiles at LEM-2 subdomain boundaries. Sliding window averages (100-bp window; 50-bp offset) of ChIP-chip z-scores for indicated histone modifications (blue) or control H3 (gray) are plotted. For comparison, sliding window averages of LEM-2 ChIP-chip MA2C scores (red) are also shown. (c) H3K9 and H3K27 methylation profiles of genes in LEM-2 subdomains or gaps. Top 20% highly expressed (Top 20% expr) or bottom 20% lowly expressed (Bot 20% expr) genes in mixed-stage embryos across the genome are separately plotted. Lines indicate sliding window averages (100-bp window; 50-bp offset) of ChIP-chip z-scores with vertical bars for 95% confidence intervals. TSS, transcript start site; TES, transcript end site.

Figure 7

Figure 7

Small gaps between LEM-2 subdomains are exceptionally transcriptionally active. (a) A representative genomic region showing enrichment of RNA polymerase II (RNAPII), H3K4me3 and HTZ-1 in gaps between LEM-2 subdomains. The top panel indicates the chromosomal position of the enlarged region. RNAPII and HTZ-1 ChIP-chip data are shown as z-scores of log2 (ChIP/Input), whereas H3K4me3 ChIP-seq data are shown as normalized base counts. (b) Average RNAPII, H3K4me3, HTZ-1 and LEM-2 levels in subdomain-gap boundaries. For RNAPII and HTZ-1, sliding window averages (100-bp window; 50-bp offset) are shown, whereas for H3K4me3 the average of each base position is plotted. (c) Expression status of genes located near the boundaries. Each dot represents a transcript, whose abundance is shown on the y-axis and the distance from a boundary is shown on the x-axis. Boundaries between LEM-2 subdomains and medium gaps (left) or between subdomains and small gaps (right) are shown. The horizontal dashed bars indicate median transcript levels across 5-kb or 10-kb regions in gaps or subdomains. (d) RNA interference (RNAi) phenotypes of genes in LEM-2 subdomains and gaps. Numbers of genes with indicated RNAi phenotypes are shown. Chi-square test was used for statistical analysis with distribution of all genes with phenotypic annotations (shown in the header) as a background probability. Phenotypes annotated for more than 500 genes are listed.

Figure 8

Figure 8

Nuclear membrane association pattern of an X;IV fusion chromosome. (a) Schematic representation of the wild-type (N2 strain) chromosomes X and IV, and the X;IV fusion chromosome mnT12. Large LEM-2 domains are indicated in dark colors. The arrow indicates the fusion point. (b) LEM-2 ChIP-chip signals on chromosomes X and IV in wild type (red, top) and on the X;IV fusion chromosome (black, bottom). Wild-type data are the average of four biological replicates. Data from the fusion strain are the average of two biological replicates. The arrow indicates the fusion point. The boxes indicate the regions shown more closely in (c-f). (c-f) LEM-2 ChIP-chip signal patterns in wild type (red) or the fusion chromosome strain (black) at the ends of chromosome X (c,d) or chromosome IV (e,f).

Figure 9

Figure 9

Model for genome-nuclear membrane associations in C. elegans. (a) A model for large-scale chromosome arrangements mediated by the nuclear membrane. The large arm regions of autosomes are attached to the nuclear membrane, whereas the central portions of the chromosomes are looped out. For the X chromosome, the left arm is attached to the nuclear membrane, and the central and right portion of the chromosome are largely unattached. (b) A model for small-scale genome-nuclear membrane associations that underlie the large association domains. The small-scale associations often occur at regions with repetitive sequences and silent genes, and leave differentially sized gaps, which are likely looped out from the nuclear membrane. Among the loops, small loops are transcriptionally highly active.

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