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. 2016 Oct;26(10):1441-1450.
doi: 10.1101/gr.202663.115. Epub 2016 Aug 16.

The time-resolved transcriptome of C. elegans

Max E Boeck  1 , Chau Huynh  2 , Lou Gevirtzman  2 , Owen A Thompson  2 , Guilin Wang  3 , Dionna M Kasper  3 , Valerie Reinke  3 , LaDeana W Hillier  2 , Robert H Waterston  2
Affiliations

The time-resolved transcriptome of C. elegans

Max E Boeck et al. Genome Res. 2016 Oct.

Abstract

We generated detailed RNA-seq data for the nematode Caenorhabditis elegans with high temporal resolution in the embryo as well as representative samples from post-embryonic stages across the life cycle. The data reveal that early and late embryogenesis is accompanied by large numbers of genes changing expression, whereas fewer genes are changing in mid-embryogenesis. This lull in genes changing expression correlates with a period during which histone mRNAs produce almost 40% of the RNA-seq reads. We find evidence for many more splice junctions than are annotated in WormBase, with many of these suggesting alternative splice forms, often with differential usage over the life cycle. We annotated internal promoter usage in operons using SL1 and SL2 data. We also uncovered correlated transcriptional programs that span >80 kb. These data provide detailed annotation of the C. elegans transcriptome.

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Figures

Figure 1.
Figure 1.
Gene expression dynamics across all stages. (A) Normalized expression across embryogenesis and post-embryonic time points clustered by the stage of maximal expression (see Methods for details). Normalized expression is colored from none (black), to low (blue), to medium (green), and to maximal (yellow) with the scale provided on the left running from 0% to 100% of maximal expression per gene. Only genes (17,401) with at least one stage with expression >0.07 dcpm (depth of coverage per base per million reads) are shown. Embryonic stages on the left half of the plot are given in minutes, post-two-cell embryo. Post-embryonic stages include the four larval stages (L1–L4), young adult (YA), dauer entry (DE), dauer (D), dauer exit (DX), adult soma (SO), and L4 stage males (M). (B) Genes up-regulated (left) and down-regulated (right) in one stage relative to the previous time point are shown for each time point in embryogenesis. Gene counts for Chromosomes I, II, III, IV, V, and X are colored red, dark orange, light orange, yellow, green, and blue, respectively. (C) The proportion of genes overlapping between maximal expression clusters (y-axis) and those genes called as up-regulated (left) or down-regulated (right; x-axis) is shown for each embryonic stage. The proportion is colored from light yellow (0) to dark blue (0.65). (D) GO term enrichments for each up-regulated (top) and down-regulated (bottom) set of genes for each time point are clustered and then plotted against the embryonic time points. The significance of the enrichment at a particular time point (negative log of the P-value) is given from zero (white) to dark purple (P = 10−110). Five larger clusters of GO terms are highlighted in rectangles. For example, the fifth cluster has a down, up, down pattern.
Figure 2.
Figure 2.
The relative abundance of novel versus known junctions in alternatively spliced pairs within WormBase gene models. For each splice junction site that could be spliced to two or more other sites, i.e., alternatively spliced, we calculated the ratio of reads for each minor form versus the major form and the numbers of reads spanning the minor form. (A) Sites where both the major and minor isoforms were both present in WormBase. (B) Sites where the major isoform was present in WormBase but the minor form was not. (C) Sites where neither the major nor minor isoform were present in WormBase. (D) Sites where the minor form was present in WormBase, but not the major isoform. For the case in which the minor form is novel and the major form is known (B), a larger fraction of the minor form junctions are rare, e.g., ≤100 reads and ≤5% of the major form, than in the case in which both forms are known (A). However, the absolute number of pairs in which the minor form is not rare is almost twice the number of junctions annotated in WormBase (6782 versus 3428), and the other two cases (C,D) add another 4527 relatively well represented alternatively spliced junctions not in WormBase. The rare junctions could represent splicing errors, but considering their overlap in representation with junctions annotated in WormBase, they could also be biologically important.
Figure 3.
Figure 3.
Alternative splicing in exons 5–9 of the transcription factor gene ceh-38. (A) In WormBase, only the topmost gene model is represented, using introns 5-6 S, 6-7, and 7-8 as illustrated. Our data show the presence of additional introns 5-6 L and 6-8. The former deletes two amino acids from exon 6, changing the spacing between the cut (in exon 5) and homebox (in exon 6) DNA binding domains. Intron 6-8 skips exon 7, but maintains the reading frame. (B) Expression data in dcpm for introns 6-7, 7-8, and 6-8 as well as exons 6, 7, and 8 indicate that although the included form is maternally inherited and then lost rapidly, the skipped form is expressed in the early zygote as well as maternally inherited. (C) Expression data in dcpm for introns 5-6 S and 5-6 L show that the shorter intron is maternally inherited and degraded rapidly. In contrast, the longer form has little maternal contribution, rises rapidly in the early embryo, and persists into the later embryo and larval stages, albeit at lower levels.
Figure 4.
Figure 4.
Operon gene regulation across development. (A) A box plot of the ratio of SL2 reads to all splice leader reads for all second genes in operons across the embryonic time series 0223. Well-expressed second genes in operons (dcpm ≥0.1) were divided into eight equally sized bins (105 genes) based on the overall SL2 fraction. Outliers may indicate stage-specific usage of the two promoters. (B) The same eight bins showing average SL1 (top) and SL2 (bottom) expression in dcpm across development for the second genes. (C) Average H3K27ac signal across the transcript start site of the first (left) and second (right) gene in operons, divided into the same eight bins as in A. Marks of open chromatin may serve to maintain open chromatin for polymerase read-through during transcription of the operon. Average H3K79me2 signal across the transcription start site of the first (left) and second (right) gene in operons, divided into the same eight bins as in A. Promoter areas of these second genes are sites of active regulation.
Figure 5.
Figure 5.
Histone expression across development. (A) The expression in dcpm of the replicative histone, his-64, an example of a pattern with a substantial maternal component. The expression is shown across embryogenesis (labeled in embryo time) for the unified series (red line, open circles), the three rRNA subtracted series (blue, orange, and green), and the poly(A)-selected series (purple) along with the single four-cell sample (red cross). Its pattern is typical of the genes located in the clusters on Chromosome IV. (B) The expression in dcpm of the replicative histone, his-6, an example of a largely zygotically expressed histone. The pattern is shown across the same time points, colored as in A. Its expression pattern is typical of the histone clusters on Chromosomes I and V. (C) The expression in dcpm of his-70, a C-terminally truncated H3.3 variant (Ooi et al. 2006), is shown across embryogenesis (left) and into larval and adult time points (right). The gene lacks an intron but apparently has a poly(A) tail. The embryogenesis time points are colored as in A, the late time points are for L1 through young adult (blue), the dauer stages (green), soma (purple), and male (orange). Both the individual samples (closed symbols) and weighted averages (open symbols) are shown. Post-embryonic time points are shown in approximate chronological order. (D) The percentage of total aligned sequence reads specific to histone genes during embryonic development (time in minutes) for the 0223 rRNA subtracted series (0223) is shown. Uniquely aligned reads are shown in light blue, whereas sequence reads aligning equally to multiple histone gene family members are shown in dark blue.
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