Condensin controls recruitment of RNA polymerase II to achieve nematode X-chromosome dosage compensation
- Howard Hughes Medical Institute, University of California, Berkeley, United States
- Cornell University, United States
Figures
Figure 1 with 12 supplements
Genome-wide annotation of Caenorhabditis elegans transcription start sites.
(A)–(E) Examples of newly annotated transcription start sites (TSSs) for protein-coding genes, non-coding RNA genes, and multigenic transcription units called operons identified using the …
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Figure 1—source data 1
Aligned reads for GRO-seq, GRO-cap, and ChIP-seq experiments.
The number of reads from each replicate of GRO-seq, GRO-cap, and ChIP-seq that uniquely aligned to the Caenorhabditis elegans genome are listed.
- https://doi.org/10.7554/eLife.00808.004
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Figure 1—source data 2
Annotation of transcription start sites for protein-coding genes.
- https://doi.org/10.7554/eLife.00808.005
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Figure 1—source data 3
Annotation of transcription start sites for non-coding RNAs.
- https://doi.org/10.7554/eLife.00808.006
Figure 1—figure supplement 1
GRO-seq profiles are reproducible between replicates.
The GRO-seq profiles of a select X-chromosome genomic region from two biological replicates of control RNAi embryos and their average GRO-seq profile are shown along with the unique mappability of …
Figure 1—figure supplement 2
Genome-wide GRO-seq signal is highly correlated between replicates.
(A), (C)–(E) Scatter plots comparing GRO-seq signal between biological replicates. (B) Scatter plot comparing GRO-seq signal between averaged replicates of wild-type embryos vs control RNAi embryos. …
Figure 1—figure supplement 3
GRO-seq signal within protein coding genes is highly correlated between replicates.
(A)–(E) Average GRO-seq expression within the gene bodies was calculated using gene models from the WormBase WS230 release. For all genes greater than 1.1 kb, the GRO-seq signal was totaled within …
Figure 1—figure supplement 4
GRO-seq expression is correlated with gene expression from microarray and RNA-seq experiments.
(A) GRO-seq experiments have a higher dynamic range than microarray experiments. Scatter plots are shown of gene expression levels determined by GRO-seq vs microarray experiments from control RNAi …
Figure 1—figure supplement 5
Genome-wide annotation of TSSs improves gene models.
(A)–(D) To gauge the improvement of our new transcription start site (TSS) calls on gene model accuracy, we plotted the average GRO-seq signal across a 2 kb window centered on the WormBase (WB) …
Figure 1—figure supplement 6
GRO-cap signal is strong at newly annotated TSSs.
(A)–(D) Because the corrected GRO-cap signal (TAP+ signal after subtracting the TAP− signal) was used to annotate transcription start sites (TSSs), we assessed whether our TSS calls coincided with …
Figure 1—figure supplement 7
Heat maps showing GRO-seq and GRO-cap signal relative to either WB starts or TSSs for developmental stages reveal improvements in gene models.
(A)–(C) The GRO-seq signal was plotted, one gene per row, for each gene of a specific developmental stage relative to the WormBase (WB) starts. The genes were ordered from top to bottom with …
Figure 1—figure supplement 8
TSSs can be far upstream of the previously annotated WB starts.
(A)–(C) Shown are GRO-seq signals, GRO-cap signals (TAP+ and TAP− or TAP− subtracted from TAP+ [E–F]), ChIP-chip signals of phospho ser 2 Pol II (from Pferdehirt et al., 2011), and ChIP-chip signals …
Figure 1—figure supplement 9
GRO-cap revealed that 21 U-RNAs have a TSS 2 bp upstream of the mature RNA.
GRO-cap readily identified the transcription start sites (TSSs) of 21 U-RNAs from L3 larvae. To map TSSs, we determined the highest GRO-cap signal (TAP+ minus TAP−) within 10 bp of the 5′ end of …
Figure 1—figure supplement 10
Features of promoters and TSSs.
(A) A transcription start site (TSS) can be far downstream of the WormBase (WB) start. The TSS for MO3C11.3 is 2510 bp downstream of the WB start in all developmental stages examined. The TSS was …
Figure 1—figure supplement 11
Distances between the TSS and WB starts of the trans-splicing acceptor site.
(A) and (B) For all genes with a transcription start site (TSS) called in wild-type embryos, the difference between the TSS and WormBase (WB) start or SL1 trans-splice acceptor site was grouped in …
Figure 1—figure supplement 12
Comparison of enhancers in Chen et al. (2013) and our annotated TSSs.
(A)–(C) Comparison of our GRO-seq and GRO-cap profiles to enhancer regions from Chen et al. (2013). (A) Chen et al. (2013) analyzed scRNA sequencing data from mixed-stage embryos to annotate …
Figure 2
GRO-cap strategy for identifying TSSs.
GRO-cap is a modified form of GRO-seq that utilizes the tagging and extensive purification of nascent RNAs from GRO-seq (Core et al., 2008) and then employs redundant enzymatic steps to enrich for …
Figure 3 with 2 supplements
Features of promoters and TSSs.
(A) Trans-spliced genes can have multiple transcription start sites (TSSs), suggesting that trans-splicing eliminates the pressure to have only one precise TSS per gene. Shown are GRO-seq and …
Figure 3—figure supplement 1
Evolutionarily conserved promoter elements.
(A) A TATA box element with one or no mismatch from the consensus (TATAWAWR) is highly enriched 15–45 bp upstream of transcription start sites (TSSs) for 391 of 4547 Caenorhabditis elegans genes. …
Figure 3—figure supplement 2
Conserved core promoter elements in promoters of microRNA genes.
(A) A TATA box element with a perfect match to the consensus derived for genes encoding microRNAs was highly enriched 29–32 bp upstream of transcription start sites (TSSs) for 15 of 57 microRNA …
Figure 4 with 3 supplements
Features of Caenorhabditis elegans transcription: 3′ Pol II pausing and divergent transcription.
(A)–(C) Pol II 3′ accumulation is prevalent in worms. 3′ End pausing ratios were calculated by dividing the highest average GRO-seq signal at the 3′ end by the average GRO-seq signal in the gene …
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Figure 4—source data 1
Gene expression, and 5′ pausing and 3′ pausing data for protein-coding genes.
- https://doi.org/10.7554/eLife.00808.024
Figure 4—figure supplement 1
The 3′ accumulation of RNA polymerase II.
(A) The 3′ accumulation of RNA Pol II is positively correlated with gene expression. The maximum average GRO-seq signal from control RNAi embryos was calculated across a 200 bp window at the 3′ end …
Figure 4—figure supplement 2
RNA polymerase II accumulation at 3′ ends compared against trans-spliced genes, non-trans-spliced genes, and U-rich regions at 3′ ends.
(A) Shown are the 3′ pausing ratios for the first and middle genes in operons (n = 949), last genes in operons (n = 625), monocistronic genes with trans-splicing (n = 603), monocistronic genes …
Figure 4—figure supplement 3
Divergent transcription in Caenorhabditis elegans.
(A) and (B) Comparison of average GRO-seq signal around promoters with a divergent gene pair vs promoters not associated with a divergent gene partner. The genes analyzed had a log2(sense/antisense) …
Figure 5 with 3 supplements
GRO-seq analysis of dosage-compensation.
(A) Genetic hierarchy for coordinate control of sex determination and dosage compensation. sdc-2 is expressed solely in XX embryos and triggers the hermaphrodite fate. sdc-2 acts together with sdc-1 …
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Figure 5—source data 1
Genome-wide changes in gene expression caused by the disruption of dosage compensation.
Genes were separated into different gene sets based on their length, origin, and chromosome (X vs autosome) to compare GRO-seq gene expression between the sdc-2 mutant and control RNAi embryos. Shown are the total number of genes, the median sdc-2 mutant/control RNAi expression ratio, the average sdc-2 mutant/control RNAi expression ratio together with the standard error of the mean, and the number of genes in each set that are more highly expressed in each condition. Across numerous protein-coding gene sets, the X chromosome is more highly expressed and the autosomes are slightly less expressed in the sdc-2 mutant. Furthermore, when X-linked genes are significantly changed in expression, they are almost exclusively increased in expression. Each gene set is separated into two sets, one containing all genes and the other containing genes that are significantly changed in expression as determined by analysis with DESeq (p<0.05) (Anders and Huber, 2010). For the first two lists (labeled with ‘≥250 bp’), average GRO-seq gene expression was calculated from the beginning to the end of either the WormBase (WB) model or the newly annotated transcription start site (TSS) gene model. WB genes had to be expressed at greater than 1 RPKM (reads per kilobase per million), and have at least 250 uniquely mappable bases in both sets. For the next set (labeled with ‘WormBase WS230 Genes ≥1.1 kb’), average GRO-seq expression was calculated for genes greater than 1.1 kb, with the first and last 300 bp of the gene excluded. The level of expression had to be ≥1 RPKM for WB genes, and have at least 250 uniquely mappable bases for a gene to be included. For the final two sets of genes (labeled with ‘≥1.1 kb’ and ‘≥1.5 kb’), expression was calculated for genes of the indicated length that have a newly annotated TSS, with the first and last 300 bp of the gene excluded. A gene had to have at least 250 uniquely mappable bases for it to be included. RNA polymerase II transcribed microRNAs are controlled by dosage compensation, while RNA polymerase III transcribed tRNAs are not. Average GRO-seq gene expression from sdc-2 mutant and control RNAi embryos was compared across ncRNAs. For microRNAs, expression values were calculated from the full length of the WB ‘primary transcript’ or re-annotated TSS gene models. For tRNAs, expression values were calculated from the beginning of the ‘mature transcript’ to 50 bp downstream of the stop. Because tRNAs are highly repetitive and transcription of highly transcribed tRNAs continues downstream of the stop, the extra 50 bp was included to increase the unique mappability of each tRNA. For a gene to be considered for analysis, it had to have at least 25 bp of uniquely mappable DNA, and to have an average expression of at least 1 RPKM in both control RNAi and sdc-2 mutant embryos. The median and mean sdc-2/control expression levels show that X-linked microRNAs are more susceptible to dosage compensation than autosomal microRNAs. X-linked tRNAs are decreased slightly in expression in the sdc-2 mutant, suggesting that its expression is not controlled by dosage compensation.
- https://doi.org/10.7554/eLife.00808.029
Figure 5—figure supplement 1
Western blot shows the reduction in SDC-2 protein levels in sdc-2(y93, RNAi) animals.
The sdc-2(y93) partial-loss-of function mutant was treated with RNAi against sdc-2 to reduce its gene activity. Extracts from wild-type and sdc-2 mutant embryos were fractionated on an SDS-PAGE gel, …
Figure 5—figure supplement 2
X-linked gene expression is selectively increased in sdc-2 mutants.
(A)–(D) Scatter plots of the average GRO-seq signal (log2 RPKM) from RNAi control and sdc-2 mutant embryos from the bodies of genes on X or autosomes. The genes had to be ≥1.1 kb and had to have at …
Figure 5—figure supplement 3
Occupancy of the DCC subunit DPY-27 in the promoter of a gene is correlated with the gene’s expression level but not its dosage compensation status.
The total DPY-27 ChIP-seq signal was calculated for the 500 bp window upstream of the transcription start sites (TSSs) for X-linked genes and compared with the expression level of the genes in …
Figure 6 with 1 supplement
Promoter-proximal RNA Pol II pausing is rare in Caenorhabditis elegans and is not the target of dosage compensation.
(A) GRO-seq and GRO-cap signals show that a gene not paused in embryos becomes paused in L1 larvae deprived of food. (B) Comparison of average GRO-seq signal from embryos, starved L1 larvae, and L3 …
Figure 6—figure supplement 1
The dosage compensation process does not control promoter-proximal pausing of Pol II.
(A)–(C) Shown are plots of the average GRO-seq signal from different developmental stages of wild-type embryos plotted across a 2 kb window centered on the transcription start sites (TSSs) …
Figure 7 with 4 supplements
The DCC condensin complex reduces X-chromosome gene expression in XX embryos by restricting Pol II recruitment to promoters.
(A) Uniform increase in GRO-seq signal across the length of X-linked genes results from disrupting dosage compensation. Metagene analysis comparing the average GRO-seq signal from 665 X-linked genes …
Figure 7—figure supplement 1
GRO-seq signal is increased along the length of individual X-linked genes when dosage compensation is disrupted.
The GRO-seq signal from sdc-2 mutant vs control RNAi embryos is shown for a representative set of 27 X-linked genes. The first vertical line in each gene shows the location of the transcription …
Figure 7—figure supplement 2
Disruption of dosage compensation causes a uniform increase in GRO-seq signal across the length of X-linked genes in different quartiles of gene expression determined from control RNAi samples.
(A)–(D) Metagene analyses comparing the average GRO-seq signal from X-linked genes ≥1.5 kb in control RNAi and sdc-2 mutant embryos. The 665 X-linked genes have been split into four quartiles of …
Figure 7—figure supplement 3
Disruption of dosage compensation causes a uniform increase in GRO-seq signal across the length of X-linked genes of different size ranges.
(A)–(C) Shown are metagene analyses comparing average GRO-seq signal from X-linked genes ≥1.5 kb in control RNAi or sdc-2 mutant embryos. The 665 X-linked genes have been split into three size …
Figure 7—figure supplement 4
The level of antisense transcription is unaffected by dosage compensation.
(A) Sense and upstream divergent transcription are coordinately increased for X-linked genes in sdc-2 mutants. Scatter plot comparing the log2(sense/antisense) ratios for sdc-2 mutant and control …
Figure 8
Gene expression is balanced between X chromosomes and autosomes.
(A) Caenorhabditis elegans has a mechanism to equalize expression between X chromosomes and autosomes. Metagene analysis comparing the average GRO-seq signal from X-linked and autosome-linked genes …
