The X-chromosome gene regulatory process called dosage compensation ensures that males (1X) and females (2X) express equal levels of X-chromosome transcripts. The mechanism in Caenorhabditis elegans has been elusive due to improperly annotated transcription start sites (TSSs). Here we define TSSs and the distribution of transcriptionally engaged RNA polymerase II (Pol II) genome-wide in wild-type and dosage-compensation-defective animals to dissect this regulatory mechanism. Our TSS-mapping strategy integrates GRO-seq, which tracks nascent transcription, with a new derivative of this method, called GRO-cap, which recovers nascent RNAs with 5′ caps prior to their removal by co-transcriptional processing. Our analyses reveal that promoter-proximal pausing is rare, unlike in other metazoans, and promoters are unexpectedly far upstream from the 5′ ends of mature mRNAs. We find that C. elegans equalizes X-chromosome expression between the sexes, to a level equivalent to autosomes, by reducing Pol II recruitment to promoters of hermaphrodite X-linked genes using a chromosome-restructuring condensin complex.
In many species, including humans, females have two X chromosomes whereas males have only one. To ensure that females do not end up with a double dose of the proteins encoded by genes on the X chromosome, animals employ a strategy called dosage compensation to control the expression of X-linked genes.
The mechanisms underlying dosage compensation vary between species, but they typically involve a regulatory complex that binds to the X chromosomes of one sex to modify gene expression. In the nematode worm Caenorhabditis elegans—which consists of hermaphrodites (XX) and males (XO)—this regulatory complex, called the dosage compensation complex (DCC), binds to both X chromosomes of XX individuals, reducing gene expression from each by 50%. DCC shares many subunits with a protein complex called condensin, which regulates the structure of chromosomes to achieve proper chromosome segregation. However, it is unclear exactly how the DCC controls the expression of X-linked genes.
For a gene to be expressed, an enzyme called RNA polymerase II must bind to the gene’s promoter—a stretch of DNA upstream of the protein-coding part of the gene—so that it can begin transcribing the DNA into RNA. Promoters have been difficult to define in C. elegans, but Kruesi et al. devised a strategy to map transcription start sites, and hence promoters, throughout the worm genome. The strategy integrates the results of two methods: One measures the extent and orientation of each gene’s transcribed region, and the other locates the distinctive cap structures that mark the true 5′ ends of newly made RNAs.
Using this new promoter information, coupled with genome-wide measurements of the levels of newly synthesized transcripts from wild-type and dosage-compensation-defective animals, they showed that C. elegans achieves dosage compensation by reducing the recruitment of RNA polymerase II to the promoters of X-linked genes in XX individuals.
Kruesi et al. also identified a second regulatory mechanism that acts in both sexes to increase the level of transcription of genes on the X chromosome. This ensures that after dosage compensation, genes on the X chromosome are expressed at a similar level to those on the autosomes (all chromosomes other than X and Y).
As well as shedding light on the mechanism by which dosage compensation occurs in C. elegans, the study by Kruesi et al. provides a valuable data set on transcription start sites in the worm, and puts forward a general strategy that could be used to map these sites in other species.