Counting X Chromosomes and Autosomes to Determine Sex
In numerous organisms, sex is determined by a counting mechanism that distinguishes one X chromosome from two. Embryos with one X chromosome develop as males, and those with two develop as females or hermaphrodites. We have used the model organism Caenorhabditis elegans to dissect one such precise chromosome-counting mechanism in molecular detail. In this nematode, at least five X-linked genes—called X signal elements (XSEs)—communicate the embryo's X chromosome dose by controlling the activity of one gene, the master sex-determination gene xol-1 (XO lethal). The two doses of X signal elements in XX animals repress xol-1, promoting the hermaphrodite fate, while the single dose in XO animals activates xol-1, promoting the male fate.
We have shown that XSEs use at least two mechanisms to repress xol-1. The first involves transcriptional repression and requires the XSEs called sex-1 (signal element on X) and ceh-39 (C. eleganshomeobox). SEX-1 protein belongs to the nuclear hormone receptor family of proteins used widely to translate environmental cues into appropriate levels of gene expression. (A similar molecule is involved in the first steps of mammalian sex determination.) CEH-39 belongs to the ONECUT family of homeodomain proteins used widely to determine an animal's body plan. SEX-1 and CEH-39 act directly on xol-1, associating with its promoter in vivo to repress xol-1 transcription in XX embryos. Thus, xol-1 is the direct molecular target of the primary sex-determination signal.
The second mechanism involves the post-transcriptional regulation of xol-1 and requires fox-1 (feminizing locus on X), which encodes an RNA-binding protein. The xol-1 pre-mRNA is alternatively processed, and FOX-1 binds an intron of xol-1, thus preventing the RNA-processing event that generates the functional xol-1 transcript.
Both the transcriptional and RNA-splicing mechanisms of xol-1 repression are essential, and the XSEs act cumulatively to repress xol-1 fully. Moreover, increasing the degree of xol-1 repression by one mechanism can compensate for loss of the other. Such multigenic regulatory mechanisms serve as models for understanding complex human genetic traits.
Rather than counting the absolute number of X chromosomes, the nematode counts X chromosome number relative to the sets of autosomes (non–X chromosomes) to determine sex. We showed that the autosomal component of this X:A signal includes a set of dose-sensitive genes, called autosomal-signal elements (ASEs), that communicate the ploidy by activating xol-1. ASEs include the T-box protein SEA-1 and the zinc finger protein SEA-2, both of which bind directly to the xol-1 promoter to turn on its transcription. How two doses of ASEs oppose the single dose of XSEs in XO animals to activate xol-1 is under investigation. These experiments will answer the long-standing issue of how a twofold difference in the concentration of molecular signals can be translated into alternative developmental fates. (A grant from the National Institutes of Health supports aspects of this work.)
Epigenetic Control of X Chromosome Dosage Compensation and Its Relationship to Chromosome Segregation
The C. elegans sex-determination mechanism causes males and hermaphrodites to differ in their dose of X chromosomes, yet both sexes require equivalent levels of X chromosome gene products. We showed that an X chromosome–wide regulatory process called dosage compensation neutralizes the difference in X chromosome dose between sexes by equalizing X chromosome transcript levels. In C. elegans and other organisms (such as flies and mammals) that also use chromosome-based mechanisms to determine sex, a specialized dosage compensation complex is targeted exclusively to the X chromosome(s) of only one sex to modulate X transcript levels in that sex. This selective recruitment of the dosage compensation machinery establishes the epigenetic regulation of X chromosomes that is maintained throughout the lifetime of the animal. Dosage compensation is essential, and failure to accomplish this global regulation causes lethality to only one sex. By studying dosage compensation, we expect to discover how the expression state of an entire chromosome can be established and maintained.
Fundamental questions are relevant to all forms of dosage compensation. (1) What is the composition of the machinery that implements dosage compensation? (2) What are the sex-specific factors that activate the dosage compensation machinery in only one sex? (3) What is the nature of cis-acting recruitment sites that target X chromosomes for regulation by the dosage compensation complex? (4) How is gene expression coordinately controlled along an entire X chromosome? (5) What is the molecular mechanism for fine-tuning X-linked gene expression by only twofold? We have used integrated genetic, biochemical, and cell biological approaches to address these basic questions in C. elegans and to dissect this regulatory process.
We showed that C. elegans dosage compensation is achieved by a complex of ~10 proteins that assembles on both X chromosomes of young hermaphrodite embryos to reduce X transcript levels by half. Five members of the complex resemble 13S condensin, a conserved complex essential for the compaction and resolution of mitotic and meiotic chromosomes from yeast to humans (Figure 1a). One member of the dosage compensation complex functions not only in regulating gene expression but also separately in mitotic and meiotic chromosome segregation. The protein partitions its roles in the two separate biological processes, gene expression and chromosome segregation, through its participation in two separate condensin-like complexes (Figure 1a). When associated with the dosage compensation complex, the protein binds to X (Figure 1b); when associated with the mitotic/meiotic condensin complex, the protein colocalizes with the centromeres of mitotic chromosomes in both sexes (Figure 1c) or with the meiotic chromosomes in diplotene/diakinesis (Figure 1d). These results suggest that the C. elegans dosage compensation process evolved by recruiting components used in other chromosome behaviors to the new task of fine-tuning gene expression.
The similarity of the dosage compensation complex to condensin and the participation of one protein in both condensin complexes suggest a common mechanism for repressing X chromosome gene expression during dosage compensation and for establishing chromosome resolution and higher-order chromosome structure during mitotic and meiotic chromosome segregation. Other members of the dosage compensation complex have retained their roles in conserved cellular processes: one regulates gene expression through its association with a complex that methylates histones; two participate in regulating the number and distribution of crossovers between homologous meiotic chromosomes.
Meiotic Crossover Control by Dosage Compensation Proteins Mediated by Chromosome Structure
During meiosis, homologous chromosomes recombine to ensure proper segregation. Crossovers are tightly controlled: meiotic crossovers display an interference pattern such that one crossover decreases the likelihood of a proximal crossover. In C. elegans, interference is extreme; only one crossover occurs per homolog pair per meiosis. This regulation could be imposed at many steps, starting with the formation of programmed double-strand breaks (DSBs), which initiate crossovers. In many organisms, the number of DSBs far exceeds the number of resultant crossovers, suggesting that much of the crossover/noncrossover decision is made after DSB formation. In C. elegans, we found that DSB formation is a critical control point for determining crossover number and distribution, and that DSB number is regulated by dosage compensation components DPY-28 and DPY-26. These proteins regulate DSB number by controlling chromosomal organization (Figure 1e), and regulation of DSB number restricts crossover formation.
Targeting the Dosage Compensation Complex to X Chromosomes Only in Hermaphrodites
Our work showed that sex determination and dosage compensation are coordinately regulated by a group of genes that respond to the primary sex-determination signal (X:A). Following a common step of regulation, sex determination and dosage compensation are separately controlled by distinct genetic pathways. Two of the coordinate control proteins, SDC-2 and SDC-3 (sex determination and dosage compensation), play pivotal roles in localizing the dosage compensation complex to X. SDC-2 is unique in localizing to X in the absence of other dosage compensation components, suggesting that SDC-2 plays a central role in X chromosome recognition and confers chromosome specificity to dosage compensation. SDC-2 also confers hermaphrodite specificity: it is the sole dosage compensation protein produced exclusively in hermaphrodites. Its activity is repressed in males by xol-1. SDC-2 is thus the pivotal sex-specific factor that triggers the hermaphrodite program of dosage compensation. Once the dosage compensation machinery assembles on X, it remains localized to X throughout the cell cycle (Figure 1f).
Recruitment and Spreading of the Dosage Compensation Complex Along Hermaphrodite X Chromosomes
To define the special features of X that distinguish it from autosomes to recruit the dosage compensation complex, we first conducted a chromosome-wide search to define large cis-acting X regions that bind the complex when detached from X. Detached X regions were found that robustly recruited the complex, weakly recruited the complex, or failed to recruit the complex. When located on the native X, all regions recruited the complex robustly. Thus, multiple, discrete, well-dispersed X sites recruit the dosage compensation complex and nucleate spreading of the complex, over short or long distances, to regions of X that lack recruitment sites, thus establishing the global epigenetic regulation of X that is maintained throughout the lifetime of hermaphrodites.
We then discovered and dissected small, cis-acting sites that mark nematode X chromosomes as targets for gene repression by the dosage compensation complex. These recruitment elements on X (rex sites) were found through a general strategy that assessed binding of the dosage compensation complex to DNA in vivo (Figure 1g). At least 30 rex sites have been identified; several have been reduced to less than 200 base pairs of DNA. These rex sites are widely dispersed along X and share at least two distinct motifs that act in combination to recruit the dosage compensation complex. Mutating these motifs severely reduces or abolishes binding of the dosage compensation complex in vivo, demonstrating the importance of primary DNA sequence in chromosome-wide regulation. Unexpectedly, the motifs are not enriched on X, but altering motif numbers within rex sites demonstrates that motif co-occurrence in unusually high densities is essential for optimal recruitment of the dosage compensation complex. Thus, X-specific repression can be established through sequences not specific to X. Rather, the distinctive distribution of common motifs within recruitment regions appears to initiate repression along an entire chromosome.
The overall strategy by which C. elegans X chromosomes attract the condensin-like dosage compensation complex has emerged. Future experiments will determine the mechanism by which the dosage compensation complex spreads along X from initial recruitment sites and whether such spreading involves histone modifications and/or noncoding RNAs, as do other forms of epigenetic regulation across a whole chromosome or within chromosome domains. Ultimately, a detailed understanding of the mechanism underlying the twofold repression in X chromosome expression will emerge.