Our studies have revealed a mechanistic link between the remodeling of X chromosome topology that underlies dosage compensation in the nematode Caenorhabditis elegans and the remodeling of higher-order chromosome structure that underlies accurate chromosome segregation. The changes in chromosome structure are mediated by distinct condensin complexes that arise from the reshuffling of interchangeable subunits to create independent molecular machines with similar architectures but distinct functions (Figures 1 and 2).
Counting Chromosomes to Determine Sex
Many organisms specify sexual fate by a chromosome-counting mechanism that distinguishes one X chromosome from two. Embryos with one X become males, while those with two become females. We dissected the C. elegans counting mechanism in molecular detail to understand how small changes in the concentrations of molecular signals are translated into different developmental fates. This nematode tallies X chromosome number relative to the ploidy, the sets of autosomes (X:A signal). It discriminates with high fidelity between tiny differences in the signal (Figure 3). We found that genes encoded on X, called X-signal elements (XSEs), communicate X chromosome dose by acting in a cumulative, dose-dependent manner to repress the master sex-determining gene xol-1, a GHMP kinase homolog (Figure 3). In contrast, genes on autosomes, called autosomal-signal elements (ASEs), communicate autosomal dose by activating xol-1 (Figure 3). In diploid embryos, two doses of XSEs counteract the two doses of ASEs to repress xol-1, permitting hermaphrodite development, while two doses of ASEs counteract the single dose of XSEs to activate xol-1 and trigger male development. XSEs (nuclear hormone receptors and homeodomain proteins) and ASEs (T-box and zinc finger proteins) bind directly to multiple sites on xol-1 to antagonize each other's activities and thereby regulate xol-1 transcription (Figure 4). XSEs likely antagonize ASEs by recruiting cofactors with reciprocal activities that induce opposite transcriptional states. Thus, multiple antagonistic molecular interactions carried out on a single promoter help explain how tiny differences in the X:A signal elicit different sexual fates. Although most XSEs repress xol-1 by regulating transcription, one XSE, an RNA-binding protein, represses xol-1 by blocking its pre-mRNA splicing, thereby generating a nonfunctional transcript with an in-frame stop codon (Figure 4). This second tier of repression enhances the fidelity of the counting process. The concept of a sex signal having competing XSEs and ASEs arose as a theory for fruit flies one century ago and became entrenched in textbooks. Ironically, recent work of others showed that the fly sex signal does not fit this simple paradigm, while our work shows the worm signal does.
Repressing X Chromosomes via Molecular Machines
Organisms that use sex chromosomes to determine sexual fate evolved the essential, chromosome-wide regulatory process called dosage compensation to balance X chromosome gene expression between the sexes. Strategies for dosage compensation differ, but invariably a regulatory complex is targeted to X chromosomes of one sex to modulate transcription along the entire chromosome. The heritable, epigenetic regulation of X chromosome expression during dosage compensation is exemplary for dissecting the coordinate regulation of gene expression over large chromosomal territories and the role of chromosome structure in regulating gene expression. We found that the C. elegans dosage-compensation complex (DCC) is homologous to condensin, a conserved protein complex that mediates the compaction, resolution, and segregation of mitotic and meiotic chromosomes from yeast to humans (Figure 1). The DCC binds to both X chromosomes of hermaphrodites to reduce transcription by half (Figure 1). Failure to reduce expression kills hermaphrodites. Most DCC condensin subunits also control the structure and function of mitotic and meiotic chromosomes by participating in two other distinct condensin complexes (Figure 2).
The DCC condensin subunits are recruited specifically to hermaphrodite X chromosomes by sex-specific DCC subunits that trigger binding to cis-acting regulatory elements on X: rex (recruitment elements on X) and dox (dependent on X) sites. rex sites recruit the DCC in an autonomous, sequence-dependent manner, using DNA motifs highly enriched on X chromosomes. The DCC spreads to dox sites, which reside in promoters of active genes and bind the DCC robustly only when linked to rex sites. Stable DCC assembly onto rex sites requires the sex-specific SUMOylation of three DCC subunits. Not only has the DCC co-opted subunits of condensin to control gene expression, it co-opted a subunit from the MLL/COMPASS complex, a histone-modifying complex, to help recruit condensin subunits to rex sites.
In principle, the DCC could control any step of transcription: recruitment of RNA polymerase II (Pol II) to the promoter, initiation of transcription, escape of Pol II from the promoter or pause sites, elongation of RNA transcripts, or termination of transcription. To understand the step of transcription controlled by the DCC, we devised a procedure to map the position, density, and orientation of transcriptionally engaged Pol II genome-wide and a strategy to identify transcription start sites (TSSs), in collaboration with John Lis (Cornell University). Nascent RNA transcripts from most nematode genes undergo rapid cotranscriptional processing in which the 5' end is replaced by a common 22-nucleotide leader RNA through a trans-splicing mechanism, thereby removing all information about TSSs and promoters. The paucity of annotated promoters made transcription regulation difficult to study. Our work established a general strategy for TSS mapping and provided an invaluable TSS data set. We found that promoter-proximal pausing is rare, unlike in other metazoans, and promoters are unexpectedly far upstream from 5' ends of mature mRNAs. By comparing the location and density of transcriptionally engaged Pol II in wild-type and dosage-compensation-defective embryos, we found that C. elegans equalizes X chromosome–wide gene expression between the sexes by reducing Pol II recruitment to the promoters of X-linked genes in XX embryos. We are dissecting the mechanisms by which the DCC limits Pol II recruitment.
Condensin-Driven Remodeling of X Chromosome Topology during Dosage Compensation
The three-dimensional organization of a genome plays a critical role in regulating gene expression, yet little is known about the machinery and mechanisms that determine higher-order chromosome structure. The involvement of bona fide condensin subunits in dosage compensation suggested that the DCC might alter the topology of X chromosomes to reduce gene expression chromosome-wide. Using genome-wide chromosome conformation capture techniques (in collaboration with Job Dekker, University of Massachusetts Medical School) with single-cell fluorescence in situ hybridization and RNA-seq to compare chromosome structure and gene expression in wild-type and dosage-compensation-defective embryos, we showed that the DCC remodels X chromosomes of hermaphrodites into a unique, sex-specific spatial conformation, distinct from autosomes, using its highest-affinity rex sites to facilitate long-range interactions across X.
Dosage-compensated X chromosomes consist of self-interacting domains (~1 Mb) resembling mammalian topologically associating domains (TADs). TADs on X have stronger boundaries and more regular spacing than those on autosomes. Many TAD boundaries on X coincide with the highest-affinity rex sites, and these boundaries become diminished or lost in mutants lacking DCC binding, causing the structure of X to resemble that of autosomes. Furthermore, deletion of an endogenous rex site at a DCC-dependent boundary disrupted the boundary, further demonstrating the condensin-driven remodeling of X chromosome topology during dosage compensation. Thus, condensin acts as a key structural element to reorganize interphase chromosomes and thereby regulate gene expression. No other molecular trigger or set of DNA-binding sites is yet known to cause a comparably strong effect on TAD structure in higher eukaryotes. Our understanding of the topology of dosage-compensated X chromosomes provides fertile ground to decipher the detailed mechanistic relationship between higher-order chromosome structure and chromosome-wide regulation of gene expression.
Targeted Genome Editing across Highly Diverged Nematode Species
Thwarted by the lack of reverse genetic approaches to enable cross-species comparisons of gene function, we established robust strategies for targeted genome editing across nematode species diverged by 300 million years. We used site-specific nucleases with engineered specificity. The first engineered nucleases contained fusions between the DNA-cleavage domain of the enzyme FokI and a custom-designed DNA-binding domain: either zinc finger motifs for zinc finger nucleases or transcription activator-like effector (TALE) domains for TALE nucleases (TALENs). We then adopted the CRISPR-associated nuclease Cas9, which uses RNA guides to program target specificity. All three nucleases induce DNA double-strand breaks (DSBs) at specifically designated loci. DSBs are repaired imprecisely by nonhomologous end joining (NHEJ) to generate random insertions and deletions or repaired precisely by homology-directed repair (HDR) from exogenously supplied templates to generate specific insertions or deletions.
Despite successful application of Cas9 technology, predicting DNA targets and guide RNAs that support efficient genome editing was problematic. We devised a strategy for high-frequency genome editing (both NHEJ and HDR) at all targets tested. The key innovation was designing guide RNAs with a GG motif at the 3' end of their target-specific sequences. This design increased the frequency of mutagenesis 10-fold. The ease of mutant recovery was further enhanced by combining this efficient guide design with a coconversion strategy, in which targets of interest are analyzed in animals exhibiting a dominant phenotype caused by Cas9-dependent editing of an unrelated target.
Evolution of X Chromosomes and cis-Acting Regulatory Sites
Mechanisms that specify sexual fate and compensate for X chromosome dose have diverged rapidly compared to other developmental processes, making it informative to study both processes over short evolutionary timescales. Application of genome-editing strategies to Caenorhabditis briggsae revealed that the core dosage-compensation machinery and key components of the genetic hierarchy that controls dosage compensation were conserved across the 30-million-year separation between C. elegans and C. briggsae. In contrast, the set of cis-acting elements on X that recruit the DCC (rex sites) has diverged, retaining no functional overlap. The evolution of these sites differs dramatically from unchanged DCC-binding sites used by equivalently diverged fruit fly species and from unchanged target sites of conserved transcription factors that control multiple developmental processes from flies to humans. Hence, the divergence in DCC-binding specificity across nematode species provides an opportunity to understand the path and timing for the concerted change in hundreds of DNA target sites and the evolution of X chromosomes.
Tethering Replicated Chromosomes via Cohesin to Ensure Genome Stability
Faithful segregation of chromosomes during cell division is essential for genome stability. Accurate chromosome segregation is required both for the proliferative cell divisions that produce daughter cells during mitosis and the two sequential divisions that produce haploid sperm and eggs from diploid germline stem cells during meiosis. Approximately 30 percent of human zygotes have abnormal chromosome content at conception due to defects in meiosis. Such aneuploidy is a leading cause of miscarriages and birth defects and arises, in part, from defects in sister chromatid cohesion (SCC). SCC tethers replicated sister chromatids prior to cell divisions to ensure proper chromosome segregation. In humans, SCC is established in the developing germ cells of a fetus and must be maintained until ovulation in adults. This long-lived SCC is established and maintained by cohesin complexes, evolutionarily conserved protein complexes structurally related to condensin (Figure 5).
Studies in budding yeast showed that mitotic and meiotic cohesins are distinct but differ only in a single subunit called the kleisin. During yeast meiosis, a single cohesin complex carries out all aspects of SCC. In contrast, our work in nematodes shows that regulation of meiotic SCC in higher eukaryotes is more complex. We found that multiple functionally specialized cohesin complexes mediate the establishment and two-step release of SCC that underlies the production of haploid gametes (Figure 5). The meiotic complexes differ by a single kleisin subunit, and the kleisin influences nearly all aspects of meiotic cohesin function: the mechanisms for loading cohesins onto chromosomes, for triggering DNA-bound cohesins to become cohesive, and for releasing cohesins in a temporal- and location-specific manner (Figure 6). One kleisin triggers cohesion just after the chromosomes replicate, as in yeast. Unexpectedly, the other triggers cohesion in a replication-independent manner, only after programmed DSBs are made during meiosis to initiate recombination between homologous maternal and paternal chromosomes. Our findings establish a new model for cohesin function in meiosis: the choreographed actions of multiple cohesins, endowed with specialized functions by their kleisins, underlie the stepwise separation of homologous chromosomes and then sister chromatids required for reduction of genome copy number. This model diverges significantly from that in yeast but likely applies to plants and mammals, which utilize similar meiotic kleisins.
Grants from the National Institutes of Health and the Miller Institute at U.C. Berkeley provided partial support for these projects.
As of March 23, 2016