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[
International Worm Meeting,
2011]
Dosage compensation is a mechanism that equalizes gene expression from the X chromosomes between heterogametic sexes. In Caenorhabditis elegans, the dosage compensation complex (DCC) binds both hermaphrodite X chromosomes to repress transcription by approximately half, to equal the level expression from the single male X. Although C. elegans and C. briggsae diverged 15-30 million years ago, our analysis has shown that dosage compensation complex (DCC) subunits are conserved between species. Each C. elegans DCC component has a homolog in C. briggsae, and the DCC components DPY-27, MIX-1, and SDC-2 have been shown to have similar functions in C. briggsae dosage compensation. However, while DCC components appear conserved, DCC binding sites appear diverged. The C. elegans consensus motif (MEX, motif enriched on X) pivotal for C. elegans DCC recruitment to X is only enriched 0.6-2-fold on C. briggsae X compared to autosomes, in contrast to the 3.8-24-fold enrichment on the C. elegans X chromosome. Furthermore, we characterized the recruitment potential of several C. elegans recruitment sites and their C. briggsae homologous regions in both species. No C. elegans or C. briggsae sequences tested were able to recruit the DCC in C. briggsae to the same degree as in C. elegans. This suggests that the cis-acting DNA recruitment sites in C. briggsae have diverged. Ongoing ChIP-seq experiments to define the C. briggsae DCC binding sites will reveal the degree of divergence. The identification of DNA binding sequences in C. briggsae will set the stage to allow us to investigate the molecular co-evolution of the DNA sequence motif and the DNA-binding domain of the DCC.
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[
International Worm Meeting,
2015]
Dosage compensation (DC) across Caenorhabditis species exemplifies an essential process that has undergone rapid co-evolution of protein-DNA interactions central to its mechanism. In C. elegans, recruitment elements on X (rex sites) recruit a condensin-like DC complex (DCC) to hermaphrodite X chromosomes to balance gene expression between the sexes. Recruitment assays in vivo showed that C. elegans rex sites do not recruit the DCC of C. briggsae, and vice versa. To understand how DC complexes and X chromosomes evolved to use different X targeting sequences, we compared DCC subunits and binding sites in C. elegans to those in three species of the C. briggsae clade (15-30 MYR diverged): C. briggsae, its close relative C. nigoni (C. sp. 9), and C. tropicalis (C. sp. 11). By raising antibodies and introducing endogenous tags with TALENs or CRISPR/Cas9, we showed that homologs of both SDC-2, the pivotal X targeting factor, and DPY-27, a DCC-specific condensin subunit, bind X chromosomes of XX animals. Although the DCC shares key components across these four species, the binding sites differ. First, ChIP-seq studies in C. briggsae and C. nigoni identified DCC binding sites that are homologous across these close relatives but differ from C. elegans sites in sequence and location. Second, C. elegans sites use motifs enriched on X (MEX and MEXII) to drive DCC binding, but these motifs are not in C. briggsae or C. nigoni DCC sites and are not X-enriched. Third, we found an X-enriched motif at DCC binding sites of C. briggsae and C. nigoni that is not X-enriched in C. elegans. An oligo with the C. briggsae motif recruits the DCC in C. briggsae, but a similar oligo lacking the motif fails to recruit, establishing the importance of the motif. Fourth, another motif was found in C. briggsae and C. nigoni that shares a few nucleotides with MEX, but its functional divergence was shown by C. elegans recruitment assays. Fifth, two endogenous C. briggsae X-chromosome regions with strong C. elegans MEX motifs fail to recruit the C. briggsae DCC, as assayed by ChIP-seq and recruitment assays. None of these DCC motifs is enriched on the C. tropicalis draft X sequence, supporting further binding site divergence within the C. briggsae clade. Ongoing ChIP-seq studies in C. tropicalis will help determine how C. elegans and C. briggsae clade motifs are evolutionarily related. Comparison of DCC targeting mechanisms across these four species allows us to characterize a rarely captured event: the recent co-evolution of a protein complex and its rapidly diverged target sequences across an entire X chromosome.
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[
International Worm Meeting,
2013]
Comparative studies have shown remarkable divergence in the conservation of developmental mechanisms. Strategies to determine sexual fate and to compensate X-chromosome dosage between sexes have evolved particularly rapidly: mammals, flies, and worms utilize different methods. Understanding such rapidly changing processes requires comparisons over shorter evolutionary time-scales, such as between C. briggsae and C. elegans. Comparison of sex determination and dosage compensation across nematode species using heritable, targeted mutagenesis protocols we developed has shown that key features of the dosage compensation complex (DCC) and the genetic pathway that coordinates sex determination and dosage compensation are conserved. Despite conservation of the DCC and its regulatory hierarchy, X-chromosome targeting mechanisms have diverged. The cis-acting DNA recruitment elements on X (rex) and their motifs are distinct. C. elegans rex sites ported to C. briggsae fail to bind the C. briggsae DCC. The reciprocal also holds: C. briggsae rex sites ported into C. elegans fail to bind the C. elegans DCC. Also, C. briggsae rex sites lack the X-enriched C. elegans DNA motifs pivotal for DCC recruitment. The divergence of DCC binding sites between C. elegans and C. briggsae prompted us to explore X targeting in C. sp. 9, which is closer to C. briggsae than to C. elegans. C. sp. 9 proteins homologous to DCC subunits of C. briggsae and C. elegans co-localize on X chromosomes of C. sp. 9 hermaphrodites. We established site-directed mutagenesis in C. sp.9 using TALENs and recovered an XX-specific lethal mutation in the key component of the regulatory hierarchy that triggers assembly of the DCC onto X. Further TALEN knockouts and ChIP-seq experiments will determine the divergence in X-targeting mechanisms. Dosage compensation provides a unique opportunity to study the co-evolution of regulator proteins and their binding sites. The evolution of DCC binding sites followed a different pattern from that of binding sites for conserved regulatory proteins that control many unrelated cellular processes. For multi-functional proteins few significant changes have occurred in their DNA binding domains and cognate DNA binding motifs. In contrast, the DCC complexes, which lack the constraints of multiple functions, exhibit robust divergence in binding sites.
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[
International Worm Meeting,
2017]
Interphase chromosome structure is regulated at multiple length scales, and each level of organization controls nuclear functions such as transcription, replication, and recombination. To learn how these structures are formed, we dissected the mechanism by which a condensin complex imposes a distinct, higher-order structure across X chromosomes of C. elegans hermaphrodites. Here our deletion analysis of the endogenous X revealed that architectural proteins can remodel chromosome-wide topology by binding to a small number of sites. The dosage compensation complex (DCC), a specialized condensin complex, is recruited to dozens of specific sites (rex sites) on both hermaphrodite X chromosomes and represses X-linked gene expression by half. The DCC establishes a higher-order structure composed of megabase-scale topologically associating domains (TADs) that is distinct from the structure of autosomes and male X chromosomes. The DCC-dependent TAD boundaries all contain a strong rex site, and the DCC promotes long-range interactions both between rex sites at TAD boundaries and between rex sites within TADs. To discern the contributions of these different rex-site interactions to TAD boundary formation, we deleted the eight rex sites at DCC-dependent TAD boundaries and examined X structure using an updated in-nucleus Hi-C protocol that detects distant interactions efficiently. In the rex-delete strain, the specific loops between adjacent DCC-dependent TAD boundaries were eliminated. All eight DCC-dependent boundaries were lost or significantly weakened, producing a structure that recapitulates the X structure of a DCC mutant. Therefore, though the DCC binds dozens of sites on X, the TAD boundaries are established by binding to these eight high-affinity sites. Disruption of TAD structure by deleting a series of cis elements uniquely allows us to assess the effects of TAD boundaries on gene expression. The rex-deleted worms lack strong dosage compensation phenotypes, indicating that TAD boundaries alone are insufficient to enact full repression of X gene expression. Ongoing sensitive gene expression assays are determining whether TAD boundary disruption causes local or subtle transcriptional changes. Intra-TAD interactions present in wild-type and rex-delete animals but absent in DCC mutants likely underlie mechanisms that control X gene expression. Furthermore, Hi-C results showed that in C. briggsae, which also uses a condensin DCC, X chromosomes have a unique TAD structure compared to that of autosomes, even though the rex sites are in different locations relative to C. elegans' sites. Hi-C also revealed errors in C. briggsae's genome assembly, which are now being corrected based on Hi-C data. This improved genome assembly facilitates studies of how condensin's role in shaping higher-order chromosome structure is maintained as its binding sites evolve.
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Meyer, Barbara, Haag, Eric, Schwarz, Erich, Schartner, Caitlin, Yin, Da, Ralston, Edward
[
International Worm Meeting,
2015]
Shifts in mating system are widespread in eukaryotes. This is expected to have pronounced consequences for population genetics, mating-related traits and the nature and intensity of sexual selection, all of which should be reflected in the genome. Self-fertility evolved at least three times in the Elegans sub-genus, within C. briggsae, C. tropicalis, and C. elegans. In each case, this trait was derived from obligately outcrossing male-female ancestors. Previous studies indicate that selfing species have smaller genomes and several thousand fewer protein-coding genes than their outcrossing ancestors. This pattern of genomic reduction may be driven by an interaction between partial selfing and segregation distortion in male meiosis favoring transmission of deletions to XX daughters. However, the structural basis of specific deletions remains unknown. To characterize the process of genome shrinkage, we have produced an Illumina-based genome assembly from the closest known outcrossing relative of C. briggsae, C. nigoni. These two species are genetically close enough that their F1 hybrids females undergo meiotic recombination and are fertile, and therefore they must have similar genomic organization. The C. nigoni genome is roughly 20 Mb (20%) larger than that of C. briggsae. By comparing C. nigoni contigs with the chromosome-level assembly for C. briggsae, we created an approximation of the C. nigoni physical map. Sequence alignments to whole C. briggsae chromosomes revealed that indels of various sizes are enriched in the high-recombination domains of the autosomes. We are now working on new genome assemblies of C. nigoni and C. wallacei using long PacBio reads, which we expect will yield de novo genomic assemblies with very high (chromosome-sized) regions of contiguity. This, in turn, should enable greatly improved comparisons between C. nigoni versus C. briggsae (and C. wallacei versus C. tropicalis, another selfing-outcrossing sibling species pair). Using C. sinica (formerly C. sp. 5) as an outgroup, we are also able to infer the identity of specific genes recently lost in the C. briggsae lineage. These genes include a family of male-specific secreted short (MSS) proteins, that have been lost from the genomes of all three selfing species. We are now using behavioral assays and genome editing tools to examine their possible effects on female behavior and/or physiology.
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Pickle, Catherine, Schartner, Caitlin, Anderson, Erika, Ralston, Ed, Gurling, Mark, Lo, Te-Wen, Meyer, Barbara J.
[
International Worm Meeting,
2013]
We have achieved targeted genome editing across nematode species diverged by 300 million years. These methods have proven to be invaluable for evolutionary studies across species that lack reverse genetic tools but have sequenced genomes. Our editing protocols work in parasitic nematodes (P. pacificus), male/female species (C. sp.9), and hermaphroditic species (C. elegans and C. briggsae). Our approach uses engineered nucleases made of fusions between the DNA cleavage domain of FokI and a custom-designed DNA-binding domain of transcription activator-like effector (TALE) repeats. Our protocols permit not only the isolation of "knock-out" mutations, but also the recovery of multiple different custom-designed "knock-in" mutations in the genomic location of choice. The various "knock-in" and "knock-out" modifications can be recovered from the progeny of a single injected animal. The entire process from TALEN design to isolation of DNA-sequence-verified homozygous mutants can be completed in three weeks, and the cost is minimal.
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Haag, Eric S., Schartner, Caitlin M., Ralston, Edward J., Yin, Da, Meyer, Barbara J., Schwarz, Erich M.
[
International Worm Meeting,
2013]
Sexual mode evolves rapidly in some eukaryotic lineages. This is expected to have pronounced consequences for population genetics, sexual differentiation and the nature and intensity of sexual selection, all of which may be reflected in the genome. C. elegans is a self-fertile species, derived recently from an obligately outcrossing male-female ancestor. This trait has evolved in at least two other species of the Elegans sub-genus, C. briggsae and C. sp. 11. Previous studies indicate that selfing species have smaller genomes and several thousand fewer protein-coding genes than their outcrossing ancestors. This reproducibility may be stimulated by an interaction between partial selfing and segregation distortion affecting large indels in male meiosis. However, the size, location, and gene content of specific deletions remain unknown for any natural system. To characterize the process of genome shrinkage, we have produced a genome assembly from the closest known outcrossing relative of C. briggsae, C. sp.9. The C. sp. 9 genome is roughly 20 Mb (20%) larger than that of C. briggsae. By comparing C. sp.9 contigs with the chromosome-level assembly for C. briggsae, we created an approximation of the C. sp.9 physical map. This allowed us to examine the size and location of C. sp.9-specific sequences with respect to chromosome, and to relate them to known domains of gene density and recombination within a chromosome. Using an outgroup, we are also able to infer the identity of specific genes recently lost in the C. briggsae lineage. In this poster we present details of these analyses, along with some of their implications.
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Meyer, Barbara J., Haag, Eric S., Schartner, Caitlin M., Ralston, Edward J., Korf, Ian, Thomas, Cristel G., Yin, Da, Schwarz, Erich M.
[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2014]
Sexual mode evolves rapidly in some eukaryotic lineages. This is expected to have pronounced consequences for population genetics, sexual differentiation and the nature and intensity of sexual selection, all of which may be reflected in the genome. C. elegans is a self-fertile species, derived recently from an obligately outcrossing male-female ancestor.This trait has evolved in at least two other species of the Elegans sub-genus, C. briggsae and C. sp. 11 (Kiontke et al. 2011). Previous studies indicate that selfing species have smaller genomes and several thousand fewer protein-coding genes than their outcrossing ancestors (Thomas et al. 2012). This reproducibility may be stimulated by an interaction between partial selfing and segregation distortion affecting large indels in male meiosis (Wang et al. 2010). However, the size, location, and gene content of specific deletions remain unknown for any natural system. To characterize the process of genome shrinkage, we have produced a genome assembly from the closest known outcrossing relative of C. briggsae, C. nigoni (formerly C.sp. 9; Felix et al. 2014). The C. nigoni genome is roughly 20 Mb (20%) larger than that of C. briggsae. By comparing C. nigoni contigs with the chromosome-level assembly for C. briggsae, we created an approximation of the C. nigoni physical map. Genome-wide sequence alignment showed the majority of the size reduction is located on the two arms of the five autosomes. Using C. sp.5 as an outgroup, we are able to identify gene family reductions, as well as specific genes recently lost in the C. briggsae lineage. Finally, we present detailed characterization of a family of rapidly evolving proteins that were independently lost in C. elegans and C. briggsae, the MSS (male-specific secreted) family, We have characterized their temporal and spatial expression, and find they are likely to be transferred to the female reproductive tract. We are now developing assays to reveal potential physiological responses to MSS proteins in females, including using calcium imaging to localize putative responder cells in female reproductive tract. References Felix, M.-A., C. Braendle, et al. (2014). "A streamlined system for species diagnosis in Caenorhabditis (Nematoda: Rhabditidae) with name designations for 15 distinct biological species." PLoS One 9:
e94723.Kiontke, K., M.-A. Felix, et al. (2011). "A phylogeny and molecular barcodes for Caenorhabditis, with numerous new species from rotting fruits." BMC Evol Biol 11: 339.Thomas, C. G., R. Li, et al. (2012). "Simplification and desexualization of gene expression in self-fertile nematodes." Curr Biol 22: 2167-2172.Wang, J., P. J. Chen, et al. (2010). "Chromosome size differences may affect meiosis and genome size." Science 329: 293.
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Zeitler, Bryan, Gregory, Philip, Miller, Jeffrey, Lo, Te-Wen, Wood, Andrew, Zhang, Lei, Meyer, Barbara, Rebar, Edward, Schartner, Caitlin, Pickle, Catherine, Lee, Andrew, Urnov, Fyodor
[
International Worm Meeting,
2011]
Genome sequencing has facilitated research beyond traditional model organisms, but the paucity of broadly effective reverse genetic tools has limited cross-species comparisons of gene function needed to explore biological mechanisms. To overcome this limitation for nematodes, we developed a strategy for heritable, targeted gene disruption using engineered nucleases: fusions between custom-designed DNA binding domains of either the C2H2 zinc-finger motifs (ZFNs) or transcription activator-like effector (TALE) repeat motifs and the endonuclease FokI. ZFNs and TALE nucleases (TALENs) induce a double-strand break at a desired locus that can be imperfectly repaired to yield small insertions and deletions. Procedures were optimized for C. elegans (Ce) using ZFNs to recover mutant lines without reliance phenotype. TALENs proved equally effective, yielding the first TALEN-induced animal gene knockouts.
We applied this technology to C. briggsae (Cbr) to study the evolution of sex determination (SD) and X-chromosome dosage compensation (DC). The DC machinery and key components of the genetic hierarchy that regulate SD and DC proved to be functionally conserved over 15-30 Myr. In contrast, recruitment elements that target the DC machinery to X chromosomes have diverged. The Ce X motifs that are enriched on X relative to autosomes and pivotal for recruiting the Ce DCC to X are not enriched on the Cbr X. Moreover, all DCC recruitment elements imported from Ce into Cbr, fail to bind the Cbr DCC. ChIP-seq confirmed that DNA target specificity has diverged, and on going experiments will identify sequence specificity for Cbr DCC binding.
Like many developmental regulatory proteins (e.g. Twist, Dorsal), the DCC controls hundreds of genes through its action on cis-acting target sites. However, the evolution of DCC recruitment sites followed a very different pattern from that of binding sites for regulatory proteins that control multiple, unrelated developmental and cellular processes. Pleitropy of Twist and Dorsal, caused the proteins to accumulate few functionally significant changes to their DNA binding domains or their cognate DNA binding motifs. In contrast, DCC complex with multiple targets but lacking the constraints of pleiotropy exhibited a divergence of binding sites. Such divergence could have been an important driver for nematode speciation.
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Chandrasekar, Sinduja, Koutsovoulos, Georgios, Yin, Da, Ralston, Edward J., Blaxter, Mark, Stevens, Lewis, Haag, Eric S., Anderson, Erika C., Meyer, Barbara J., Schartner, Caitlin M., Schwarz, Erich M.
[
International Worm Meeting,
2019]
Hermaphroditism has independently evolved at least three times within the Caenorhabditis genus, and six times in Pristionchus. This has often coincided with substantial losses of protein-coding genes, which are often implicated in male reproduction. However, the hermaphrodite C. tropicalis challenges this pattern. Although C. tropicalis has a substantially reduced genome (83 Mb in size, versus ~130 Mb in several male-female Caenorhabditis species), its closest male-female relative (C. wallacei) has an almost equally small genome (85 Mb). One explanation might be that genome shrinkage in C. tropicalis arose independently of hermaphroditism; this would fit the recent discovery of male-female Caenorhabditis with remarkably compact genomes, such as C. sulstoni with 65 Mb. An alternative explanation might be that C. wallacei reverted to male-female sexuality after hermaphroditism had already shrunk the genome of its shared tropicalis/wallacei ancestor. To begin testing these hypotheses, we used PacBio, Illumina, and Hi-C sequencing to produce third-generation genome assemblies for C. tropicalis and C. wallacei, each having six complete chromosomal scaffolds. Both assemblies are 98.6%-98.7% complete as scored by BUSCO, which matches the score for C. elegans (98.6%). In hermaphroditic C. briggsae versus its male-female sister species C. nigoni, ~7,000 genes lost in C. briggsae disproportionately include small genes with male-biased gene expression, such as the male secreted short (mss) gene family; the mss family encodes sperm surface glycoproteins, found only in outcrossing species, that are required for sperm competitiveness in mating. In contrast, C. tropicalis has only ~1,400 fewer protein-coding genes than C. wallacei (19,722 versus 21,017), 20% the disparity of C. briggsae vs. C. nigoni. Two clustered multigene families with male-biased expression conserved widely in male-female species (mss and a CAP-domain family that includes CRE28795) are absent not only in C. tropicalis but also in C. wallacei. More generally, gene families with conserved XO- or XX-biased expression have consistently fewer members in C. wallacei than in male-female species C. nigoni, C. remanei, or C. brenneri, and the diminished gene numbers of C. wallacei approach or equal those seen in hermaphrodites (for XO-biased and XX-biased gene families, respectively). These data suggest that C. wallacei might indeed be an atypical male-female Caenorhabditis species that underwent a temporary period of hermaphroditism, and jettisoned male reproductive genes during that period.