Author response: X-chromosome target specificity diverged between dosage compensation mechanisms of two closely related Caenorhabditis species

Abstract

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract An evolutionary perspective enhances our understanding of biological mechanisms. Comparison of sex determination and X-chromosome dosage compensation mechanisms between the closely related nematode species Caenorhabditis briggsae (Cbr) and Caenorhabditis elegans (Cel) revealed that the genetic regulatory hierarchy controlling both processes is conserved, but the X-chromosome target specificity and mode of binding for the specialized condensin dosage compensation complex (DCC) controlling X expression have diverged. We identified two motifs within Cbr DCC recruitment sites that are highly enriched on X: 13 bp MEX and 30 bp MEX II. Mutating either MEX or MEX II in an endogenous recruitment site with multiple copies of one or both motifs reduced binding, but only removing all motifs eliminated binding in vivo. Hence, DCC binding to Cbr recruitment sites appears additive. In contrast, DCC binding to Cel recruitment sites is synergistic: mutating even one motif in vivo eliminated binding. Although all X-chromosome motifs share the sequence CAGGG, they have otherwise diverged so that a motif from one species cannot function in the other. Functional divergence was demonstrated in vivo and in vitro. A single nucleotide position in Cbr MEX can determine whether Cel DCC binds. This rapid divergence of DCC target specificity could have been an important factor in establishing reproductive isolation between nematode species and contrasts dramatically with the conservation of target specificity for X-chromosome dosage compensation across Drosophila species and for transcription factors controlling developmental processes such as body-plan specification from fruit flies to mice. Editor's evaluation This important study uses state-of-the-art methods to explore the evolution of dosage compensation between two closely related nematode species. The evidence supporting the rapid evolution of the recruitment motifs on the X chromosome, despite a general conservation of the dosage compensation machinery, is compelling. This work will be of broad interest to cell biologists and evolutionary biologists. https://doi.org/10.7554/eLife.85413.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Comparative studies have shown that different facets of metazoan development exhibit remarkably different degrees of conservation across species (Carroll, 2008). At one extreme, homeobox-containing Hox genes and Wnt-pathway signaling genes play conserved roles in body plan formation (Hox) and cell-fate determination, neural patterning, or organogenesis (Wnt) across clades diverged by more than 600 million years (MYR) (Malicki et al., 1990; De Kumar and Darland, 2021; Rim et al., 2022). Distant orthologous genes within these ancestral pathways can substitute for each other. For example, both the mouse Small eye (Pax-6) gene (Hill et al., 1991) and the fruit fly eyeless (ey) gene (Quiring et al., 1994; Halder et al., 1995) control eye morphogenesis and encode a transcription factor that includes a paired domain and a homeodomain. Ectopic expression of mouse Pax-6 in different fruit fly imaginal disc primordia can induce morphologically normal ectopic compound eye structures on fruit fly wings, legs, and antennae (Halder et al., 1995). Hence, at a deep level, eye morphogenesis is under related genetic and molecular control in vertebrates and insects, despite profound differences in eye morphology and mode of development. At the other extreme are aspects of development related to sex. For example, chromosomal strategies to determine sexual fate in mice, fruit flies, and nematodes (XY or XO males and XX females or hermaphrodites) and the mechanism needed to compensate for the consequent difference in X-chromosome dose between sexes have diverged greatly. To balance X gene expression between sexes, female mice randomly inactivate one X chromosome (Yin et al., 2021; Loda et al., 2022), while male fruit flies double expression of their single X chromosome (Samata and Akhtar, 2018; Rieder et al., 2019), and hermaphrodite worms halve expression of both X chromosomes (Meyer, 2022a; Meyer, 2022b). The divergence in these pathways is so great that comparisons among animals of the same genus can provide useful evolutionary context for understanding the developmental mechanisms that distinguish the sexes. Therefore, we determined the genetic and molecular specification of sexual fate and X-chromosome dosage compensation in the nematode C. briggsae and compared it to the wealth of knowledge amassed about these processes in C. elegans. These two species have diverged by 15–30 MYR (Cutter, 2008). In C. elegans, the sex determination and dosage compensation pathways are linked by genes that coordinately control both processes. For example, in XX embryos, the switch gene sdc-2 sets the sex determination pathway to the hermaphrodite mode and triggers the binding of a DCC onto both X chromosomes to reduce X gene expression by half and thereby match X expression with that from XO males (Meyer, 2022a). The DCC shares subunits with condensin, a protein complex that controls the structure, resolution, and segregation of mitotic and meiotic chromosomes from yeast to humans (Yatskevich et al., 2019; Meyer, 2022b). We determined the extent to which the sex-specific gene regulatory hierarchy is conserved between C. elegans and C. briggsae and the extent to which subunits of the C. briggsae DCC correspond to those of the C. elegans DCC. We also defined the cis-acting regulatory sites that confer X-chromosome specificity and recruit the C. briggsae DCC. We found that the DCC itself and the regulatory hierarchy that determines sex and directs the DCC to X have been conserved, but remarkably, both the X-chromosome target specificity of the C. briggsae DCC and its mode of binding to X have diverged. Results Conservation between C. briggsae and C. elegans of the core dosage compensation machinery and genetic hierarchy that regulates dosage compensation The pivotal hermaphrodite-specific regulatory protein that coordinately controls both sex determination and dosage compensation in C. elegans is a 350 kDa protein called SDC-2. It directs the DCC to both X chromosomes of XX embryos to achieve dosage compensation and also activates the hermaphrodite program of sexual differentiation (Chuang et al., 1996; Dawes et al., 1999; Chu et al., 2002; Pferdehirt et al., 2011). Loss of Cel sdc-2 causes XX-specific lethality due to excessive X-chromosome gene expression and masculinization of escaper animals (Nusbaum and Meyer, 1989; Kruesi et al., 2013). SDC-2 has no known homologs outside of nematodes and only a coiled-coil domain as a predicted structural feature (Meyer, 2022a). Among five Caenorhabditis species compared, the entire SDC-2 protein has 23–29% identity and 38–45% similarity (Figure 1—figure supplement 2A). Between Cbr and Cel, the entire SDC-2 protein shows 26% identity and 43% similarity (Figure 1—figure supplements 1 and 2A). To assess the conservation of gene function, we deployed genome-editing technology in C. briggsae to knockout sdc-2. Using a PCR-based molecular strategy to identify insertions and deletions induced by DNA repair following directed mutagenesis with zinc finger nucleases, we recovered several independent Cbr sdc-2 mutant lines (Figure 1—figure supplement 3). Homozygous Cbr sdc-2 mutations caused extensive XX-specific lethality, consistent with a defect in dosage compensation and the conservation of gene function (Figure 1A). Nearly all Cbr sdc-2 hermaphrodites died as embryos or young larvae; rare XX survivors exhibited slow growth and masculinization. Cbr sdc-2 males were viable (Figure 1A) and had wild-type body morphology. Figure 1 with 4 supplements see all Download asset Open asset Conservation of X-chromosome dosage compensation machinery between C. briggsae and C. elegans. (A) sdc-2 mutations cause XX-specific lethality in C. briggsae. Graph shows percent viability of wild-type and Cbr sdc-2 mutant XX and XO adults. Viability of homozygous XX and hemizygous XO Cbr sdc-2 mutants is expressed as the percentage of live adults for each karyotype relative to the number expected (shown in parentheses) in the progeny of a cross if all mutant animals were viable. Crosses and calculations are described in Materials and methods. Sequence changes of sdc-2 mutations derived from genome editing using zinc-finger nucleases are shown in Figure 1—figure supplement 3A. (B) Schematic of the C. elegans dosage compensation complex (left) and C. briggsae orthologs identified by BLASTP (right). The C. elegans dosage compensation complex (DCC) includes homologs of all core condensin subunits (MIX-1, DPY-27, DPY-26, DPY-28, and CAPG-1). C. briggsae DCC components identified and characterized in this study are shown in color; other orthologs are in gray. DPY-27 and MIX-1 belong to the SMC (Structural Maintenance of Chromosomes) family of chromosomal ATPases. Each has nucleotide-binding domains (NBDs) at its N- and C-termini that are linked by two long coiled-coil domains separated by a hinge domain. Each SMC protein folds back on itself to form a central region of two anti-parallel coiled coils flanked by the NBDs and the hinge. DPY-27 and MIX-1 dimerize through interactions between their hinge domains and their NBD domains. The globular NBDs bind to the three non-SMC condensin DCC subunits (DPY-26, DPY-28, and CAPG-1) (See Meyer, 2022a). (C) Condensin subunit DPY-27 binds X chromosomes and mediates dosage compensation in C. briggsae. Confocal images of C. briggsae hermaphrodite gut nuclei co-stained with the DNA dye DAPI (gray), antibodies to Cbr DPY-27 (green), and FISH probes to either 5% of X (red, top), or 1% of chromosome III (red, bottom) show that Cbr DPY-27 co-localizes with X but not III, consistent with a role in dosage compensation. Scale bars, 1 μm. (D) Confocal images of C. briggsae gut nuclei from dpy-27(+) or dpy-27(y436) mutant XX adult hermaphrodites co-stained with DAPI (blue) and the Cbr DPY-27 rabbit antibody (red). DPY-27 shows subnuclear localization in a dpy-27(+) gut nucleus (top), as expected for X localization. The mutant gut nucleus (bottom) shows diffuse nuclear distribution of DPY-27, as anticipated for a mutant SMC-4 condensin ortholog that lacks most of the N-terminal part of the ATPase domain and, therefore, has no ATP binding or hydrolysis. Scale bars, 1 μm. (E) Confocal images of a C. briggsae gut nucleus from wild-type adult hermaphrodites co-stained with DAPI (gray) and antibodies to Cbr DPY-27 (green) and Cbr MIX-1 (red) show that Cbr MIX-1 co-localizes with Cbr DPY-27 on X in wild-type hermaphrodites. Scale bars, 1 μm. (F) Association of Cbr MIX-1 (red) with X found in a dpy-27(+) nucleus (top) is disrupted in a Cbr dpy-27(y436) nucleus (bottom), in accord with participation of Cbr MIX-1 in a protein complex with Cbr DPY-27. Scale bars, 1 μm. (G) Viability of dpy-27 mutant XX C. briggsae animals. The left panel shows the genetic scheme to characterize the effect of maternal genotype on viability of dpy-27 null XX mutants. Comparison is made between homozygous null dpy-27 progeny from heterozygous or homozygous non-Dpy mutant mothers. The genotype of non-DPY mothers was established through PCR analysis. The right panel shows the percent viability of progeny from wild-type hermaphrodites and heterozygous or homozygous dpy-27 mutant hermaphrodites. The maternal genotype, number of broods, total number of embryo progeny from all broods, and average brood size are provided for two null alleles of dpy-27. Molecular characterization of mutations is shown below the graph and in Figure 1—figure supplement 3B. Almost all progeny of dpy-27 null mutant mothers are dead; a homozygous dpy-27 null strain cannot be propagated. More than 20% of progeny of dpy-27/+heterozygous mutant mothers are very Dpy or dead, indicating that a wild-type DPY-27 maternal contribution has minimal effect on suppressing the deleterious effect of the homozygous null zygotic genotype. The complete XX lethality is consistent with a major role for condensin subunit DPY-27 in dosage compensation. To determine whether the hermaphrodite-specific lethality of Cbr sdc-2 mutants was caused by defects in dosage compensation, we first identified components of the C. briggsae DCC and then asked whether DCC binding to X is disrupted by mutation of Cbr sdc-2, as it is by mutation of Cel sdc-2. In C. elegans, five of the ten known DCC proteins are homologous to subunits of condensin, an evolutionarily conserved protein complex required to restructure and resolve chromosomes in preparation for cell divisions in mitosis and meiosis (Figure 1B; Chuang et al., 1994; Lieb et al., 1996; Lieb et al., 1998; Chan et al., 2004; Tsai et al., 2008; Csankovszki et al., 2009; Mets and Meyer, 2009; Yatskevich et al., 2019; Meyer, 2022a). The evolutionary time scale over which condensin subunits were co-opted for dosage compensation in nematodes had not been explored. Several lines of evidence indicate that a condensin complex mediates dosage compensation in C. briggsae as well. First, BLASTP searches revealed C. briggsae orthologs of all known C. elegans DCC condensin subunits (Figure 1B). Alignment of DPY-27 protein revealed 38% identity and 56% similarity between C. elegans and C. briggsae (Figure 1—figure supplement 2B). Immunofluorescence experiments using antibodies against Cbr DPY-27, the SMC4 ortholog of the only Cel DCC condensin subunit (Cel DPY-27) not associated with mitotic or meiotic condensins (Chuang et al., 1994), revealed X chromosome-specific localization in hermaphrodites, but not males, indicating conservation of function (Figure 1C and Figure 2A and B). Specificity of DPY-27 antibodies was demonstrated by Western blot analysis (Figure 1—figure supplement 4A). Figure 2 Download asset Open asset Conserved genetic hierarchy targets the C. briggsae dosage compensation complex (DCC) to the X chromosomes of hermaphrodites. (A–E) Schematic depiction of the genetic hierarchy controlling sex-specific DCC recruitment to C. briggsae X chromosomes (left) paired with representative immunofluorescence experiments exemplifying DCC localization (right). Scale bars, 5 μm. Gut nuclei (A, B, C, E) or embryos (D) were co-stained with DAPI (red) and antibodies to Cbr DPY-27 (green). In wild-type XX, but not XO gut nuclei (A, B), DPY-27 co-localizes with X chromosomes, consistent with a role for condensin subunit DPY-27 in dosage compensation (see also Figure 1C). (C) SDC-2 is required for recruitment of DPY-27 to the X chromosomes of hermaphrodites. Failure of the DCC to bind X chromosomes of sdc-2 XX mutants underlies the XX-specific lethality. Shown is the gut nucleus of a rare XX sdc-2 mutant escaper near death. sdc-2 mutant XX escaper animals are masculinized. (D) Lethality of Cbr xol-1(y430) XO animals corresponds to inappropriate binding of the DCC to the single X in embryos. (E) Mutation of the DCC recruitment factor Cbr sdc-2 in a Cbr xol-1 XO mutant prevents DCC recruitment to X and suppresses the XO lethality. See Figure 3B for quantification. Second, disruption of Cbr dpy-27 conferred hermaphrodite-specific lethality, with rare XX escaper animals exhibiting a dumpy (Dpy) phenotype, like the disruption of Cel dpy-27 (Figure 1G). Immunofluorescence experiments with Cbr DPY-27 antibodies revealed diffuse nuclear distribution of DPY-27 in Dpy escapers of dpy-27(y436) mutants instead of X localization, consistent with lethality (Figure 1D). Third, co-immunoprecipitation of proteins with rabbit Cbr DPY-27 antibodies followed by SDS-PAGE and mass spectrometry of excised trypsinized protein bands identified Cbr MIX-1 (Table 1; Materials and methods), the SMC2 condensin subunit ortholog found in the Cel DCC complex (Lieb et al., 1998; Figure 1B). Both DPY-27 and MIX-1 belong to the SMC family of chromosomal ATPases that dimerize and participate in condensin complexes (Figure 1B). Table 1 MALDI-TOF identification of Cbr MIX-1 peptides. m/z SubmittedMH+ MatchedDelta ppmPeptideMissedCleavageDatabase Sequence916.47916.469.5674–6800(K)YHENVVR(L)1163.591163.583.3375–3841(K)LRGELEGMSR(G)1214.651214.66–3.6631–6410(R)VLIESQCLPGR(R)1224.631224.628.8713–7231(R)EVAYTDGVKSR(T)1263.741263.74–0.87524–5340(R)DVEGLVLHLIR(L)1285.691285.69–2.8631–6410(R)VLIESQCLPGR(R)1350.691350.70–8.9656–6660(R)YTIINDQSLQR(A)1881.971881.98–2.3134–1500(R)GVGLNVNNPHFLIMQGR(I)1886.891886.91–6.886–1010(K)QSPFGMDHLDELVVQR(H)2064.012064.003.4460–4770(K)ITQQVQSLGYNADEDVQR(R)2377.182377.165.6385–4151(R)GTVTNDKGEHVSLETYIQETR(A) This table lists the mass-to-charge ratio (m/z) of measured peptides, the predicted masses (MH+ Matched), and the deviation from predicted masses (Delta ppm). The ID of each measured peptide is described by the residue range within full-length MIX-1 (Peptide) and its corresponding amino acid sequence (Database Sequence). The number of uncut tryptic peptide bonds is listed for each peptide (Missed Cleavage). In addition to MIX-1, MALDI-TOF analysis of excised protein bands in the molecular weight range of condensin subunits excised from an SDS-PAGE gel revealed peptides corresponding to four common high-molecular weight contaminants: the three vitellogenin yolk proteins VIT-2, VIT-4, VIT-5, and CBG14234, an ortholog of VIT-4. No protein bands corresponding to the molecular weights of SDC-2 or SDC-3 were visible on the SDS-PAGE gel. Fourth, immunofluorescence experiments using Cbr MIX-1 antibodies (Figure 1—figure supplement 4B) revealed co-localization of Cbr MIX-1 with Cbr DPY-27 on hermaphrodite X chromosomes (Figure 1E). Cbr MIX-1 protein did not bind to X chromosomes in Cbr dpy-27(y436) mutant animals (Figure 1F). Instead, MIX-1 exhibited diffuse nuclear distribution, like DPY-27, consistent with the two proteins participating in a complex and the dependence of MIX-1 on DPY-27 for its binding to X (Figure 1F). These data demonstrate that condensin subunits play conserved roles in the dosage compensation machinery of both C. briggsae and C. elegans. In contrast to DPY-27, MIX-1 shows 55% identity and 72% similarity between C. elegans and C. briggsae. Not only does MIX-1 participate in the DCC, it also participates in two other distinct Caenorhabditis condensin complexes that are essential for the proper resolution and segregation of mitotic and meiotic chromosomes (Mets and Meyer, 2009; Csankovszki et al., 2009). Conserved roles in chromosome segregation complexes would constrain MIX-1 sequence divergence, thereby explaining its greater conservation between species. Evidence that DCC binding defects underlie the XX-specific lethality caused by Cbr sdc-2 mutations is our finding that neither Cbr DPY-27 (Figure 2C) nor Cbr MIX-1 (not shown) binds to X chromosomes in Cbr sdc-2 mutant hermaphrodites. Instead, we found a low level of diffuse nuclear staining. Thus, the role of sdc-2 in the genetic hierarchies that activate dosage compensation is also conserved. We next explored why maternally supplied DCC subunits fail to bind to the single X chromosome of C. briggsae males. In C. elegans XO embryos, the master switch gene xol-1 (XO lethal) represses the hermaphrodite-specific sdc-2 gene required for DCC binding to X and thereby prevents other DCC subunits from functioning in males (Miller et al., 1988; Rhind et al., 1995; Dawes et al., 1999; Meyer, 2022a). Loss of Cel xol-1 activates Cel sdc-2 in XO embryos, causing DCC binding to X, reduction in X-chromosome gene expression, and consequent death. We isolated the null mutant allele Cbr xol-1(y430) by PCR screening of a C. briggsae deletion library (Supplementary file 1). We found that the Cbr xol-1 mutation caused inappropriate binding of the DCC to the single X of XO embryos (Figure 2D) and fully penetrant male lethality (Figure 3B), as expected from the disruption of a gene that prevents the DCC machinery from functioning in C. briggsae males. Cbr xol-1 mutant XX hermaphrodites appeared wild-type. Figure 3 Download asset Open asset sdc-2 controls dosage compensation and sex determination in C. briggsae. (A) Diagram of the screening strategy to recover Cbr sdc-2 mutations as suppressors of the XO-specific lethality caused by a xol-1 mutation. Cbr xol-1 XX hermaphrodites were mated with males carrying a gfp-marked X chromosome to allow F1 XO males to be monitored for the parental origin of the X chromosome. Animals with mating plugs (indicating successful mating) were injected with mRNAs to sdc-2 zinc-finger nucleases, and all F1 males were examined for GFP fluorescence. Non-green males necessarily inherited an X chromosome carrying a Cbr-xol-1 mutation and, assuming conservation of the dosage compensation complex (DCC) regulatory hierarchy, would be inviable without a concomitant Cbr sdc-2 mutation. GFP-positive males arose at low frequency from fertilization of nullo-X oocytes (caused by non-disjunction of the maternal X chromosome) with gfp-X-bearing sperm. These false positives were discarded from further study. (B) Cbr sdc-2 mutations rescue Cbr xol-1(y430) XO lethality. Graph shows percent viability of wild-type XO animals and mutant XO animals carrying combinations of Cbr xol-1 and Cbr sdc-2 mutations. The % XO viability is expressed as the percentage of live XO adults relative to the number expected (shown in parentheses) in the progeny of the cross. Formulae for viability calculations are given in the Materials and methods. Sequence changes of sdc-2 mutations are shown in Figure 1—figure supplement 3C and D. (C) sdc-2 activates the program for Cbr hermaphrodite sexual development. DIC images show the comparison of tail morphologies for Cbr L4 animals of different genotypes. sdc-2 mutations, but not dpy-27 mutations, cause masculinization of XX animals. Scale bar, 20 μm. (D) DIC images show tail morphologies of wild-type or doubly mutant Cbr adults. An sdc-2 mutation suppresses both the XO lethality and feminization caused by a xol-1 mutation, consistent with a role for sdc-2 in controlling both dosage compensation and sex determination. xol-1 sdc-2 XO animals are viable, fertile males, indicating that the sdc-2 mutation suppressed the lethality and feminization caused by xol-1 mutations in XO animals. A dpy-27 mutation suppresses the XO lethality but not feminization caused by a xol-1 mutation, consistent with a role for dpy-27 in dosage compensation but not sex determination. dpy-27; xol-1 XO animals are fertile hermaphrodites. Scale bar, 20 μm. To investigate the hierarchical relationship between Cbr xol-1 and Cbr sdc-2, we asked whether a Cbr sdc-2 mutation could suppress the male lethality caused by a Cbr xol-1 mutation. Both genes are closely linked in C. briggsae, prompting us to use genome editing technology to introduce de novo mutations in cis to pre-existing lesions without relying on genetic recombination between closely linked genes. If Cbr xol-1 controls Cbr sdc-2, then mutation of Cbr sdc-2 should rescue the male lethality of Cbr xol-1 mutants (Figure 2E). This prediction proved to be correct. XO males were observed among F1 progeny from mated Cbr xol-1 hermaphrodites injected with ZFNs targeting Cbr sdc-2 (Figure 3A, B and D). Insertion and deletion mutations were found at the Cbr sdc-2 target site in more than twenty tested F1 males (examples are in Figure 1—figure supplement 3C and D). Quantification of male viability in four different xol-1 sdc-2 mutant lines revealed nearly full rescue (Figure 3B), with a concomitant absence of DCC binding on the single X chromosome (Figure 2E). Therefore, Cbr xol-1 functions upstream of Cbr sdc-2 to repress it and thereby prevents DCC binding to the male X chromosome. In summary, not only is the core condensin dosage compensation machinery conserved between Caenorhabditis species, but so also are the key features of the genetic hierarchy that confers sex-specificity to the dosage compensation process. Conservation between C. briggsae and C. elegans of the genetic hierarchy that regulates early stages of sex determination Mechanisms controlling sex determination and differentiation are dynamic over evolutionary time; major differences can exist even within an individual species. For example, males within the house fly species Musca domestica can utilize one of many different male-determining factors on autosomes and sex chromosomes to determine sex depending on a factor’s linkage to other beneficial traits (Meisel et al., 2016). Within the Caenorhabditis genus, similarities and differences occur in the genetic pathways governing the later stages of sex determination and differentiation (Haag, 2005). For example, three sex-determination genes required for C. elegans hermaphrodite sexual differentiation but not dosage compensation, the transformer genes tra-1, tra-2, and tra-3, are conserved between C. elegans and C. briggsae and play very similar roles. Mutation of any one gene causes virtually identical masculinizing somatic and germline phenotypes in both species (Kelleher et al., 2008). Moreover, the DNA binding motif for both Cel and Cbr TRA-1 (Berkseth et al., 2013), a Ci/GL1 zinc-finger transcription factor that acts as the terminal regulator of somatic sexual differentiation (Zarkower and Hodgkin, 1992), is conserved between the two species. At the opposite extreme, the mode of sexual reproduction, hermaphroditic versus male/female, dictated the genome size and reproductive fertility of Caenorhabditis species diverged by only 3.5 million years (Yin et al., 2018; Cutter et al., 2019). Species that evolved self-fertilization (e.g. C. briggsae or C. elegans) lost 30% of their DNA content compared to male/female species (e.g. C. nigoni or C. remanei), with a disproportionate loss of male-biased genes, particularly the male secreted short (mss) gene family of sperm surface glycoproteins (Yin et al., 2018). The mss genes are necessary for sperm competitiveness in male/female species and are sufficient to enhance it in hermaphroditic species. Thus, sex has a pervasive influence on genome content. In contrast to these later stages of sex determination and differentiation, the earlier stages of sex determination and differentiation had not been analyzed in C. briggsae. Therefore, we asked whether xol-1 and sdc-2 control sexual fate as well as dosage compensation in C. briggsae, as they do in C. elegans, over the 15–30 MYR that separates them. Our analysis of Cbr sdc-2 XX mutant phenotypes revealed intersexual tail morphology in the rare animals that survived to the L3/L4 stage (Figure 3C), indicating a role for Cbr sdc-2 in sex determination. Sexual transformation to the male fate was unlikely to have resulted from a disruption in dosage compensation since such transformation was never observed in Cbr dpy-27 XX mutants (Figure 3C). Analysis of sexual phenotypes in double mutant strains confirmed that Cbr sdc-2 controls sex determination. Specifically, Cbr xol-1 Cbr sdc-2 double mutant XO animals develop as males, whereas Cbr dpy-27; Cbr xol-1 double mutant XO animals develop as hermaphrodites (Figure 3C and D). That is, both Cbr sdc-2 and Cbr dpy-27 mutations suppress the XO lethality caused by a xol-1 mutation, but only Cbr sdc-2 mutations also suppress the sexual transformation of XO animals into hermaphrodites. These results show that both sdc-2 and dpy-27 function in C. briggsae dosage compensation, but only sdc-2 also functions in sex determination. Thus, the two master regulatory genes that control the earliest stages of both sex determination and X-chromosome dosage compensation, xol-1 and sdc-2, are conserved between C. briggsae and C. elegans. DCC recruitment sites isolated from C. briggsae X chromosomes fail to bind the C. elegans DCC Discovery that the dosage compensation machinery and the gene regulatory hierarchy that controls sex determination and dosage compensation are functionally conserved between C. briggsae and C. elegans raised the question of whether the cis-acting regulatory sequences that recruit dosage compensation proteins to X chromosomes are also conserved. In C. elegans, the DCC binds to recruitment elements on X (rex) sites and then spreads across X to sequences lacking autonomous recruitment ability (Csankovszki et al., 2004; Jans et al., 2009; Pferdehirt et al., 2011; Albritton et al., 2017; Anderson et al., 2019). Within rex sites, combinatorial clustering of three DNA sequence motifs directs synergistic binding of the DCC (Fuda et al., 2022). To compare X-recruitment mechanisms between species, DNA binding sites for the Cbr DCC recruitment protein SDC-2 and the Cbr DCC condensin subunit DPY-27 were defined by chromatin immuno-precipitation experiments followed by sequencing of captured DNA (ChIP-seq experiments) (Figure 4A). SDC-2 sites were obtained with anti-FLAG antibodies from a genome-engineered Cbr strain encoding a FLAG-tagged version of endogenous SDC-2. DPY-27 sites were obtained from either a wild-type Cbr strain with DPY-27 antibodies or from a genome-engineered strain encoding endogenous FLAG-tagged DPY-27 with anti-FLAG antibodies. Figure 4 with 1 supplement see all Download asset Open asset Identification of C. briggsae dosage compensation complex (DCC) recruitment elements on X. (A) ChIP-seq profiles of Cbr SDC-2 and Cbr DPY-27 binding to X chromosomes. ChIP-seq experiments were performed using an anti-FLAG antibody to immunoprecipitate SDC-2 from a strain encoding FLAG-tagged SDC-2, and the same anti-FLAG antibody was used in ChIP-seq experime