X-chromosome target specificity diverged between dosage compensation mechanisms of two closely related Caenorhabditis species
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eLife assessment
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 recognition motifs on the X chromosome, despite a general conservation of the mechanism, is compelling. Provided the discussion on the evolutionary aspect of the findings is improved, this work will be of broad interest to cell biologists and evolutionary biologists.
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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.
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Author Response
Reviewer #1 (Public Review):
Sex determination and dosage compensation are two fundamental mechanisms in organisms with distinct sexes. These mechanisms vary greatly across the various model organisms in which they have been studied. Comparisons across more closely related members of the same genus have already proven productive in the past, to understand how these essential mechanisms evolve. In this study, the authors compare some aspects of the dosage compensation and sex determination mechanisms across two Caenorhabditis species that diverged ~15-30 MYA.
Previously, the authors have studied dosage compensation and sex determination extensively in C. elegans. Here, they first identify the homologs of some key factors in C. briggsae, a species that independently evolved hermaphroditism. The authors show that some …
Author Response
Reviewer #1 (Public Review):
Sex determination and dosage compensation are two fundamental mechanisms in organisms with distinct sexes. These mechanisms vary greatly across the various model organisms in which they have been studied. Comparisons across more closely related members of the same genus have already proven productive in the past, to understand how these essential mechanisms evolve. In this study, the authors compare some aspects of the dosage compensation and sex determination mechanisms across two Caenorhabditis species that diverged ~15-30 MYA.
Previously, the authors have studied dosage compensation and sex determination extensively in C. elegans. Here, they first identify the homologs of some key factors in C. briggsae, a species that independently evolved hermaphroditism. The authors show that some of the key players in these processes play the same roles in C. briggsae as they do in C. elegans. Namely, they show that the nematode-specific SDC-2 protein plays a role in both dosage compensation and sex determination also in C. briggsae, they find the homologs of some of the SMC protein complex that performs dosage compensation also in C. elegans and they study the binding specificity on the X chromosome.
Overall, the work is thorough and compelling and is very clearly presented. The authors generate a number of genetic tools in C. briggsae and the careful genetic analyses together with a number of binding assays in vivo and in vitro, support the authors' main conclusions: that the main players and genetic regulatory hierarchy are conserved between these two nematodes, but the binding sites for the DCC on the X chromosome have diverged and the mode of binding has changed as well. Whereas in C. elegans the DCC binds sites in the X chromosome that contain multiple sequence motifs in a synergistic manner, in briggsae they seem to do so additively. This latter point is supported by the data, but it could be explored a bit more deeply using the available ChIP-seq data that the authors have generated. In addition, it would be interesting to discuss the possible implications of this difference.
One minor weakness of this work is that it could be better put in the context of other related comparisons of these mechanisms. For example, the comparison of sex determination pathway by Haag et al. in Genetics 2008, and the comparison of dosage compensation across Drosophila species (Ellison and Bachtrog, Plos Genetics, 2019), and possibly others. The other point that the authors could provide deeper insight into, is the rate of divergence of proteins like SDC-2 (which is thought to be the protein that contacts DNA), versus some other proteins in the DCC and in general other proteins not involved in sex determination or dosage compensation (this doesn't need to be limited to comparing elegans and briggsae as there are numerous Caenorhabditis genomes available). This would provide a more complete view of the evolution of these processes.
Regarding the comparison of our studies to those of the C. briggsae sex determination pathway described by Haag and others, we have included the following in our revised manuscript:
Pages 8-9. "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 zincfinger 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."
Regarding the comparison to Drosophila dosage compensation, including the work of Ellison and Bachtrog (2019), we included the following in the Discussion of our revised manuscript (page 22) and included related remarks in the abstract.
"In contrast to the divergence of X-chromosome target specificity between Caenorhabditis species, X-chromosome target specificity has been conserved among Drosophila species. A 21-bp GA-rich sequence motif on X is utilized across Drosophila species to recruit the dosage compensation machinery, although it may not be the sole source of X target specificity (Alekseyendo, 2008; Kuzu, 2016, Ellison, 2008; Alekseyendo, 2013)."
Regarding a comparison of our work to that of other rapidly evolving processes, we have made the following revision to our Discussion (page 22):
"Conservation of DNA target specificity among species is also a common theme among developmental regulatory proteins that participate in multiple, unrelated developmental processes, such as Drosophila Dorsal in body-plan specification (Schloop et al., 2020) or Caenorhabditis TRA-1 in hermaphrodite sexual differentiation and male neuronal differentiation (Berkseth et al., 2013; Bayer et al., 2020). Typically, for such multi-purpose proteins, target-site specificity is evolutionarily constrained: protein function is changed far more by changes in the number and location of conserved cis-acting target sequences than by changes in the target sequences themselves (Carroll, 2008; Nitta et al., 2015). Hence, the divergence in X-chromosome target specificity across the Caenorhabditis genus is atypical among developmental regulatory complexes with highly diverse target genes and could have been an important factor for establishing reproductive isolation between species. Our finding is reminiscent of the discovery that centromeric sequences and their corresponding centromere-binding proteins have co-evolved rapidly as a consequence of hybrid incompatibilities (Malik and Henikoff, 2001; Henikoff et al., 2001; Talbert and Henikoff, 2022). Occurrence of rapidly changing DNA targets and their corresponding DNA-binding proteins (see also Lienard et al., 2016; Ting et al., 1998; Ting et al., 2004; Sun et al., 2004) is an increasingly dominant theme contributing to reproductive isolation."
A brief comment about all three comparisons is also made in the beginning of the Discussion on page 18.
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eLife assessment
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 recognition motifs on the X chromosome, despite a general conservation of the mechanism, is compelling. Provided the discussion on the evolutionary aspect of the findings is improved, this work will be of broad interest to cell biologists and evolutionary biologists.
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Reviewer #1 (Public Review):
Sex determination and dosage compensation are two fundamental mechanisms in organisms with distinct sexes. These mechanisms vary greatly across the various model organisms in which they have been studied. Comparisons across more closely related members of the same genus have already proven productive in the past, to understand how these essential mechanisms evolve. In this study, the authors compare some aspects of the dosage compensation and sex determination mechanisms across two Caenorhabditis species that diverged ~15-30 MYA.
Previously, the authors have studied dosage compensation and sex determination extensively in C. elegans. Here, they first identify the homologs of some key factors in C. briggsae, a species that independently evolved hermaphroditism. The authors show that some of the key players in …
Reviewer #1 (Public Review):
Sex determination and dosage compensation are two fundamental mechanisms in organisms with distinct sexes. These mechanisms vary greatly across the various model organisms in which they have been studied. Comparisons across more closely related members of the same genus have already proven productive in the past, to understand how these essential mechanisms evolve. In this study, the authors compare some aspects of the dosage compensation and sex determination mechanisms across two Caenorhabditis species that diverged ~15-30 MYA.
Previously, the authors have studied dosage compensation and sex determination extensively in C. elegans. Here, they first identify the homologs of some key factors in C. briggsae, a species that independently evolved hermaphroditism. The authors show that some of the key players in these processes play the same roles in C. briggsae as they do in C. elegans. Namely, they show that the nematode-specific SDC-2 protein plays a role in both dosage compensation and sex determination also in C. briggsae, they find the homologs of some of the SMC protein complex that performs dosage compensation also in C. elegans and they study the binding specificity on the X chromosome.
Overall, the work is thorough and compelling and is very clearly presented. The authors generate a number of genetic tools in C. briggsae and the careful genetic analyses together with a number of binding assays in vivo and in vitro, support the authors' main conclusions: that the main players and genetic regulatory hierarchy are conserved between these two nematodes, but the binding sites for the DCC on the X chromosome have diverged and the mode of binding has changed as well. Whereas in C. elegans the DCC binds sites in the X chromosome that contain multiple sequence motifs in a synergistic manner, in briggsae they seem to do so additively. This latter point is supported by the data, but it could be explored a bit more deeply using the available ChIP-seq data that the authors have generated. In addition, it would be interesting to discuss the possible implications of this difference.
One minor weakness of this work is that it could be better put in the context of other related comparisons of these mechanisms. For example, the comparison of sex determination pathway by Haag et al. in Genetics 2008, and the comparison of dosage compensation across Drosophila species (Ellison and Bachtrog, Plos Genetics, 2019), and possibly others. The other point that the authors could provide deeper insight into, is the rate of divergence of proteins like SDC-2 (which is thought to be the protein that contacts DNA), versus some other proteins in the DCC and in general other proteins not involved in sex determination or dosage compensation (this doesn't need to be limited to comparing elegans and briggsae as there are numerous Caenorhabditis genomes available). This would provide a more complete view of the evolution of these processes.
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Reviewer #2 (Public Review):
This study by Yang & al. explores the mechanism of X dosage compensation in the nematode species C. briggsae; which is a close relative of C. elegans. The mechanism is well described in C. elegans, and the authors have asked whether the same condensin-like complex (DCC) is responsible for the silencing of the X and which motifs on the X are responsible for this binding specificity in C. briggsae. They discovered that although the general principle of X inactivation is conserved between these 2 species, and ortholog proteins of the pathway (xol-1, sdc-2, and the genes encoding the DCC complex) are conserved, the sequences on the X that are recognized by the DCC complex have evolved very rapidly. The motifs of C. briggsae are not recognized by the C. elegans proteins and vice versa. The authors have …
Reviewer #2 (Public Review):
This study by Yang & al. explores the mechanism of X dosage compensation in the nematode species C. briggsae; which is a close relative of C. elegans. The mechanism is well described in C. elegans, and the authors have asked whether the same condensin-like complex (DCC) is responsible for the silencing of the X and which motifs on the X are responsible for this binding specificity in C. briggsae. They discovered that although the general principle of X inactivation is conserved between these 2 species, and ortholog proteins of the pathway (xol-1, sdc-2, and the genes encoding the DCC complex) are conserved, the sequences on the X that are recognized by the DCC complex have evolved very rapidly. The motifs of C. briggsae are not recognized by the C. elegans proteins and vice versa. The authors have accumulated very solid data, both in vitro and in vivo, to support this conclusion.
Overall, the results are very convincing and extremely interesting, for the chromatin field but also from an evolutionary perspective. This finding is comparable to the discovery that centromeric sequences and centromere proteins, despite their essential function in cells, evolve extremely rapidly. The reason is that they are involved in genetic conflicts, are a perfect target to generate hybrid incompatibilities during crosses, and therefore, under such selective pressure, evolve super fast. Most examples of hybrid incompatibilities rely on chromatin conflicts, and with this study, it appears that the dosage compensation system could be one other way to generate hybrid breakdown.
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Reviewer #3 (Public Review):
In this manuscript, Meyer and colleagues characterized the conserved dosage compensation complex (DCC) and its recruitment mechanisms to X chromosomes in C. briggsae. This paper features comparative analyses of the dosage compensation mechanisms between C. briggsae and C. elegans, which are separated by 15-30 million years in evolution. While the dosage compensation machinery and the regulatory hierarchy are conserved, the target specificity of the DCC complex, the density of the recruiting motifs, and the mode of recruitment have diverged between the two species. The authors speculated that the divergence of the X chromosome DCC target sites could have been a factor for nematode speciation.
Overall, this is a thorough work demonstrating how the dosage compensation mechanisms in C. briggsae compare with …
Reviewer #3 (Public Review):
In this manuscript, Meyer and colleagues characterized the conserved dosage compensation complex (DCC) and its recruitment mechanisms to X chromosomes in C. briggsae. This paper features comparative analyses of the dosage compensation mechanisms between C. briggsae and C. elegans, which are separated by 15-30 million years in evolution. While the dosage compensation machinery and the regulatory hierarchy are conserved, the target specificity of the DCC complex, the density of the recruiting motifs, and the mode of recruitment have diverged between the two species. The authors speculated that the divergence of the X chromosome DCC target sites could have been a factor for nematode speciation.
Overall, this is a thorough work demonstrating how the dosage compensation mechanisms in C. briggsae compare with those in C. elegans. By employing a series of complementary assays, the authors provided compelling evidence, establishing how C. briggsae and C. elegans have diverged DCC recruitment sites and motifs, while the composition of the DCC and the regulatory hierarchy are conserved. The manuscript is clearly written, and all the experiments are rigorously performed with proper controls. The figures are also effective and nicely illustrate the experimental designs and the results. The conclusions drawn from the current work are compelling, and I have no major concerns.
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