Parallel evolution of X chromosome-specific SMC complexes in two nematode lineages

Abstract

Mechanisms of X chromosome dosage compensation have been studied extensively in three model organisms that represent distinct clades. The diversity within each clade as a function of sex chromosome evolution though is largely unknown. Here, we anchor ourselves to the nematode Caenorhabditis elegans, where dosage compensation is accomplished by an X chromosome specific condensin that belongs to the family of structural maintenance of chromosomes (SMC) complexes. By combining a phylogenetic analyses of the C. elegans dosage compensation complex with a comparative analysis of its epigenetic signatures, such as X-specific topologically associating domains (TADs) and enrichment of H4K20me1, we show that the condensin-mediated mechanism evolved recently in the lineage leading to Caenorhabditis following an SMC-4 duplication. Unexpectedly, we found an independent duplication of SMC-4 in Pristionchus pacificus along with the presence of X-specific TADs and H4K20me1 enrichment, which suggests that condensin-mediated dosage compensation evolved more than once in nematodes. Differential expression analysis between sexes in several nematode species indicates that dosage compensation itself precedes the evolution of X-specific condensins. In Rhabditina, X-specific condensins may have evolved in the presence of an existing mechanism linked to H4K20 methylation as Oscheius tipulae X chromosomes are enriched for H4K20me1 without SMC-4 duplication or TADs. In contrast, Steinernema hermaphroditum lacks H4K20me1 enrichment, SMC-4 duplication, and TADs. Together, our results indicate that dosage compensation mechanisms continue to evolve in species with shared X chromosome ancestry, and SMC complexes may have been coopted repeatedly in nematodes, suggesting that the process of evolving chromosome wide gene regulatory mechanisms are constrained.

Keywords: H4K20me1; Hi-C; SMC complexes; Structural Maintenance of Chromosomes; TAD; X chromosome; caenorhabditis; condensin; dosage compensation; evolution; nematodes; oscheius; parallel evolution; pristionchus; sex chromosomes; steinernema; transcription.

Figure 1.. Independent duplications of SMC-4 in Caenorhabditis and Pristionchus generated novel SMC-4 proteins.

(A) Condensin I-DC and five non-condensin proteins (green) make the C. elegans dosage compensation complex (DCC). Condensin I-DC shares all but one subunit, DPY-27, with the canonical condensin I. (B) Proteomes with BUSCO scores > 85% were run in OrthoFinder and grouped as orthogroups. SDC-2 and SDC-3 only appear in Caenorhabditis, whereas SDC-1, DPY-21, and DPY-30 are conserved among these three clades. *trioecious. (C) OrthoFinder results were used to generate a maximum-likelihood phylogeny of SMC-4. DPY-27 is an SMC-4 paralog that arose in the lineage leading to Caenorhabditis (orange circle). An independent duplication of SMC-4 occurred in Pristionchus (blue circle). For simplicity, species names were substituted for gene names. Gene names can be found in Table 2. Numbers indicate bootstrap values. Scale indicates branch length. (D) Multiple sequence alignment of the N-terminal ATPase domain highlights conserved and distinct amino acid sequences in DPY-27 and SMC-4. Within this region, example amino acid changes that support the duplication and divergence of DPY-27 in Caenorhabditis are shown in orange, and changes that support the independent duplication of SMC-4 in Caenorhabditis and Pristionchus are shown in blue.

Figure 2.. X chromosome specific topologically associating domains are present in P. pacificus, but are absent in S. hermaphroditum, O. tipulae, and O. onirici.

(A) Schematic diagram of loop-anchored topologically associating domains (TADs) on the C. elegans X chromosome. DNA contacts are higher within TADs than between. C. elegans TAD boundaries are formed by strong rex sites, reminiscent of mammalian CTCF binding elements, and are proposed to function by loading and blocking one-sided DNA loop extrusion of condensin I-DC. (B) Hi-C matrices of representative X and autosome windows at 5 kb resolution with insulation scores below (150 kb window). Empty bins result in insulation score dips that can be tracked by lack of data in the Hi-C matrix (white lines). P. pacificus X chromosomes display loop-anchored TADs phenotypic of C. elegans dosage compensation along with dips in insulation score at TAD boundaries (tick marks). S. hermaphroditum, O. tipulae and O. onirici do not display X chromosome specific TADs.

Figure 3.. Distance decay curve in P. pacificus supports the presence of an X-specific loop extruder similar to the C. elegans dosage compensation condensin.

Distance decay curves in each species show the contact probability, P(s), as a function of separation, s, for each chromosome at 5 kb resolution (top panel). Mean loop sizes for each chromosome were computed by taking the local maxima of the slope (bottom panel, tick marks). Similar to C. elegans, the P. pacificus hermaphrodite X chromosomes displays a local maxima shifted to the left (smaller loop size) when compared to the autosomes, indicative of an X chromosome specific loop extruding factor. O. tipulae does not show a difference in mean loop size between the X chromosome and autosomes, consistent with a lack of X-specific loop extruding activity.

Figure 4.. X chromosomes are consistently dosage compensated in multiple nematode lineages.

(A) Log2 fold difference in gene expression between females (or hermaphrodites) and males is plotted for expressed genes (mean TPM of replicates > 1 in both sexes) on autosomes and X chromosomes (top panel), and in soma enriched genes (bottom panel). (B) Log2 fold difference between wild type C. elegans and a dosage compensation mutant, e428, showing the expected level of expression increases on the X chromosomes in the absence of dosage compensation. (C) Distribution of sex-biased gene expression from adults plotted as |log2(hermaphrodite/male)| in O. tipulae. Similar to C. elegans, a bimodal distribution of sex-biased gene expression in O. tipulae results from germline-enriched genes with higher sex-biased gene expression. (D) -log10(p-value) from Wilcoxon rank sum tests comparing the mean log2 fold difference of a chromosome to the mean of the rest (e.g., mean of X and mean of I-V). p-values in all but the e428 mutant cluster, suggesting no statistical difference between each chromosome in each wild type species. Inf = infinite. (E) Log2 fold difference between females and males in S. carpocapsae in soma enriched genes separated by scaffold/chromosome does not support chromosome-wide dosage compensation of the X in this species. Scaffold 15 is colored orange because of its predicted location on the X chromosome.

Figure 5.. P. pacificus and O. tipulae X chromosomes are enriched for H4K20me1.

(A) H4K20me1 ChIP-seq enrichment tracks (ChIP minus input) are shown for the entire X chromosome and a representative autosome, III in S. hermaphroditum, P. pacificus, C. elegans, and O. tipulae hermaphrodite larvae. (B) Representative 50–100 kb windows are shown to highlight the distribution of H4K20me1, H3K4me3, and IgG ChIP-seq tracks (ChIP minus input) relative to gene locations. (C) H4K20me1 ChIP enrichment across gene bodies (z-scored log2(ChIP/input) from transcription start to end site) was plotted for each chromosome. H4K20me1 levels are higher on the X chromosomes of P. pacificus, C. elegans, and O. tipulae when compared to autosomes. In contrast, H4K20me1 is depleted on the X chromosomes of S. hermaphroditum. Black dot represents the mean. (D) −log10(p-value) from Wilcoxon rank sum tests comparing the mean log2(ChIP/input) of a chromosome to the mean of the rest (e.g., mean of X and mean of I-V).

Figure 6.. Model for the evolution of dosage compensation in nematodes.

Nematodes display significant changes to X chromosome content resulting from autosome to sex chromosome fusions (column 1). Dosage compensation is as old as the common ancestor of Spirurina, Tylenchina, and Rhabditina, which is supported by its presence in Brugia, Pristionchus, Caenorhabditis, Haemonchus, and Oscheius (column 2). Here, we postulate that it is likely as old as the XO sex determination in nematodes. The canonical SMC-4 is found in all nematodes sampled (column 3). DPY-27, the condensin I-DC specific subunit, is an SMC-4 paralog found only in Caenorhabditis (column 4), and the duplication event occurred in the lineage leading to Caenorhabditis (orange circle). A second, independent SMC-4 duplication occurred in Pristionchus (column 4, dark blue circle). X chromosome specific loop-anchored TADs are a signature of condensin-mediated dosage compensation in C. elegans, and are also observed in P. pacificus and C. remanei, but not in S. hermaphroditum, O. tipulae and O. onirici (column 5). Here, we postulate that X specific condensins in nematodes evolved in parallel in Caenorhabditis and Pristionchus. The repressive histone mark, H4K20me1 is enriched on the hermaphrodite X chromosomes without SMC-4 duplication or TADs in O. tipulae, suggesting it precedes condensins as a mechanism common to Rhabditina (column 6, light blue circle). -, not found; *blank space*, not checked; , nigon element; , dosage compensation; , X specific TADS; no TADs on X chromosome; , X specific enrichment of H4K20me1.