The mole genome reveals regulatory rearrangements associated with adaptive intersexuality

Abstract

Linking genomic variation to phenotypical traits remains a major challenge in evolutionary genetics. In this study, we use phylogenomic strategies to investigate a distinctive trait among mammals: the development of masculinizing ovotestes in female moles. By combining a chromosome-scale genome assembly of the Iberian mole, Talpa occidentalis, with transcriptomic, epigenetic, and chromatin interaction datasets, we identify rearrangements altering the regulatory landscape of genes with distinct gonadal expression patterns. These include a tandem triplication involving CYP17A1, a gene controlling androgen synthesis, and an intrachromosomal inversion involving the pro-testicular growth factor gene FGF9, which is heterochronically expressed in mole ovotestes. Transgenic mice with a knock-in mole CYP17A1 enhancer or overexpressing FGF9 showed phenotypes recapitulating mole sexual features. Our results highlight how integrative genomic approaches can reveal the phenotypic impact of noncoding sequence changes.

Fig. 1. Mole genome and epigenetic and transcriptional study of ovotestis development.

(A) An Iberian mole (Talpa occidentalis) and an adult mole ovotestis. Scale bar represents 500 μm. (B) Genome assembly of T. occidentalis and gene annotation statistics. (C) Phylogenetic tree, based on four-fold degenerate sites, between selected species. (D) Venn diagram of active enhancers (data S1). (E) PC analysis of RNA-seq datasets of P7 mole gonads.

Fig. 2. Identification of genes with altered 3D chromatin regulatory landscapes.

(A) Strategy used to identify genes with altered 3D chromatin organization as a result of species-specific rearrangements. (B) Strategy used to assign regulatory elements to candidate genes. Number of active enhancers are correlated to gene expression levels for each tissue. (C) Correlation between the percentage of active enhancers and gene expression per tissue (orange=ovary part, green=testis part, blue=male testis) for selected candidates (full gene dataset in fig. S4 and data S7). STRA6, FGF9 and CYP17A1 display the highest positive correlation, ATM shows negative correlation.

Fig. 3. Duplication of regulatory elements at the CYP17A1 locus and associated increase in androgen production and strength.

(A) Comparative genomics at the CYP17A1 locus. (B) CYP17A1 expression (RNA-seq) in mole and mouse adult gonads (n=2). (C) Expression profile (RNA-seq, top), enhancer marks (H3K27Ac, center) and open chromatin (ATAC-seq, bottom) for testis part and testis at P7 gonads. Segmentation for active enhancers for testis part (green bars) and testis (blue bars). BLAT Sequence homology is represented in gray boxes. Duplicated enhancer (A-B) results from fusion of enhancer A and B. (D) Up: Integration of the mole CYP17A1 duplicated enhancer into the mouse Cyp17a1 locus. Down: Expression analysis of Cyp17a1 (RT-qPCR) in adult mouse mutant gonads and wild-type controls (n ≥ 5). (E) Circulating testosterone levels in adult mouse mutants and wild-type controls (n=7). (F) Grip strength test in adult mouse mutants and wild-type controls (n=7). Bars represent mean and SD. Two-sided Student’s t-test, n.s = non-significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Fig. 4. An inversion altering the regulatory landscape of the FGF9 mole locus.

(A) Comparative genomics at the FGF9 locus. (B) Hi-C maps for human and mole displaying synteny break (discontinuous line) and TAD prediction. Below, CTCF ChIP-seq (mole P7 gonads) with peak orientation. 4C-seq using mole FGF9 promoter as viewpoint shows contact extension beyond synteny break. Zoom of FGF9 interacting region shows active ovarian enhancers (asterisks). H3K27Ac, ATAC-seq and segmentation for active enhancer tracks are displayed in orange. (C) FGF9 expression (RNA-seq) in mice and moles at different timepoints. Bars represent mean and SD (n ≥ 2). Two-sided Student's t-test, n.s = non-significant, *P ≤ 0.05, **P ≤ 0.01.

Fig. 5. FGF9 sustained expression delays meiosis and promotes XX gonadal masculinization.

(A) Spatio-temporal expression of FGF9 and the meiotic marker SYCP3 (immunostaining, green, DAPI in blue). Insets display zoomed regions from OP. Scale bars represent 100 μm. (B) Volcano plot from RNA-seq of XX mutant versus wild-type gonads (E13.5) and gene ontology analysis. (C) Hematoxylin and Eosin staining of XX gonads of adult mutants and wild-type controls. Cord-like structures in mutants denote XX-to-XY sex reversal. Inset shows SOX9 expression (immunostaining, green, DAPI in blue). Scale bars represent 200 μm.