The impact of differential transposition activities of autonomous and nonautonomous hAT transposable elements on genome architecture and gene expression in Caenorhabditis inopinata

Abstract

Transposable elements are DNA sequences capable of moving within genomes and significantly influence genomic evolution. The nematode Caenorhabditis inopinata exhibits a much higher transposable element copy number than its sister species, Caenorhabditis elegans. In this study, we identified a novel autonomous transposable element belonging to the hAT superfamily from a spontaneous transposable element-insertion mutant in C. inopinata and named this transposon Ci-hAT1. Further bioinformatic analyses uncovered 3 additional autonomous hAT elements-Ci-hAT2, Ci-hAT3, and Ci-hAT4-along with over 1,000 copies of 2 nonautonomous miniature inverted-repeat transposable elements, mCi-hAT1 and mCi-hAT4, likely derived from Ci-hAT1 and Ci-hAT4 through internal deletion. We tracked at least 3 sequential transpositions of Ci-hAT1 over several years. However, the transposition rates of the other 3 autonomous hAT elements were lower, suggesting varying activity levels. Notably, the distribution patterns of the 2 miniature inverted-repeat transposable element families differed significantly: mCi-hAT1 was primarily located in the chromosome arms, a pattern observed in the transposable elements of other Caenorhabditis species, whereas mCi-hAT4 was more evenly distributed across chromosomes. Additionally, interspecific transcriptome analysis indicated that C. inopinata genes with upstream or intronic these miniature inverted-repeat transposable element insertions tend to be more highly expressed than their orthologous genes in C. elegans. These findings highlight the significant role of de-silenced transposable elements in driving the evolution of genomes and transcriptomes, leading to species-specific genetic diversity.

Keywords: Caenorhabditis inopinata; hAT superfamily; gene expression; genome evolution; transposable element.

Fig. 1.

Identification of a novel active hAT transposon in C. inopinata. a) Adult female and male wild-type (WT) and blistered (Bli) mutants of C. inopinata. Arrowheads indicate blisters present on the cuticle. Scale bars represent 100 μm. b) PCR analysis of the Cin-bli-1 gene (Sp34_20288200) in the WT and Bli mutants. c) Schematic gene structure of Cin-bli-1 alleles. Compared to the reference allele (Kanzaki et al. 2018), tjTi120 and tj182 had insertions of 3,144 and 14 bp, respectively, within the third exon. Insertions are indicated in red font. d) DNA sequences of the Cin-bli-1 alleles. In the tjTi120 allele, 8-bp target site duplications (TSDs) and 18-bp terminal inverted repeats (TIRs) of the transposon Ci-hAT1 were found at both ends of the insertion. In the tj182 allele, 2 TSDs (with a single base deletion) and an additional 8-bp insertion were identified as the transposon footprint. The target site and TSDs are highlighted in red letters, and the transposon and the additional 8-bp sequence are shown in bold black letters. e) The predicted BLI-1 amino acid sequence encoded by each allele. Frameshift mutations and premature termination codons were observed in tjTi120 and tj182.

Fig. 2.

Characterization of the Ci-hAT1 transposon. a) Schematic diagram of the Ci-hAT1 transposon. Two highly conserved nucleotides and subterminal repeats in the 5′-TIR are shown in red and blue, respectively. b) Dot plot alignment of Ci-hAT1 sequences. c) Diagram showing subterminal repeats at both ends of Ci-hAT1. The repeats on the top and bottom strands of Ci-hAT1 are depicted by blue circles above and below the transposon, respectively. d) GC content of Ci-hAT1 sequence. The AT-rich regions at both ends are indicated by gray squares. e) Domain organization of the predicted Ci-hAT1 transposase (Sp34_10071920). The DDE motifs are marked in red. f) Alignment of the amino acid sequences of RNase H-like domains in 8 known active hAT transposases and 6 Caenorhabditis hAT transposases. The distances between conserved blocks are indicated by the number of amino acid residues. Conserved amino acids are indicated by arrowheads. g) Transcripts per million (TPM) represent the abundance of Sp34_10071920 at both developmental stages. The transposases used in this analysis are listed in Supplementary Table 4.

Fig. 3.

Frequent transposition of Ci-hAT1 within the C. inopinata genome. a) Structure of 2 alleles around the Ci-hAT1 insertion site on chromosome 1. The position and distance of each primer set are indicated. The red arrowhead below the chromatogram indicates the ligation site after the deletion of 10,707 bp in tj180. b) PCR analysis of each primer set in (a) for the genome of the wild-type population. c) Sequence variation at each Ci-hAT1 insertion site. Ci-hAT1 are shown as white pentagons, with acute angles indicating the coding orientation of the transcript. The TSDs and additional sequences are shown in red and black, respectively. The deleted region in chromosome 1 is indicated by a gray square. The populations in which each allele was observed are indicated next to the sequences. d) Predicted order of Ci-hAT1 transpositions in the C. inopinata genome.

Fig. 4.

Additional identification of hAT superfamily TEs in C. inopinata. a) Dot plot alignments of the Ci-hAT2, Ci-hAT3, and Ci-hAT4 sequences. b) PCR validation of the alleles at the Ci-hAT2, Ci-hAT3, and Ci-hAT4 insertion sites in the WT population. c) Sequences of the Ci-hAT2, Ci-hAT3, and Ci-hAT4 insertion sites. Transposons are indicated by white pentagons with acute angles indicating the coding orientation of the transcript. The TSDs are indicated in red. B.I.: before insertion.

Fig. 5.

Phylogenetic tree of hAT superfamily transposases. Phylogenetic tree of transposase sequences from the Caenorhabditis genus and other groups of hAT elements. Sixty transposases were used to draw a maximum-likelihood tree. Bootstrap values are shown to the right of each branch. Each transposase name is colored according to the taxonomic group: red for animals, green for plants, and blue for fungi. The sequence of IpTip100 was used as the outgroup.

Fig. 6.

Two groups of Ci-hAT-derived nonautonomous TEs. a) Histogram showing the size distribution of elements extracted from the C. inopinata genome by “CAGTGT” search (red) or “TAGTGT” search (blue). b) Sequence logos of TSDs of “CAGTGT” and “TAGTGT”-extracted elements. (c, d) Sequence logos of 30 bp at both ends of “CAGTGT” (c) and “TAGTGT”-extracted elements (d). The black logos indicate the queries. Arrows and dotted lines above the logos indicate the range of the Ci-hAT1 or Ci-hAT4 TIRs and subterminal repeats, respectively. (e, f) Graphical distribution of blast hits in Ci-hAT1 and Ci-hAT4 (E-value < 0.01). A total 731 “CAGTGT”-extracted elements were plotted on the Ci-hAT1 (e) and 237 “TAGTGT”-extracted elements were plotted on the Ci-hAT4 (f). g) Comparison of Ci-hAT1 and mCi-hAT1 (left) and a hypothetical diagram of MITE formation by abortive gap repair (right). The formation of a stem-loop structure between the subterminal repeats of single-stranded Ci-hAT1 during gap repair causes only the terminal region of Ci-hAT1 to elongate in the homologous chromosomes and generate MITEs. Black triangles and gray boxes indicate TIRs and subterminal regions, respectively.

Fig. 7.

Chromosomal distribution and evolutionary distances of mCi-hAT1 and mCi-hAT4 in the C. inopinata genome. a) The top row for each chromosome plot shows the histogram and kernel density estimation (KDE) of protein-coding genes (black), the middle row shows mCi-hAT1 (red), and the bottom row shows mCi-hAT4 (blue). The x-axis is scaled in megabase pairs (Mbp), and the y-axis on the left indicates the density (×10⁻⁷) of each element for the KDE plot, while the y-axis on the right indicates the count of each element for the histogram. KDE plots beyond the data limits have been truncated. The numbers at the top of each plot indicate the total number of the corresponding elements on each chromosome. The gray lines at the bottom of the plots for mCi-hAT1 and mCi-hAT4 represent the relative chromosome lengths, and the overlapping rug plots show the position of each MITE on the chromosome. b) The distribution of evolutionary distances of 2 MITE types, mCi-hAT1 (red) and mCi-hAT4 (blue), from their consensus sequences. The evolutionary distance on the x-axis is calculated using the Kimura 2-parameter model, indicating the level of base sequence variation per site from the consensus. The right y-axis shows the number of MITE sequences corresponding to each distance in the histograms, while the left y-axis shows the density as estimated by kernel density estimation (KDE).

Fig. 8.

Analysis of differentially expressed genes (DEGs) between C. elegans and C. inopinata. a) Interspecific DEGs in the one-to-one orthologs between C. elegans and C. inopinata at 2 developmental stages. Orange dots indicate interspecific DEGs highly expressed in C. inopinata (ino), yellow dots indicate interspecific DEGs highly expressed in C. elegans (ele), and gray dots indicate non-DEGs. b) The proportion of interspecific DEGs at the young adult stage with or without MITEs (mCi-hAT1 or mCi-hAT4) at each gene location (upstream, intron, and downstream). Each bar indicates genes with (with) and without (without) MITEs, and includes a number indicating the number of genes in each category. Yellow indicates interspecific DEGs highly expressed in C. elegans, orange indicates interspecific DEGs highly expressed in C. inopinata, and gray indicates all other genes (including non-DEGs). Odds ratios (OR) and P-values shown at the top of each section were calculated using Fisher's exact test to indicate statistical differences between categories; P-values < 0.05 are highlighted in red.