Remarkable compartmentalization of transposable elements and pseudogenes in the heterochromatin of the Tetraodon nigroviridis genome

Abstract

Tetraodon nigroviridis is among the smallest known vertebrate genomes and as such represents an interesting model for studying genome architecture and evolution. Previous studies have shown that Tetraodon contains several types of tandem and dispersed repeats, but that their overall contribution is >10% of the genome. Using genomic library hybridization, fluorescent in situ hybridization, and whole genome shotgun and directed sequencing, we have investigated the global and local organization of repeat sequences in Tetraodon. We show that both tandem and dispersed repeat elements are compartmentalized in specific regions that correspond to the short arms of small subtelocentric chromosomes. The concentration of repeats in these heterochromatic regions is in sharp contrast to their paucity in euchromatin. In addition, we have identified a number of pseudogenes that have arisen through either duplication of genes or the retro-transcription of mRNAs. These pseudogenes are amplified to high numbers, some with more than 200 copies, and remain almost exclusively located in the same heterochromatic regions as transposable elements. The sequencing of one such heterochromatic region reveals a complex pattern of duplications and inversions, reminiscent of active and frequent rearrangements that can result in the truncation and hence inactivation of transposable elements. This tight compartmentalization of repeats and pseudogenes is absent in large vertebrate genomes such as mammals and is reminiscent of genomes that remain compact during evolution such as Drosophila and Arabidopsis.

Figure 1

One satellite and four transposable element probes were hybridized to a 10-genome equivalent Tetraodon BAC library, and a large fraction of clones in common were identified. (A) The four transposable elements include Dr-Line, a LINE element similar to a Danio rerio (zebrafish) LINE; Dm-Line, which is a different LINE element homologous to Drosophila factor I; a TC1-like, and a copia-like sequences. Together the five probes (10-mer satellite, Dr-Line, Dm-Line, TC1-like, and Copia-like) identify 1,743 clones, of which 1,562 are identified with at least one transposable element and 76 clones hybridize with all four elements. The Dm-Line (B) and the TC1-like (C) probes were cohybridized by FISH to the same Tetraodon metaphase chromosome preparations and show distinct signals in the same pairs of small subtelocentric chromosomes. (Magnification: ×750.)

Figure 2

Structure and annotation of BAC clone C0AA029L14 positive with all four transposable elements tested on the BAC library. Analysis of duplicated sequences larger than 1,000 bases with miropeats (45) shows that >34% of the sequence is not unique. Arched lines join the beginning and end of duplicated regions that are represented as thick horizontal lines. The Trapeze pseudogene (filled box) and the six iSET pseudogenes (open boxes) are represented on either side of the scale bar (forward and reverse strand).

Figure 3

Exon–intron structure of the Trapeze pseudogene (GenBank accession no. AJ313483; Middle) and the native Tetraodon EZH2 gene (GenBank accession no. AJ313481, Top) and TRAPα genes (GenBank accession no. AJ313482, Bottom). Filled exons indicate EZH2 exons both in the native gene and Trapeze, and open exons indicate TRAPα exons, both in Trapeze and the native gene. Exon 9 of Trapeze is the result of a fusion between part of exon 9 of EZH2 and part of exon 5 of TRAPα. The Tetraodon EZH2 gene spans <4 kb, a 10-fold reduction in size compared with the human EZH2 gene (46). Underlined exons were used to evaluate the number of copies of each gene in the Tetraodon genome by sequence comparison. The region in Trapeze underlined with a thin dashed line was used as probe in FISH and BAC library hybridization, and the region underlined with a thick dashed line was used for deletion rate measurements. The dashed box spans the exons that code for the SET domain of EZH2.

Figure 4

Hybridization results from the iSET domain and the Trapeze pseudogene, in comparison with those of transposable elements. Of the 1,136 BAC clones identified by iSET, 1,028 were already identified by one of the four transposable elements previously hybridized to the library. Similarly, of the 396 clones positive with Trapeze, 368 are also positive with iSET, indicating that both sequences are always physically associated in the genome.