Novel gene acquisition on carnivore Y chromosomes

Abstract

Despite its importance in harboring genes critical for spermatogenesis and male-specific functions, the Y chromosome has been largely excluded as a priority in recent mammalian genome sequencing projects. Only the human and chimpanzee Y chromosomes have been well characterized at the sequence level. This is primarily due to the presumed low overall gene content and highly repetitive nature of the Y chromosome and the ensuing difficulties using a shotgun sequence approach for assembly. Here we used direct cDNA selection to isolate and evaluate the extent of novel Y chromosome gene acquisition in the genome of the domestic cat, a species from a different mammalian superorder than human, chimpanzee, and mouse (currently being sequenced). We discovered four novel Y chromosome genes that do not have functional copies in the finished human male-specific region of the Y or on other mammalian Y chromosomes explored thus far. Two genes are derived from putative autosomal progenitors, and the other two have X chromosome homologs from different evolutionary strata. All four genes were shown to be multicopy and expressed predominantly or exclusively in testes, suggesting that their duplication and specialization for testis function were selected for because they enhance spermatogenesis. Two of these genes have testis-expressed, Y-borne copies in the dog genome as well. The absence of the four newly described genes on other characterized mammalian Y chromosomes demonstrates the gene novelty on this chromosome between mammalian orders, suggesting it harbors many lineage-specific genes that may go undetected by traditional comparative genomic approaches. Specific plans to identify the male-specific genes encoded in the Y chromosome of mammals should be a priority.

Figure 1. Radiation Hybrid Maps of Feline Y and X Chromosomes

(A) Radiation hybrid map of single-copy, X-degenerate genes on the domestic cat Y chromosome. The scale is given in centirays (given in 50 cR₅₀₀₀ units). Markers in bold are positioned with odds of ≥1,000:1. (B) Radiation hybrid map of the domestic cat X chromosome, showing the position of X-homologous sequences of TETY2 and CUL4BY. The markers are placed in their most likely intervals relative to the existing feline X chromosome RH map [13]. Red lines with arrows show the comparative position of these genes on an ideogram of the human X chromosome. Additional red dashed lines affirm the overall conservation of gene order observed between the feline and human X chromosomes [13,29,43,44]. Colored blocks on the left demarcate the relative boundaries of four of the five known human X chromosome evolutionary strata [4,26,27,51]. Note that CUL4BX is clearly nested in Stratum 1, consistent with its divergence from CUL4BY prior to the divergence of eutherians, marsupials, and monotremes approximately 230 million years ago [52] (see Table S2).

Figure 2. Alignment of Predicted Amino Acid Sequences for Three Feline FLJ36031Y Genes and Human Autosomal FLJ36031

Above the alignments, asterisks indicate sites that are conserved. Below the alignments are histograms displaying level of amino acid conservation across the alignment. Note that the human FLJ36031 protein contains a long stretch of mostly unique amino acids from position 40 to 111, whereas the carboxyl terminus of the feline FLJ36031Y proteins (positions 232–318) is unique.

Figure 3. Retention Frequency of Different Classes of Cat Y Chromosome Genes in the Feline RH Panel

The retention frequency of single copy X-degenerate genes (boxed in green) is similar to the genome-wide average of 0.39 [–44]. Single copy X-degenerate genes close to the centromere (see Figure 5 for SRY FISH results) and potentially low–copy number genes (e.g., HSFY), are slightly elevated (boxed in blue). The five multicopy genes (boxed in red) have considerably higher retention frequencies than the single-copy genes, confirmed by the broad Yq chromosomal distributions determined by FISH (Figure 5).

Figure 4. Male-Specific Amplification of TETY2 and CUL4BY STS Primers in Cat and Dog

A fragment of both genes has been PCR amplified with Y chromosome STS markers in matched cat and dog male and female genomic DNA samples. A 1-kilobase (kb) DNA ladder is shown to the left and right. PCR products are of the expected size in male DNAs, whereas no or nonspecific amplification is observed in female DNAs. The third lane in each set of primers (−) is a no-DNA control reaction. Dog ESTCO610012 is orthologous to cat TETY2.

Figure 5. Multicopy Distribution of Novel Y Chromosome cDNAs Confirmed by FISH

FISH of feline cDNA probes on male domestic cat metaphase preparations. For each novel gene, the signal is detected only on the Y chromosome and is generally restricted to the long arm. Note that no hybridization signals were found on the distal short arm, which is heterochromatic [30]. For multicopy genes we also show the hybridization results on interphase nuclei. An SRY-containing BAC clone hybridized to the short arm of the Y chromosome near the centromere. A magnified view of the reverse-DAPI banded Y chromosome is shown in the upper left corner of each image, both with (right) and without (left) hybridization signals.

Figure 6. Testis-Specific Transcription of Feline Multicopy Y Chromosome Genes

Amplification results of PCR primers specific for nine feline genes/sequences are shown in a panel of six adult domestic cat mRNA samples: Te, testis; Br, brain (cerebrum); Mu, muscle; He, heart; Ki, kidney; Lu, lung; −, no mRNA control; +, male domestic cat genomic DNA control. Each reaction contains 10 ng of mRNA. The gene being assayed is listed to the left of each frame. No background genomic DNA amplification was detected using a noncoding Y chromosome genomic STS marker. Negative RT reactions were also run for each STS marker and showed no amplification (unpublished data). All feline multicopy genes show testis-only expression, with the exception of TETY2, which also showed weak expression in kidney.