ZNF280BY and ZNF280AY: autosome derived Y-chromosome gene families in Bovidae

Abstract

Background: Recent progress in exploring the Y-chromosome gene content in humans, mice and cats have suggested that "autosome-to-Y" transposition of the male fertility genes is a recurrent theme during the mammalian Y-chromosome evolution. These transpositions are lineage-dependent. The purpose of this study is to investigate the lineage-specific Y-chromosome genes in bovid.

Results: We took a direct testis cDNA selection strategy and discovered two novel gene families, ZNF280BY and ZNF280AY, on the bovine (Bos taurus) Y-chromosome (BTAY), which originated from the transposition of a gene block on the bovine chromosome 17 (BTA17) and subsequently amplified. Approximately 130 active ZNF280BY loci (and ~240 pseudogenes) and ~130 pseudogenized ZNF280AY copies are present over the majority of the male-specific region (MSY). Phylogenetic analysis indicated that both gene families fit with the "birth-and-death" model of evolution. The active ZNF280BY loci share high sequence similarity and comprise three major genomic structures, resulted from insertions/deletions (indels). Assembly of a 1.2 Mb BTAY sequence in the MSY ampliconic region demonstrated that ZNF280BY and ZNF280AY, together with HSFY and TSPY families, constitute the major elements within the repeat units. The ZNF280BY gene family was found to express in different developmental stages of testis with sense RNA detected in all cell types of the seminiferous tubules while the antisense RNA detected only in the spermatids. Deep sequencing of the selected cDNAs revealed that different loci of ZNF280BY were differentially expressed up to 60-fold. Interestingly, different copies of the ZNF280AY pseudogenes were also found to differentially express up to 10-fold. However, expression level of the ZNF280AY pseudogenes was almost 6-fold lower than that of the ZNF280BY genes. ZNF280BY and ZNF280AY gene families are present in bovid, but absent in other mammalian lineages.

Conclusions: ZNF280BY and ZNF280AY are lineage-specific, multi-copy Y-gene families specific to Bovidae, and are derived from the transposition of an autosomal gene block. The temporal and spatial expression patterns of ZNF280BYs in testis suggest a role in spermatogenesis. This study offers insights into the genomic organization of the bovine MSY and gene regulation in spermatogenesis, and provides a model for studying evolution of multi-copy gene families in mammals.

Figure 1

Tissue expression profiles of ZNF280B/ZNF280BY and ZNF280A/ZNF280AY. Both ZNF280BY and ZNF280AY were proved to be Y-specific genes via the male-specific PCR (lane marked with ♂). ZNF280BY is expressed predominantly in the testes and slightly in liver, adrenal gland and lymph node while the autosomal ZNF280B is expressed specifically in the testis. The ZNF280AY is expressed predominantly in the spleen and low in testis and brain, while the expression of the ZNF280A was not detected among the eight tissues studied. The β-actin gene was used as positive control. Te, testis; Li, liver; Ki, kidney; Spl, spleen; Br, brain (cerebrum); Ad, adrenal gland; Mu, muscle; Ly, lymph node; Ov, ovary; ♂, bovine male genomic DNA control; ♀, bovine female genomic DNA control; -, water; M, 1 kb DNA ladder.

Figure 2

Genome structures of the bovine ZNF280BY. Schematic representation of the three major genomic structures of ZNF280BY, denoted as type A, B and C. Type A is the normal form with an estimated 113 copies on BTAY. Type B is resulted from a 13 bp insertion while Type C resulted from an 8 bp deletion in the coding region. The coding segments (CDS) are shaded in black. The number denotes the length of exons, introns and CDS in bp. The non-homologous exon 1 of ZNF280BY type C is shaded in grey. The polyA [(A)_n] sites are indicated.

Figure 3

Locus-specific expression of ZNF280BY and ZNF280AY in testis. The alignment of read pairs derived from the deep sequencing of the selected cDNAs against unique coding regions of the ZNF280BY (A) and ZNF280AY (B) gene families. The left side Y-axis indicates the normalized number of read pairs, and the right side Y-axis indicates the identical copy number for a particular gene locus on BTAY. The blue columns indicate the perfectly matched read-pairs, while the yellow columns show the matched read-pairs with one nucleotide mismatch. The deep sequencing reads counting results are detailed in Additional file 3.

Figure 4

Sequence analysis of a 1.2 Mb ampliconic region of BTAY. This region was assembled from nine Y-BACs (see Additional file 4). A. Triangular dot-plot analysis of the 1.2 Mb BTAY sequence. Each dot represents a perfect match in a word size of 10 nucleotides [81]. The plot indicates that the BTAY ampliconic region is enriched with internally repetitive units. B. Two direct repeats (DR1 and DR2) and two inverted repeats (IR1 and IR2) were detected in the region. The regions with high arm-to-arm similarity are highlighted in red and blue for DR and IR, respectively. The corresponding repeat regions are also highlighted in the dot-plot. The dark red regions highlight the DRs with similarity over 99.5%; the dark blue regions highlight the IRs with similarity over 99.5%. The light red regions are extended direct repeats with similarity over 99% and the light blue regions are extended inverted repeats with similarity over 98.5%. C. The predicted transcribed regions of ZNF280BY, ZNF280AY, HSFY and TSPY are depicted in different tracks with different colors as indicated in the up-right corner. Scale: bar = 100 kb.

Figure 5

Phylogenetic trees of ZNF280B/ZNF280BY and ZNF280A/ZNF280AY gene families. A. ZNF280B/ZNF280BY tree. B. ZNF280A/ZNF280AY tree. The bovine ZNF280BY and ZNF280AY gene families are consistently clustered with their autosomal paralog on chromosome 17, supporting that these two gene families originated from the transposition of the autosomal gene block. The trees were built based on the Neighboring-joining method implemented in MEGA4 software [77]. The trees are drawn to scale and the evolutionary distances were computed using the Maximum Composite Likelihood method. The bootstrap values (1000 replicates) are shown next to the branches [83]. The filled triangle indicates the condensed branch for each of the gene families. Scale: bar = 0.05 unit of the number of base substitutions per site. HSA = Human; PTR = Chimpanzee; MMUL = Macaque; MMU = Mouse; RNO = Rat; CFA = Dog; ECA = Horse; SSC = Pig; ORA = Sheep; BTA = Cattle.

Figure 6

Tissue localization of the bovine ZNF280BY transcript in adult testis. The sense and antisense RNA of ZNF280BY are expressed in adult testis. A. The ZNF280BY sense RNA is expressed widely and evenly across all cell types in the seminiferous tubules. B. The antisense RNA of ZNF280BY was only detected in spermatids. Sense (A) and antisense (B) RNA of ZNF280BY were detected by the corresponding DIG-labeled cRNA probes. C. The bovine Protamine gene was used as the positive control, and there is no antisense mRNA of Protamine detected in the bovine testis [34]. D. The Haematoxylin and Eosin (H&E) staining was shown. Scale: bar = 200 μm.

Figure 7

Expression of ZNF280BY sense and antisense RNA at different developmental stages of testis. The relative expression levels of the ZNF280BY sense and antisense RNA at different ages of testis (X-axis), measured by the strand-specific qPCR, were normalized by the 18S rRNA (Y-axis). Values are means ± SD of triplicates. The expression of sense RNA increased gradually with age. In contrast, the ZNF280BY antisense RNA had a stable expression level except for a decrease in 3 month old testis. The significant expression difference was identified when p ≤ 0.05 by the ANOVA analysis and denoted by (a,b) where the gene expression at stages of (b) is significantly different from that at (a).