Large palindromes on the primate X Chromosome are preserved by natural selection.

Mammalian sex chromosomes carry large palindromes that harbor protein-coding gene families with testis-biased expression. However, there are few known examples of sex-chromosome palindromes conserved between species. We identified 26 palindromes on the human X Chromosome, constituting more than 2% of its sequence, and characterized orthologous palindromes in the chimpanzee and the rhesus macaque using a clone-based sequencing approach that incorporates full-length nanopore reads. Many of these palindromes are missing or misassembled in the current reference assemblies of these species' genomes. We find that 12 human X palindromes have been conserved for at least 25 million years, with orthologs in both chimpanzee and rhesus macaque. Insertions and deletions between species are significantly depleted within the X palindromes' protein-coding genes compared to their noncoding sequence, demonstrating that natural selection has preserved these gene families. The spacers that separate the left and right arms of palindromes are a site of localized structural instability, with seven of 12 conserved palindromes showing no spacer orthology between human and rhesus macaque. Analysis of the 1000 Genomes Project data set revealed that human X-palindrome spacers are enriched for deletions relative to arms and flanking sequence, including a common spacer deletion that affects 13% of human X Chromosomes. This work reveals an abundance of conserved palindromes on primate X Chromosomes and suggests that protein-coding gene families in palindromes (most of which remain poorly characterized) promote X-palindrome survival in the face of ongoing structural instability.

Sequence analysis in Bos taurus reveals pervasiveness of X-Y arms races in mammalian lineages.

Studies of Y Chromosome evolution have focused primarily on gene decay, a consequence of suppression of crossing-over with the X Chromosome. Here, we provide evidence that suppression of X-Y crossing-over unleashed a second dynamic: selfish X-Y arms races that reshaped the sex chromosomes in mammals as different as cattle, mice, and men. Using super-resolution sequencing, we explore the Y Chromosome of Bos taurus (bull) and find it to be dominated by massive, lineage-specific amplification of testis-expressed gene families, making it the most gene-dense Y Chromosome sequenced to date. As in mice, an X-linked homolog of a bull Y-amplified gene has become testis-specific and amplified. This evolutionary convergence implies that lineage-specific X-Y coevolution through gene amplification, and the selfish forces underlying this phenomenon, were dominatingly powerful among diverse mammalian lineages. Together with Y gene decay, X-Y arms races molded mammalian sex chromosomes and influenced the course of mammalian evolution.

A gene deriving from the ancestral sex chromosomes was lost from the X and retained on the Y chromosome in eutherian mammals.

BACKGROUND: The mammalian X and Y chromosomes originated from a pair of ordinary autosomes. Over the past ~180 million years, the X and Y have become highly differentiated and now only recombine with each other within a short pseudoautosomal region. While the X chromosome broadly preserved its gene content, the Y chromosome lost ~92% of the genes it once shared with the X chromosome. PRSSLY is a Y-linked gene identified in only a few mammalian species that was thought to be acquired, not ancestral. However, PRSSLY's presence in widely divergent species-bull and mouse-led us to further investigate its evolutionary history. RESULTS: We discovered that PRSSLY is broadly conserved across eutherians and has ancient origins. PRSSLY homologs are found in syntenic regions on the X chromosome in marsupials and on autosomes in more distant animals, including lizards, indicating that PRSSLY was present on the ancestral autosomes but was lost from the X and retained on the Y in eutherian mammals. We found that across eutheria, PRSSLY's expression is testis-specific, and, in mouse, it is most robustly expressed in post-meiotic germ cells. The closest paralog to PRSSLY is the autosomal gene PRSS55, which is expressed exclusively in testes, involved in sperm differentiation and migration, and essential for male fertility in mice. Outside of eutheria, in species where PRSSLY orthologs are not Y-linked, we find expression in a broader range of somatic tissues, suggesting that PRSSLY has adopted a germ-cell-specific function in eutherians. Finally, we generated Prssly mutant mice and found that they are fully fertile but produce offspring with a modest female-biased sex ratio compared to controls. CONCLUSIONS: PRSSLY appears to be the first example of a gene that derives from the mammalian ancestral sex chromosomes that was lost from the X and retained on the Y. Although the function of PRSSLY remains to be determined, it may influence the sex ratio by promoting the survival or propagation of Y-bearing sperm.

Mammalian Y chromosomes retain widely expressed dosage-sensitive regulators.

The human X and Y chromosomes evolved from an ordinary pair of autosomes, but millions of years ago genetic decay ravaged the Y chromosome, and only three per cent of its ancestral genes survived. We reconstructed the evolution of the Y chromosome across eight mammals to identify biases in gene content and the selective pressures that preserved the surviving ancestral genes. Our findings indicate that survival was nonrandom, and in two cases, convergent across placental and marsupial mammals. We conclude that the gene content of the Y chromosome became specialized through selection to maintain the ancestral dosage of homologous X-Y gene pairs that function as broadly expressed regulators of transcription, translation and protein stability. We propose that beyond its roles in testis determination and spermatogenesis, the Y chromosome is essential for male viability, and has unappreciated roles in Turner's syndrome and in phenotypic differences between the sexes in health and disease.

Strict evolutionary conservation followed rapid gene loss on human and rhesus Y chromosomes.

The human X and Y chromosomes evolved from an ordinary pair of autosomes during the past 200-300 million years. The human MSY (male-specific region of Y chromosome) retains only three percent of the ancestral autosomes' genes owing to genetic decay. This evolutionary decay was driven by a series of five 'stratification' events. Each event suppressed X-Y crossing over within a chromosome segment or 'stratum', incorporated that segment into the MSY and subjected its genes to the erosive forces that attend the absence of crossing over. The last of these events occurred 30 million years ago, 5 million years before the human and Old World monkey lineages diverged. Although speculation abounds regarding ongoing decay and looming extinction of the human Y chromosome, remarkably little is known about how many MSY genes were lost in the human lineage in the 25 million years that have followed its separation from the Old World monkey lineage. To investigate this question, we sequenced the MSY of the rhesus macaque, an Old World monkey, and compared it to the human MSY. We discovered that during the last 25 million years MSY gene loss in the human lineage was limited to the youngest stratum (stratum 5), which comprises three percent of the human MSY. In the older strata, which collectively comprise the bulk of the human MSY, gene loss evidently ceased more than 25 million years ago. Likewise, the rhesus MSY has not lost any older genes (from strata 1-4) during the past 25 million years, despite its major structural differences to the human MSY. The rhesus MSY is simpler, with few amplified gene families or palindromes that might enable intrachromosomal recombination and repair. We present an empirical reconstruction of human MSY evolution in which each stratum transitioned from rapid, exponential loss of ancestral genes to strict conservation through purifying selection.

Reduced expression of the Kinesin-Associated Protein 3 (KIFAP3) gene increases survival in sporadic amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a degenerative disorder of motor neurons that typically develops in the 6th decade and is uniformly fatal, usually within 5 years. To identify genetic variants associated with susceptibility and phenotypes in sporadic ALS, we performed a genome-wide SNP analysis in sporadic ALS cases and controls. A total of 288,357 SNPs were screened in a set of 1,821 sporadic ALS cases and 2,258 controls from the U.S. and Europe. Survival analysis was performed using 1,014 deceased sporadic cases. Top results for susceptibility were further screened in an independent sample set of 538 ALS cases and 556 controls. SNP rs1541160 within the KIFAP3 gene (encoding a kinesin-associated protein) yielded a genome-wide significant result (P = 1.84 x 10(-8)) that withstood Bonferroni correction for association with survival. Homozygosity for the favorable allele (CC) conferred a 14.0 months survival advantage. Sequence, genotypic and functional analyses revealed that there is linkage disequilibrium between rs1541160 and SNP rs522444 within the KIFAP3 promoter and that the favorable alleles of rs1541160 and rs522444 correlate with reduced KIFAP3 expression. No SNPs were associated with risk of sporadic ALS, site of onset, or age of onset. We have identified a variant within the KIFAP3 gene that is associated with decreased KIFAP3 expression and increased survival in sporadic ALS. These findings support the view that genetic factors modify phenotypes in this disease and that cellular motor proteins are determinants of motor neuron viability.

SHIMS 3.0: Highly efficient single-haplotype iterative mapping and sequencing using ultra-long nanopore reads.

The reference sequence of structurally complex regions can only be obtained through a highly accurate clone-based approach that we call Single-Haplotype Iterative Mapping and Sequencing (SHIMS). In recent years, improvements to SHIMS have reduced the cost and time required by two orders of magnitude, but internally repetitive clones still require extensive manual effort to transform draft assemblies into reference-quality finished sequences. Here we describe SHIMS 3.0, using ultra-long nanopore reads to augment the Illumina data from SHIMS 2.0 assemblies and resolve internally repetitive structures. This greatly minimizes the need for manual finishing of Illumina-based draft assemblies, allowing a small team with no prior finishing experience to sequence challenging targets with high accuracy. This protocol proceeds from clone-picking to finished assemblies in 2 weeks for about $80 (USD) per clone. We recently used this protocol to produce reference sequence of structurally complex palindromes on chimpanzee and rhesus macaque X chromosomes. Our protocol provides access to structurally complex regions that would otherwise be inaccessible from whole-genome shotgun data or require an impractical amount of manual effort to generate an accurate assembly.

Locating and Characterizing a Transgene Integration Site by Nanopore Sequencing.

The introduction of foreign DNA into cells and organisms has facilitated much of modern biological research, and it promises to become equally important in clinical practice. Locating sites of foreign DNA incorporation in mammalian genomes has proven burdensome, so the genomic location of most transgenes remains unknown. To address this challenge, we applied nanopore sequencing in search of the site of integration of Tg(Pou5f1-EGFP)2Mnn (also known as Oct4:EGFP), a widely used fluorescent reporter in mouse germ line research. Using this nanopore-based approach, we identified the site of Oct4:EGFP transgene integration near the telomere of Chromosome 9. This methodology simultaneously yielded an estimate of transgene copy number, provided direct evidence of transgene inversions, revealed contaminating E. coli genomic DNA within the transgene array, validated the integrity of neighboring genes, and enabled definitive genotyping. We suggest that such an approach provides a rapid, cost-effective method for identifying and analyzing transgene integration sites.

A common haplotype within the PON1 promoter region is associated with sporadic ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disorder of upper and lower motor neurons. Genetic variants in the paraoxonase gene cluster have been associated with susceptibility to sporadic ALS. Because these studies have yielded conflicting results, we have further investigated this association in a larger data set. Twenty SNPs spanning the paraoxonase gene cluster were genotyped on a panel of 597 case and 692 control samples and tested for association with risk of sporadic ALS and with ALS sub-phenotypes. Our study revealed two SNPs, rs987539 and rs2074351, within the paraoxonase gene cluster that are associated with susceptibility to sporadic ALS (uncorrected p=6.47E-04 and 7.87E-04, respectively). None of the 20 SNPs displayed significant associations with age of onset, site of onset or disease survival. Using a sliding window approach, we have also identified a 5-SNP haplotype that is significantly associated with risk of sporadic ALS (p=2.75E-05). We conclude that a common haplotype within the PON1 promoter region is associated with susceptibility to sporadic ALS.

Cost-effective high-throughput single-haplotype iterative mapping and sequencing for complex genomic structures.

The reference sequences of structurally complex regions can be obtained only through highly accurate clone-based approaches. We and others have successfully used single-haplotype iterative mapping and sequencing (SHIMS) 1.0 to assemble structurally complex regions across the sex chromosomes of several vertebrate species and to allow for targeted improvements to the reference sequences of human autosomes. However, SHIMS 1.0 is expensive and time consuming, requiring resources that only a genome center can provide. Here we introduce SHIMS 2.0, an improved SHIMS protocol that allows even a small laboratory to generate high-quality reference sequence from complex genomic regions. Using a streamlined and parallelized library-preparation protocol, and taking advantage of inexpensive high-throughput short-read-sequencing technologies, a small laboratory with both molecular biology and bioinformatics experience can sequence and assemble 192 large-insert bacterial artificial chromosome (BAC) or fosmid clones in 1 week. In SHIMS 2.0, in contrast to other pooling strategies, each clone is sequenced with a unique barcode, thus enabling clones containing nearly identical sequences to be multiplexed in a single sequencing run and assembled separately. Relative to SHIMS 1.0, SHIMS 2.0 decreases the required cost and time by two orders of magnitude while preserving high sequencing accuracy.

Avian W and mammalian Y chromosomes convergently retained dosage-sensitive regulators.

After birds diverged from mammals, different ancestral autosomes evolved into sex chromosomes in each lineage. In birds, females are ZW and males are ZZ, but in mammals females are XX and males are XY. We sequenced the chicken W chromosome, compared its gene content with our reconstruction of the ancestral autosomes, and followed the evolutionary trajectory of ancestral W-linked genes across birds. Avian W chromosomes evolved in parallel with mammalian Y chromosomes, preserving ancestral genes through selection to maintain the dosage of broadly expressed regulators of key cellular processes. We propose that, like the human Y chromosome, the chicken W chromosome is essential for embryonic viability of the heterogametic sex. Unlike other sequenced sex chromosomes, the chicken W chromosome did not acquire and amplify genes specifically expressed in reproductive tissues. We speculate that the pressures that drive the acquisition of reproduction-related genes on sex chromosomes may be specific to the male germ line.