Biology subjects

Skaletsky, H.

Publications and source records attributed to Skaletsky, H..

11 recordsLinked to original sources

bioRxiv2025

The SHR/Akr Y chromosome reveals repeated turnover of the rat pseudoautosomal region

Crossing-over between Chr X and Chr Y was first observed 90 years ago, in the brown rat, Rattus norvegicus. However, the sequence of the rat pseudoautosomal region (PAR) has remained a mystery. We produced a near-complete sequence of Chr Y from the SHR strain, along with nearly a megabase of sequence from both telomeres of Chr X. Both telomeric ends of Chr Y display extensive homology to Chr X, but no homology to the ancestral PAR of placental mammals. Using rat Y BACs probes for FISH on meiotic cells, we show that pairing almost always occurs between the tips of Yq and Xp, which are virtually identical in nucleotide sequence, but contain no protein-coding genes. Homology at the other ends of Chr X and Chr Y is likely the result of a recent transposition of five genes from Xq to Yp. These sequences are only 99.5% identical and pair infrequently, but show signs that it may have been pseudoautosomal in the ancestor of the rat genus. The rat Chr Y sequence presents opportunities for experimental studies of meiotic phenomena in a tractable model organism. The short PAR, with a correspondingly high recombination rate, represents a unique substrate for molecular studies of crossing-over. Likewise, the absence of extensively co-amplified testis gene families on the rat X and Y suggests they might serve as control for the intense competition between selfish elements that completely remodeled the mouse sex chromosomes.

genomics↗

bioRxiv2024

Independent effects of testosterone, estradiol, and sex chromosomes on gene expression in immune cells of trans- and cisgender individuals

The origins of sex differences in human disease are elusive, in part because of difficulties in separating the effects of sex hormones and sex chromosomes. To separate these variables, we examined gene expression in four groups of trans- or cisgender individuals: XX individuals treated with exogenous testosterone (n=21), XY treated with exogenous estradiol (n=13), untreated XX (n=20), and untreated XY (n=15). We performed single-cell RNA-sequencing of 358,426 peripheral blood mononuclear cells. Across the autosomes, 8 genes responded with a significant change in expression to testosterone, 34 to estradiol, and 32 to sex chromosome complement with no overlap between the groups. No sex-chromosomal genes responded significantly to testosterone or estradiol, but X-linked genes responded to sex chromosome complement in a remarkably stable manner across cell types. Through leveraging a four-state study design, we successfully separated the independent actions of testosterone, estradiol, and sex chromosome complement on genome-wide gene expression in humans.

genomics↗

bioRxiv2024

Where is the boundary of the human pseudoautosomal region?

A recent publication describing the assembly of the Y chromosomes of 43 males was remarkable not only for its ambitious technical scope, but also for the startling suggestion that the boundary of the pseudoautosomal region 1 (PAR1), where the human X and Y chromosomes engage in crossing-over during male meiosis, lies 500 kb distal to its previously reported location1. Where is the boundary of the human PAR1? We first review the evidence that mapped the PAR boundary, or PAB, before the human genome draft sequence was produced, then examine post-genomic datasets for evidence of crossing-over between the X and Y, and lastly re-examine the sequence assemblies presented by Hallast and colleagues to see whether they support a more distal PAB. We find ample evidence of X-Y crossovers throughout the 500 kb in question, some as close as 246 bp to the previously reported PAB. Our new analyses, combined with previous studies over the past 40 years, provide overwhelming evidence to support the original position, and narrow the probable location of the PAB to a 201-bp window.

genetics↗

bioRxiv2024

Stable and robust Xi and Y transcriptomes drive cell-type-specific autosomal and Xa responses in vivo and in vitro in four human cell types

Recent in vitro studies of human sex chromosome aneuploidy showed that the Xi ("inactive" X) and Y chromosomes broadly modulate autosomal and Xa ("active" X) gene expression in two cell types. We tested these findings in vivo in two additional cell types. Using linear modeling in CD4+ T cells and monocytes from individuals with one to three X chromosomes and zero to two Y chromosomes, we identified 82 sex-chromosomal and 344 autosomal genes whose expression changed significantly with Xi and/or Y dosage in vivo. Changes in sex-chromosomal expression were remarkably constant in vivo and in vitro across all four cell types examined. In contrast, autosomal responses to Xi and/or Y dosage were largely cell-type-specific, with up to 2.6-fold more variation than sex-chromosomal responses. Targets of the X- and Y-encoded transcription factors ZFX and ZFY accounted for a significant fraction of these autosomal responses both in vivo and in vitro. We conclude that the human Xi and Y transcriptomes are surprisingly robust and stable across the four cell types examined, yet they modulate autosomal and Xa genes - and cell function - in a cell-type-specific fashion. These emerging principles offer a foundation for exploring the wide-ranging regulatory roles of the sex chromosomes across the human body.

genomics↗

bioRxiv2023

The human Y and inactive X chromosomes similarly modulate autosomal gene expression

Somatic cells of human males and females have 45 chromosomes in common, including the "active" X chromosome. In males the 46th chromosome is a Y; in females it is an "inactive" X (Xi). Through linear modeling of autosomal gene expression in cells from individuals with zero to three Xi and zero to four Y chromosomes, we found that Xi and Y impact autosomal expression broadly and with remarkably similar effects. Studying sex-chromosome structural anomalies, promoters of Xi- and Y-responsive genes, and CRISPR inhibition, we traced part of this shared effect to homologous transcription factors - ZFX and ZFY - encoded by Chr X and Y. This demonstrates sex-shared mechanisms by which Xi and Y modulate autosomal expression. Combined with earlier analyses of sex-linked gene expression, our studies show that 21% of all genes expressed in lymphoblastoid cells or fibroblasts change expression significantly in response to Xi or Y chromosomes.

genetics↗

bioRxiv2023

A Four "Core Genotypes" rat model to distinguish mechanisms underlying sex-biased phenotypes and diseases

BackgroundPrevious research on Four Core Genotypes and XY* mice has been instrumental in establishing important effects of sex-chromosome complement that cause sex differences in physiology and disease. We have generated rat models using similar modifications of the testis-determining gene Sry, to produce XX and XY rats with the same type of gonad, as well as XO, XXY and XYY rats with varying gonads. The models permit discovery of novel sex-chromosome effects (XX vs. XY) that contribute to sex differences in any rat phenotype, and test for effects of different numbers of X or Y chromosomes. MethodsXY rats were created with an autosomal transgene of Sry, producing XX and XY progeny with testes. In other rats, CRISPR-Cas9 technology was used to remove Y chromosome factors that initiate testis differentiation, producing fertile XY gonadal females. Interbreeding of these lines produced rats with interesting combinations of sex chromosomes and gonads: XO, XX, XY, XXY rats with ovaries; and XO, XX, XY, XXY, and XYY rats with testes. These groups can be compared to detect sex differences caused by sex-chromosome complement (XX vs. XY) and/or by gonadal hormones (rats with testes vs. ovaries). Other comparisons detect the effects of X or Y chromosome number (in gonadal females: XO vs. XX, XX vs. XXY, XO vs. XY, XY vs. XXY; in gonadal males: XY vs. XXY, XY vs. XYY; XX vs. XXY, XO vs. XY). ResultsWe measured numerous phenotypes to characterize these models, including gonadal histology, breeding performance, anogenital distance, levels of reproductive hormones, body and organ weights, and central nervous system sexual dimorphisms. Serum testosterone levels were comparable in adult XX and XY gonadal males. Phenotypes previously known to be sexually differentiated by the action of gonadal hormones were found to be similar in XX and XY rats with the same type of gonad, suggesting that XX and XY rats with the same type of gonad have comparable levels of gonadal hormones at various stages of development. ConclusionThe results establish powerful new models to discriminate sex-chromosome and gonadal hormone effects that cause sexual differences in rat physiology and disease. Plain English SummaryThe Four Core Genotypes and XY* mouse models have been broadly useful for determining if sex differences in any mouse phenotype are caused by gonadal hormones, or by sex-chromosome complement (XX vs. XY), and if sex-chromosome effects are caused by X- or Y-linked mechanisms. Using gene knockout and transgenic methods, we have produced laboratory rat models that offer similar capabilities. The new rat models allow investigators to test with relative ease, for the first time, if a sex difference in a rat trait is caused by effects of XX vs. XY sex chromosomes, not mediated by effects of gonadal hormones, and to narrow the search for X or Y genes that have that role. The models produce XO, XX, XY, and XXY rats with ovaries, and XO, XX, XY, XXY, and XYY rats with testes. The four XX and XY groups represent a Four Core Genotypes rat model, comparison of which tests for sex-chromosome and gonadal hormonal effects that cause female and male rats to have different physiological or disease traits. Moreover, comparison of rats with different numbers of X chromosomes, or of Y chromosomes, but with the same type of gonad, provides evidence regarding the effects of X or Y dosage on rat traits. The new models will improve understanding of the impact of sex chromosomes on diseases or traits that are best modeled in rats. They will also improve understanding of the evolution of functional roles of sex chromosomes. HighlightsIt is advantageous to establish the factors that cause sex differences in diseases, because those factors mitigate or exacerbate diseases. We have produced new laboratory rats that have different types and numbers of sex chromosomes but the same type of gonad, allowing investigation of the role of sex chromosomes in causing sex differences in physiology and disease. The new rat lines allow comparison of XX and XY rats with the same type of gonad, to detect sex differences caused in part by the sex chromosomes. Other comparisons of rats with the same gonad but with different numbers of X chromosomes (XO vs. XX, XY vs XXY) or of Y chromosomes (XO vs. XY, XX vs. XXY, XY vs. XYY) detect effects of X or Y chromosome number. These resources can uncover sex-chromosome effects on any rat phenotype.

genetics↗

bioRxiv2021

A gene-by-gene mosaic of dosage compensation strategies on the human X chromosome

Researchers have presumed that the "inactive" X chromosome (Xi) has little impact, in trans, on the "active" X (Xa). To test this, we quantified Xi and Xa gene expression in individuals with one Xa and zero to three Xis. Our linear modeling revealed modular Xi and Xa transcriptomes and significant Xi-driven expression changes for 38% (162/423) of expressed X-chromosome genes. By integrating allele-specific analyses, we found that modulation of Xa transcript levels by Xi contributes to many of these Xi-driven changes ([≥] 121 genes). By incorporating metrics of evolutionary constraint, we identified 10 X-chromosome genes most likely to drive sex differences in common disease, and sex chromosome aneuploidy syndromes. We conclude that human X chromosomes are regulated both in cis, through Xi-wide transcriptional attenuation, and in trans, through positive or negative modulation of individual Xa genes by Xi. The sum of cis and trans effects differs widely among genes.

genetics↗

bioRxiv2021

GC-biased gene conversion in X-Chromosome palindromes conserved in human, chimpanzee, and rhesus macaque

Gene conversion is GC-biased across a wide range of taxa. Large palindromes on mammalian sex chromosomes undergo frequent gene conversion that maintains arm-to-arm sequence identity greater than 99%, which may increase their susceptibility to the effects of GC-biased gene conversion. Here, we demonstrate a striking history of GC-biased gene conversion in 12 palindromes conserved on the X chromosomes of human, chimpanzee, and rhesus macaque. Primate X-chromosome palindrome arms have significantly higher GC content than flanking single-copy sequences. Nucleotide replacements that occurred in human and chimpanzee palindrome arms over the past 7 million years are one-and-a-half times as GC-rich than the ancestral bases they replaced. Using simulations, we show that our observed pattern of nucleotide replacements is consistent with GC-biased gene conversion with a magnitude of 70%, similar to previously reported values based on analyses of human meioses. However, GC-biased gene conversion explains only a fraction of the observed difference in GC content between palindrome arms and flanking sequence, suggesting that additional factors are required to explain elevated GC content in palindrome arms. This work supports a greater than 2:1 preference for GC bases over AT bases during gene conversion, and demonstrates that the evolution and composition of mammalian sex chromosome palindromes is strongly influenced by GC-biased gene conversion.

genomics↗

bioRxiv2020

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 non-coding sequence, demonstrating that natural selection has preserved these gene families. Unexpectedly, the spacers that separate the left and right arms of palindromes are a site of localized structural instability, with 7 of 12 conserved palindromes showing no spacer orthology between human and rhesus macaque. Analysis of the 1000 Genomes Project dataset 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.

genomics↗

bioRxiv2020

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, an extension of our SHIMS sequencing strategy, 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 dollars per clone. We recently used this protocol to produce reference sequence of structurally complex palindromes on chimpanzee and rhesus macaque X chromosomes; as a further demonstration of the capabilities of SHIMS 3.0, we finish the TSPY array on the human Y chromosome, which could not be resolved by previous sequencing methods. 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.

genomics↗

bioRxiv2017

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

Reference sequence of structurally complex regions can only be obtained through highly accurate clone-based approaches. We and others have successfully employed Single-Haplotype Iterative Mapping and Sequencing (SHIMS 1.0) to assemble structurally complex regions across the sex chromosomes of several vertebrate species and in targeted improvements to the reference sequences of human autosomes. However, SHIMS 1.0 was expensive and time consuming, requiring the resources that only a genome center could command. Here we introduce SHIMS 2.0, an improved SHIMS protocol to allow 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 high-throughput, inexpensive, short-read sequencing technologies, a small group can sequence and assemble hundreds of clones in a week. Relative to SHIMS 1.0, SHIMS 2.0 reduces the cost and time required by two orders of magnitude, while preserving high sequencing accuracy.

genomics↗