Biology subjects

Bellott, D. W.

Publications and source records attributed to Bellott, D. W..

12 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

Post-transcriptional cross- and auto-regulation buffer expression of the human RNA helicases DDX3X and DDX3Y

The Y-linked gene DDX3Y and its X-linked homolog DDX3X survived the evolution of the human sex chromosomes from ordinary autosomes. DDX3X encodes a multi-functional RNA helicase, with mutations causing developmental disorders and cancers. We find that, among X-linked genes with surviving Y homologs, DDX3X is extraordinarily dosage-sensitive. Studying cells of individuals with sex chromosome aneuploidy, we observe that when the number of Y chromosomes increases, DDX3X transcript levels fall; conversely, when the number of X chromosomes increases, DDX3Y transcript levels fall. In 46,XY cells, CRISPRi knockdown of either DDX3X or DDX3Y causes transcript levels of the homologous gene to rise. In 46,XX cells, chemical inhibition of DDX3X protein activity elicits an increase in DDX3X transcript levels. Thus, perturbation of either DDX3X or DDX3Y expression is buffered - by negative cross-regulation of DDX3X and DDX3Y in 46,XY cells, and by negative auto-regulation of DDX3X in 46,XX cells. DDX3X-DDX3Y cross-regulation is mediated through mRNA destabilization - as shown by metabolic labeling of newly transcribed RNA - and buffers total levels of DDX3X and DDX3Y protein in human cells. We infer that post-transcriptional auto-regulation of the ancestral (autosomal) DDX3 gene transmuted into auto- and cross-regulation of DDX3X and DDX3Y as these sex-linked genes evolved from ordinary alleles of their autosomal precursor.

genetics↗

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↗

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↗

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↗

bioRxiv2019

Locating 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)2Mnm (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.

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↗

bioRxiv2017

Preexisting heterogeneities in gene dosage sensitivity shaped sex chromosome evolution in mammals and birds

Mammalian X and Y chromosomes evolved from an ordinary autosomal pair; genetic decay decimated the Y, which in turn necessitated X chromosome inactivation (XCI). Genes of the ancestral autosomes are often assumed to have undertaken these transitions on uniform terms, but we hypothesized that they varied in their dosage constraints. We inferred such constraints from conservation of microRNA (miRNA)-mediated repression, validated by analysis of experimental data. X-linked genes with a surviving Y homolog have the most conserved miRNA target sites, followed by genes with no Y homolog and subject to XCI, and then genes with no Y homolog but escaping XCI; this heterogeneity existed on the ancestral autosomes. Similar results for avian Z-linked genes, with or without a W homolog, lead to a model of XY/ZW evolution incorporating preexisting dosage sensitivities of individual genes in determining their evolutionary fates, and ultimately shaping the mammalian and avian sex chromosomes.

genetics↗

bioRxiv2017

Preexisting heterogeneities in gene dosage sensitivity shaped sex chromosome evolution in mammals and birds

Mammalian X and Y chromosomes evolved from an ordinary autosomal pair. Genetic decay of the Y led to X chromosome inactivation (XCI) in females, but some Y-linked genes were retained during the course of sex chromosome evolution, and many X-linked genes did not become subject to XCI. We reconstructed gene-by-gene dosage sensitivities on the ancestral autosomes through phylogenetic analysis of microRNA (miRNA) target sites and compared these preexisting characteristics to the current status of Y-linked and X-linked genes in mammals. Preexisting heterogeneities in dosage sensitivity, manifesting as differences in the extent of miRNA-mediated repression, predicted either the retention of a Y homolog or the acquisition of XCI following Y gene decay. Analogous heterogeneities among avian Z-linked genes predicted either the retention of a W homolog or gene-specific dosage compensation following W gene decay. Genome-wide analyses of human copy number variation indicate that these heterogeneities consisted of sensitivity to both increases and decreases in dosage. We propose a model of XY/ZW evolution incorporating such preexisting dosage sensitivities in determining the evolutionary fates of individual genes. Our findings thus provide a more complete view of the role of dosage sensitivity in shaping the mammalian and avian sex chromosomes, and reveal an important role for post-transcriptional regulatory sequences (miRNA target sites) in sex chromosome evolution.

genetics↗