Biology subjects

Hughes, J. F.

Publications and source records attributed to Hughes, J. F..

6 recordsLinked to original sources

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

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↗

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↗

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↗