Biology subjects

Varsally, W.

Publications and source records attributed to Varsally, W..

8 recordsLinked to original sources

bioRxiv2026

Genome editing of marsupial RSX reveals conserved and divergent principles in mammalian X-inactivation

X-chromosome inactivation balances X-gene dosage between the sexes in eutherians and marsupials. The lncRNA Xist mediates X-inactivation in eutherians but is absent from marsupials. An evolutionarily unrelated lncRNA, RSX, has been identified in marsupials, but its role in X-inactivation is unresolved because genome editing in these mammals has only recently become feasible. Using CRISPR-Cas9 in the opossum, we show that RSX is required for the initiation of X-inactivation in marsupial embryos. RSX deletion later, in differentiated cells, causes modest X-gene de-repression, with repressive chromatin marks H3K27me3 and H3K9me3 retained. H2AK119ub is not enriched on the opossum inactive X, revealing divergent Polycomb-mediated regulation in therians. Our findings reveal stage-specific roles of RSX in marsupial X-inactivation and identify conserved and divergent features of X-dosage compensation in mammals.

genomics↗

bioRxiv2025

A human induced pluripotent stem cell toolbox for studying sex chromosome effects

Sex chromosomes shape male (XY) - female (XX) differences in development and disease. These differences can be modelled in vitro by comparing XY and XX human induced pluripotent stem cells (hiPSCs). However, in this system, inter-individual autosomal variation and unstable X-dosage compensation can confound identification of sex chromosomal effects. Here, we utilise sex chromosome loss in XXY fibroblasts to generate XX and XY hiPSCs that are autosomally isogenic and exhibit stable X-dosage compensation. We also create X-monosomic (XO) hiPSCs, to investigate X-Y dosage effects. Using these autosomally isogenic lines, we examine sex differences in pluripotent stem cell expression. Transcriptional differences between XX and XY hiPSCs are surprisingly modest. However, X-haploinsufficiency induces transcriptional deregulation predominantly affecting autosomes. This effect is mediated by Y-genes with broad housekeeping functions that have X-homologues escaping X-inactivation. Our isogenic hiPSC lines provide a resource for exploring sex chromosome effects on development and disease in vitro.

genetics↗

bioRxiv2024

Sex chromosomes shape the transcriptional landscape of the preimplantation mouseembryo

Sex chromosomes are emerging as key regulators of adult health and disease in males (XY) and females (XX), but their impact on embryo development is poorly understood. Using single-cell RNA sequencing (scRNA-seq) on wild type and aneuploid mouse embryos, we show that sex chromosomes significantly shape the preimplantation embryo transcriptional landscape. A hierarchy of effects are identified, distinctly mediated by the Y chromosome, the dosage of X chromosomes, X-chromosome imprinting, and by Xist, the non-coding RNA that initiates X-inactivation. The sex chromosomes have strong trans effects on autosomal gene expression throughout preimplantation development. The Y chromosome has an unexpectedly pronounced impact on the trophectoderm, the precursor of the placenta, and this property is shared with genes expressed from the inactive X chromosome. The paternal and maternal X chromosomes differentially promote preimplantation growth, and we identify multiple novel candidate X-linked imprinted genes mediating this effect. Our findings show that sex chromosomes impact the embryo from the beginning of life, long before the appearance of overt sex differences.

developmental biology↗

bioRxiv2024

Systematic identification of Y-chromosome gene functions in mouse spermatogenesis

The mammalian Y chromosome is essential for male fertility, but how individual Y genes regulate spermatogenesis is poorly understood. To resolve this question, we generate a deletion series of the mouse Y chromosome, creating thirteen Y-deletant mouse models and conducting exhaustive reproductive phenotyping. Eight Y genes, including several that are deeply conserved and exhibit testis-specific expression, are dispensable for spermatogenesis. For others, we uncover novel functions, including a role for Uty in establishment and differentiation of spermatogonia, and for Zfy2 in ensuring meiotic pairing and reciprocal recombination between the sex chromosomes. We also generate the first mouse equivalent of the human infertility AZFa deletion, revealing cumulative detrimental effects of Y-gene loss on spermatogenesis. We use single nuclei RNAseq to identify candidate mechanisms by which Y genes regulate the germ cell transcriptome and reveal an unexpected impact of Y genes on testis supporting cells. Our study represents a paradigm for the complete functional dissection of a mammalian Y chromosome and advances our knowledge of human infertility and Y-chromosome evolution.

genetics↗

bioRxiv2024

Marsupial single-cell transcriptomics provides an atlas of developmental heterochrony

Single-cell transcriptomics has revealed conserved and divergent programmes of organogenesis in mammals, but existing studies have focused on eutherians. Marsupials exhibit short gestation and complete development externally, necessitating accelerated differentiation of anterior features required for locomotion and feeding. As such, they represent a unique outgroup with which to understand temporal shifts in development, known as heterochrony. Here, we generate the first single-cell transcriptomic atlas of gastrulation and early organogenesis in a marsupial, the opossum Monodelphis domestica. We find that anterior prioritisation is achieved by earlier initiation and shorter duration of transcriptional programmes relative to eutherians. The result is uncoupling of transcriptional and morphological progression, revealing unforeseen diversity in the order of developmental sequences in mammals. We uncover novel tissues for which heterochrony has not previously been documented. Our findings indicate that accelerated activation of transcriptional programmes facilitates the rapid growth needed for survival of the marsupial neonate.

developmental biology↗

bioRxiv2021

A novel enhancer that regulates Bdnf expression in developing neurons

Brain derived neurotrophic factor (BDNF) is a critical secreted peptide that promotes neuronal differentiation and survival, and its downregulation is implicated in many neurological disorders. Here, we investigated the regulation of the mouse Bdnf gene in cortical neurons and identified a novel enhancer that promotes the expression of many Bdnf transcript variants during differentiation, increasing total Bdnf mRNA levels. Enhancer activity contributes to Bdnf-mediated effects on neuronal clustering and activity-dependent dendritogenesis. During Bdnf activation, enhancer-promoter contacts increase, and the region moves away from the repressive nuclear periphery. Our findings suggest that changes in nuclear structure may contribute to the expression of essential growth factors during neuronal development.

molecular biology↗

bioRxiv2021

STAG proteins promote cohesin ring loading at R-loops.

Most studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in the localization of cohesin to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin, cluster in 3D and interact with CTCF, as well as with a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA and are localised to R-loops where they contribute to R-loop regulation. Our results place SA1 within R-loop domains upstream of the cohesin complex and reveal a role for SA1 in cohesin loading which is independent of NIPBL, the canonical cohesin loader. We propose that SA1 takes advantage of structural R-loop platforms to link cohesin loading and chromatin structure with diverse functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease.

cell biology↗

bioRxiv2021

The cohesin regulator Stag1 promotes cell plasticity through heterochromatin regulation.

Several studies have shown a role for Stag proteins in cell identity. Our understanding of how Stag proteins contribute to cell identity have largely been focused on its roles in chromosome topology as part of the cohesin complex and the impact on protein-coding gene expression. Furthermore, several Stag paralogs exist in mammalian cells with non-reciprocal chromosome structure and cohesion functions. Why cells have so many Stag proteins and what specific functions each Stag protein performs to support a given cell state are poorly understood. Here we reveal that Stag1 is the dominant paralog in mouse embryonic stem cells (mESC) and is required for pluripotency. Through the discovery of diverse, naturally occurring Stag1 isoforms in mESCs, we shed new light not only on the unique ends of Stag1 but also the critical role that their levels play in stem cell identity. Furthermore, we revel a new role for Stag1, and specifically its unique N-terminal end, in regulating nucleolar integrity and safeguarding mESCs from totipotency. Stag1 is localised to repressive perinucleolar regions, bound at repeats and interacts with Nucleolin and TRIM28. Loss of the Stag1 N-terminus, leads to decreased LINE-1 and rRNA expression and disruption of nucleolar structure and function which consequently leads to activation of the two-cell-like (2C-LC)-specific transcription factor DUX and conversion of pluripotent mESCs to totipotent 2C-LCs. Our results move beyond protein-coding gene regulation via chromatin loops into a new role for Stag1 in repeat regulation and nucleolar structure, and offer fresh perspectives on how Stag proteins contribute to cell identity and disease.

cell biology↗