Dosage compensation of sex- and autosomal chromosomes

<p dir="ltr">Gene dosage imbalances that arise from imbalanced chromosome copy numbers (aneuploidy) are disruptive to organismal fitness and homeostasis. An exception to that can be found in the case of heteromorphic sex chromosome systems such as the mammalian XX/XY and avian ZZ/ZW. Both of these systems independently evolved from distinct pairs of ancient autosomal chromosomes following the gain of a sex-benefitting factor on one of the two copies and eventually, the degeneration of the sex-specific chromosome (i.e. the mammalian male-specific Y chromosome and the avian female- specific W chromosome). This degeneration therefore rendered the heterogametic mammalian XY males and avian ZW females practically monosomic for the X and Z chromosomes, respectively, creating significant dosage imbalance between the single large chromosome and the pairs of autosomes. Pioneering geneticist, Susumu Ohno, hypothesized that such dosage imbalances are resolved through the transcriptional hyperactivation of the single large sex chromosome. While Ohno's hypothesis has been shown to be true in many species, sex chromosome dosage compensating mechanisms have been revealed to be complex and often, to rely on the master regulatory functions of long non-coding RNAs (lncRNAs), such as roX1 and roX2 in Drosophila, RSX in marsupials and the newly identified MAYEX and FEREX in the green anole lizard.</p><p dir="ltr">In mammals, two X-chromosome dosage compensating mechanisms are present: X- chromosome inactivation, mediated by the lncRNA Xist, which results in the transcriptional silencing of one of the two X-chromosomes in female cells and X- chromosome upregulation, which transcriptionally upregulates the single active X chromosome in both male and female cells. However, the regulatory link between these processes and the dynamics of how they act in concert to maintain the correct X-linked dosage during development remain largely uncharacterised. On the other hand, avian sex chromosome dosage compensation does not rely on a Z-chromosome inactivation mechanism. While some genes on the female Z chromosome have been proposed to be upregulated to compensate for the dosage discrepancy, avian dosage compensating mechanisms have been suggested to be either absent or highly inefficient, begging the question of how the stoichiometric imbalance is resolved.</p><p dir="ltr">Similarly, sex chromosome dosage compensation in the vast majority of non-model animals and alternative sex chromosome systems remains elusive. Finally, whether any similar dosage compensating mechanisms may act to correct for autosomal chromosome aneuploidies to any degree in karyotypic disorders such as cancer, remains largely uncharacterised.</p><p dir="ltr">In <b>Paper I</b>, we investigate the dynamics of X-chromosome upregulation in vivo during mouse pre-and peri-implantation embryonic development and in vitro during mouse embryonic stem cell priming. Using allele-resolved single-cell RNA-sequencing data and multi-modal joint single-cell RNA and ATAC-seq data, we dissect the separate effects of X-chromosome inactivation and X-chromosome upregulation on RNA levels during mouse development. We find that X-chromosome upregulation is an elastic, dosage- compensating mechanism tuning expression dosage in a sex- and lineage-specific manner in concert with the degree of X-chromosome inactivation. Furthermore, we report that while male blastocyst cells achieve X-chromosome upregulation upon zygotic genome activation, female cells experience two waves of X-upregulation, one during imprinted X-inactivation and one during random X-inactivation, with ablation of Xist impeding X-upregulation. In contrast to the conventional and widely believed model of X- upregulation proposing that X-inactivation acts to silence one of the already hyperactive X chromosomes, we challenge this model, finding that naïve female cells lack X- upregulation, which only initiates following the initiation of X-inactivation. These results demonstrate that X-upregulation is an elastic process, dynamically controlling X- chromosome dosage during development.</p><p dir="ltr">In <b>Paper II</b>, we characterise the presence, degree and mechanistic aspects of avian Z- chromosome upregulation. While the avian ZZ/ZW sex chromosome system has been widely considered to lack efficient dosage-compensating mechanisms, we find that the female Z chromosome is dosage compensated at multiple regulatory layers. Using an array of allele-resolved multi-omics approaches, we report that the single female Z chromosome is transcriptionally hyperactivated through increased transcriptional burst frequency mechanistically resembling mammalian X-chromosome upregulation. Furthermore, ribosomal profiling and mass spectrometry-based proteomics revealed that this upregulation is additionally enhanced via elevated translational efficiency of Z- linked transcripts, achieving significant, yet incomplete rebalancing between the heterogametic ZW female and the homogametic ZZ males.</p><p dir="ltr">In <b>Paper III</b>, we explore the potential dosage compensating mechanisms employed by the newly identified cephalopod ZZ/Z0 sex chromosome system. Using RNA-sequencing data from two Octopus species, Octopus vulgaris and Octopus sinensis, we find evidence for partial sex chromosome dosage compensation between the sexes and identify a novel a male-specific Z-linked long non-coding RNA we termed "Zmast", conserved in both species. Furthermore, using high-sensitivity, bulk RNA-seq data in O. vulgaris paralarvae, we determine that partial Z-chromosome dosage compensation may be achieved by transcriptional upregulation of the single female Z chromosome and identify a second, female-biased long non-coding RNA, we termed "Zfest", conserved in both Octopus species, and displaying sex-specific splicing patterns. These findings suggest that long non-coding RNAs may be implicated in the regulation of the ancient cephalopod sex chromosome dosage compensation, mirroring some of the mechanisms found in mammals and other non-mammalian species.</p><p dir="ltr">In <b>Paper IV,</b> we explore if, and how, autosomal chromosome aneuploidies might be dosage compensated. Using allele-resolved, high-sensitivity, single-cell RNA-sequencing in monoclonal fibroblast lines with different degrees of aneuploidy, we determine that autosomal chromosome loss is transcriptionally compensated through burst-frequency- mediated transcriptional upregulation of the remaining intact allele. We find that this effect operates in a region-specific manner in both complete and segmental (partial) aneuploidies, highlighting the previously unappreciated flexibility of this mechanism. Proteomics measurements further revealed an additional layer of dosage compensation, resulting in considerable stoichiometric rebalancing across autosomal aneuploid chromosomes. These findings highlight that dosage compensation is not a sex- chromosome-specific feature, but a fundamental, genome-wide molecular mechanism in response to gene dosage imbalance.</p><h3>List of scientific papers</h3><p dir="ltr">I. Antonio Lentini, Huaitao Cheng, J.C. Noble, <b>Natali Papanicolaou</b>, Christos Coucoravas, Nathanael Andrews, Qiaolin Deng, Martin Enge & Björn Reinius#. Elastic dosage compensation by X-chromosome upregulation. Nat Commun 13, 1854 (2022). <a href="https://doi.org/10.1038/s41467-022-29414-1" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41467-022-29414-1</a></p><p dir="ltr">II. <b>Natali Papanicolaou</b>*, Antonio Lentini*, Sebastian Wettersten, Michael Hagemann- Jensen, Annika Kruger, Jilin Zhang, Christos Coucoravas, Ioannis Petrosian, Xian Xin, Ilhan Ceyhan, Joanna Rorbach, Dominic Wright & Björn Reinius#. Multi-layered dosage compensation of the avian Z chromosome by increased transcriptional burst frequency and elevated translational rates. Nat Commun 16, 9088 (2025). <a href="https://doi.org/10.1038/s41467-025-64817-w" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41467-025-64817-w</a></p><p dir="ltr">III. <b>Natali Papanicolaou</b>#, Sebastian Wettersten, Alexander Kloosterman, Eduardo Almansa, Eve Seuntjens & Björn Reinius#. Z-chromosome dosage compensation and sex-specific long non-coding RNAs in octopus. <a href="https://doi.org/10.1101/2024.12.09.627507" rel="noreferrer" target="_blank">https://doi.org/10.1101/2024.12.09.627507</a> [Manuscript Preprint]</p><p dir="ltr">IV. <b>Natali Papanicolaou</b>*, Sebastian Wettersten*, Guilherme Maia, Antonio Lentini# & Björn Reinius#. Dosage responses of aneuploid autosomal chromosomes. <a href="https://doi.org/10.1101/2025.09.18.677044" rel="noreferrer" target="_blank">https://doi.org/10.1101/2025.09.18.677044</a> [Manuscript Preprint]</p><p dir="ltr">*Equal contribution</p><p dir="ltr">#Corresponding author</p>