Maria Felicia Basilicata

Background: Rising global temperatures are expected to increase the spread of infectious diseases as warming allows disease-carrying mosquitoes such as Anopheles to survive and reproduce in new regions. To interrogate mosquito gene function rapidly and at scale, we here establish baculovirus as a gene delivery tool in Anopheles cells. We then use this system to express and study the dominant male sex-determining factor Yob, whose molecular function is unknown. Methods: We engineered plasmids with mosquito promoters spanning different strengths, driving genes-of-interest and selection markers, and used them to generate baculoviruses. An. gambiae Ag55 cells were infected over a range of virus doses and exposure durations. Infection efficiency, cell viability, and proliferation were characterized by flow cytometry. We profiled host and viral transcriptional responses by RNA-seq; assessed Yob chromatin association and localization by CUT&Tag and microscopy; and mapped protein-level changes and interactors by proteomics and immunoprecipitation. Results: We found that baculovirus infection blocks cell cycle progression and induces pronounced transcriptome changes, while Yob expression adds an additional male-biased signature. Yob localized to the nucleus but did not directly associate with chromatin by CUT&Tag, suggesting that it does not act as a transcription factor. Yob expression increased the abundance of nuclear RNA-metabolism factors, including DDX5-like helicases and CCR4-NOT-complex subunits, and Yob-HA co-immunoprecipitated with RNA-metabolism proteins. Conclusions: Together, these results demonstrate that the baculovirus system provides a versatile platform for studying mosquito gene biology in vitro. Our results imply that Yob’s mode of action is not classical DNA binding; rather, Yob is a small nuclear factor that modulates RNA-metabolism machinery, inducing a male transcriptional state. Graphical

Gene duplication underlies evolutionary innovation, yet many paralogues remain highly similar, raising questions about their functional divergence and physiological relevance. The spliceosomal Sm core protein SNRPB and its mammalian-specific paralogue SNRPN share over 90% sequence identity, but their distinct expression patterns - SNRPB being ubiquitous and SNRPN confined to the brain - suggest specialized functions. Why mammals have two different spliceosomes has remained obscure. Here, we generated isogenic human cell lines expressing ectopically either SNRPB or SNRPN exclusively and found that SNRPN stabilizes transcripts involved in energy metabolism and mitochondrial function, leading to increased mitochondrial abundance and oxygen consumption. Despite similar spliceosomal interactomes, SNRPN more strongly associates with the PRMT5 methylosome complex and exhibits dynamic arginine methylation in its C-terminal region that is sensitive to translation inhibition and amino acid availability. The SNRPN-dependent transcriptome responds to translation inhibition by stabilizing long, intron-rich genes involved in amino acid and energy metabolism. Our findings reveal a nutrient-sensitive, methylation-dependent mechanism that differentiates the two paralogues. This suggests that SNRPN functions as a metabolic-specialized spliceosomal subunit thereby providing tissue-specific adaptation of RNA processing in mammals.

While random X-chromosome inactivation in female cells of placental mammals silences one allele of the majority of X-chromosomal genes, a considerable fraction is only incompletely and variably inactivated. Human model systems to study the dynamics of incomplete X-inactivation are limited mostly to postmortem tissue, thereby disregarding developmental trajectories. Here, we used clonal human female induced pluripotent stem cells to track allele-specific expression of X-chromosomal genes along neural differentiation. We discovered dynamic reactivation and late-silencing of gene expression from the inactive X-chromosome leading to differentiation-induced locus- and lineage-specific usage of the two X-chromosomal alleles. In brain organoids modeling Opitz BBB/G syndrome, an X-linked neurodevelopmental disorder, reactivation of alleles from the inactive X-chromosome rescued cellular phenotypes and led to intermediate manifestations in female tissue. Taken together, our data demonstrate that alleles on the inactive X-chromosome can serve as a critical reservoir dynamically used during differentiation, thereby enhancing resilience of female neural tissue.

Sex chromosomes impact chromatin organization and histone modification dynamics differently between males and females, particularly those involved in dosage compensation (DC). The evolutionary diversity, as well as the tissue- and age-dependent variations of DC mechanisms are incompletely understood. Here, we investigate the occurrence of histone H4 lysine 16 acetylation (H4K16ac), previously known for its role in sex chromosome DC in the male-heterogametic fruit fly Drosophila melanogaster and the green anole lizard Anolis carolinensis. By sampling multiple arthropods, we find the convergent evolution of H4K16ac for DC in a female-heterogametic (ZW) species, the crustacean Artemia franciscana. CUT&Tag analysis demonstrates that H4K16ac is confined to the non-recombining stratum of the Z chromosome in females. H4K16ac-mediated DC is established during embryogenesis. In aged individuals, we observe an overall decline in nuclear organization, disrupted H4K16ac territories and increased variability in local acetylation levels on the female Z chromosome. Our findings shed light on the evolutionary diversity of DC across species and raise the possibility of sex-specific histone acetylation contributing to male-female differences in lifespan.

Sex chromosomes impact chromatin organization and histone modification dynamics differently between males and females, particularly those involved in dosage compensation (DC). The stability of DC mechanisms may shape sex-specific phenotypes and traits such as lifespan. However, the tissue- and age-dependent variations as well as the evolutionary diversity of DC mechanisms are incompletely understood. Here, we investigate the occurrence of histone H4 lysine 16 acetylation (H4K16ac), previously known for its role in sex chromosome DC in the male-heterogametic fruit fly Drosophila melanogaster and the green anole lizard Anolis carolinensis . By sampling multiple arthropods, we find the convergent evolution of H4K16ac for DC in a female-heterogametic (ZW) species, the crustacean Artemia franciscana . CUT&Tag analysis demonstrates that H4K16ac is confined to the non-recombining stratum of the Z chromosome in females. H4K16ac-mediated DC is established during embryogenesis, sustained until adulthood, but then becomes reconfigured and variable in aged females. Interfering with acetylation alleviates male-female differences in lifespan. Our findings shed light on the evolutionary diversity of DC across species. They highlight histone acetylation as a sex-specific vulnerability linked to the chromatin landscape of sex chromosomes and its potential role in driving male-female differences.

The X and Y sex chromosomes represent one of the key cellular differences between male and female. Here, we introduce the unique, silent nature of the inactive X in female cells (and its exceptions) and summarise recent insights to the molecular processes taking place on the X chromosome. We are curious to learn how these mechanisms drive sex differences in neurodevelopmental disorders.

Sex-specific differences in lifespan and ageing are observed in various species. In humans, women generally live longer but are frailer and suffer from different age-related diseases compared to men. The hallmarks of ageing, such as genomic instability, telomere attrition or loss of proteostasis, exhibit sex-specific patterns. Sex chromosomes and sex hormones, as well as the epigenetic regulation of the inactive X chromosome, have been shown to affect lifespan and age-related diseases. Here we review the current knowledge on the biological basis of sex-biased ageing. While our review is focused on humans, we also discuss examples of model organisms such as the mouse, fruit fly or the killifish. Understanding these molecular differences is crucial as the elderly population is expected to double worldwide by 2050, making sex-specific approaches in the diagnosis, treatment, therapeutic development and prevention of age-related diseases a pressing need.

In diploid organisms, biallelic gene expression enables the production of adequate levels of mRNA^1,2. This is essential for haploinsufficient genes, which require biallelic expression for optimal function to prevent the onset of developmental disorders^1,3. Whether and how a biallelic or monoallelic state is determined in a cell-type-specific manner at individual loci remains unclear. MSL2 is known for dosage compensation of the male X chromosome in flies. Here we identify a role of MSL2 in regulating allelic expression in mammals. Allele-specific bulk and single-cell analyses in mouse neural progenitor cells revealed that, in addition to the targets showing biallelic downregulation, a class of genes transitions from biallelic to monoallelic expression after MSL2 loss. Many of these genes are haploinsufficient. In the absence of MSL2, one allele remains active, retaining active histone modifications and transcription factor binding, whereas the other allele is silenced, exhibiting loss of promoter–enhancer contacts and the acquisition of DNA methylation. Msl2-knockout mice show perinatal lethality and heterogeneous phenotypes during embryonic development, supporting a role for MSL2 in regulating gene dosage. The role of MSL2 in preserving biallelic expression of specific dosage-sensitive genes sets the stage for further investigation of other factors that are involved in allelic dosage compensation in mammalian cells, with considerable implications for human disease.

The Anopheles mosquito is one of thousands of species in which sex differences play a central part in their biology, as only females need a blood meal to produce eggs. Sex differentiation is regulated by sex chromosomes, but their presence creates a dosage imbalance between males (XY) and females (XX). Dosage compensation (DC) can re-equilibrate the expression of sex chromosomal genes. However, because DC mechanisms have only been fully characterized in a few model organisms, key questions about its evolutionary diversity and functional necessity remain unresolved¹. Here we report the discovery of a previously uncharacterized gene (sex chromosome activation (SOA)) as a master regulator of DC in the malaria mosquito Anopheles gambiae. Sex-specific alternative splicing prevents functional SOA protein expression in females. The male isoform encodes a DNA-binding protein that binds the promoters of active X chromosomal genes. Expressing male SOA is sufficient to induce DC in female cells. Male mosquitoes lacking SOA or female mosquitoes ectopically expressing the male isoform exhibit X chromosome misregulation, which is compatible with viability but causes developmental delay. Thus, our molecular analyses of a DC master regulator in a non-model organism elucidates the evolutionary steps that lead to the establishment of a chromosome-specific fine-tuning mechanism.