The sex-specific factor SOA controls dosage compensation in Anopheles mosquitoes.

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.

Systematic discovery of protein interaction interfaces using AlphaFold and experimental validation.

Structural resolution of protein interactions enables mechanistic and functional studies as well as interpretation of disease variants. However, structural data is still missing for most protein interactions because we lack computational and experimental tools at scale. This is particularly true for interactions mediated by short linear motifs occurring in disordered regions of proteins. We find that AlphaFold-Multimer predicts with high sensitivity but limited specificity structures of domain-motif interactions when using small protein fragments as input. Sensitivity decreased substantially when using long protein fragments or full length proteins. We delineated a protein fragmentation strategy particularly suited for the prediction of domain-motif interfaces and applied it to interactions between human proteins associated with neurodevelopmental disorders. This enabled the prediction of highly confident and likely disease-related novel interfaces, which we further experimentally corroborated for FBXO23-STX1B, STX1B-VAMP2, ESRRG-PSMC5, PEX3-PEX19, PEX3-PEX16, and SNRPB-GIGYF1 providing novel molecular insights for diverse biological processes. Our work highlights exciting perspectives, but also reveals clear limitations and the need for future developments to maximize the power of Alphafold-Multimer for interface predictions.

The good, the bad, and the ugly: Evolutionary and pathological aspects of gene dosage alterations.

Diploid organisms contain a maternal and a paternal genome complement that is thought to provide robustness and allow developmental progression despite genetic perturbations that occur in heterozygosity. However, changes affecting gene dosage from the chromosome down to the individual gene level possess a significant pathological potential and can lead to developmental disorders (DDs). This indicates that expression from a balanced gene complement is highly relevant for proper cellular and organismal function in eukaryotes. Paradoxically, gene and whole chromosome duplications are a principal driver of evolution, while heteromorphic sex chromosomes (XY and ZW) are naturally occurring aneuploidies important for sex determination. Here, we provide an overview of the biology of gene dosage at the crossroads between evolutionary benefit and pathogenicity during disease. We describe the buffering mechanisms and cellular responses to alterations, which could provide a common ground for the understanding of DDs caused by copy number alterations.

RNA stability controlled by m6A methylation contributes to X-to-autosome dosage compensation in mammals.

In mammals, X-chromosomal genes are expressed from a single copy since males (XY) possess a single X chromosome, while females (XX) undergo X inactivation. To compensate for this reduction in dosage compared with two active copies of autosomes, it has been proposed that genes from the active X chromosome exhibit dosage compensation. However, the existence and mechanisms of X-to-autosome dosage compensation are still under debate. Here we show that X-chromosomal transcripts have fewer m6A modifications and are more stable than their autosomal counterparts. Acute depletion of m6A selectively stabilizes autosomal transcripts, resulting in perturbed dosage compensation in mouse embryonic stem cells. We propose that higher stability of X-chromosomal transcripts is directed by lower levels of m6A, indicating that mammalian dosage compensation is partly regulated by epitranscriptomic RNA modifications.

Distinct mechanisms mediate X chromosome dosage compensation in Anopheles and Drosophila.

Sex chromosomes induce potentially deleterious gene expression imbalances that are frequently corrected by dosage compensation (DC). Three distinct molecular strategies to achieve DC have been previously described in nematodes, fruit flies, and mammals. Is this a consequence of distinct genomes, functional or ecological constraints, or random initial commitment to an evolutionary trajectory? Here, we study DC in the malaria mosquito Anopheles gambiae The Anopheles and Drosophila X chromosomes evolved independently but share a high degree of homology. We find that Anopheles achieves DC by a mechanism distinct from the Drosophila MSL complex-histone H4 lysine 16 acetylation pathway. CRISPR knockout of Anopheles msl-2 leads to embryonic lethality in both sexes. Transcriptome analyses indicate that this phenotype is not a consequence of defective X chromosome DC. By immunofluorescence and ChIP, H4K16ac does not preferentially enrich on the male X. Instead, the mosquito MSL pathway regulates conserved developmental genes. We conclude that a novel mechanism confers X chromosome up-regulation in Anopheles Our findings highlight the pluralism of gene-dosage buffering mechanisms even under similar genomic and functional constraints.

Understanding 'Non-genetic' Inheritance: Insights from Molecular-Evolutionary Crosstalk.

Understanding the evolutionary and ecological roles of 'non-genetic' inheritance (NGI) is daunting due to the complexity and diversity of epigenetic mechanisms. We draw on insights from molecular and evolutionary biology perspectives to identify three general features of 'non-genetic' inheritance systems: (i) they are functionally interdependent with, rather than separate from, DNA sequence; (ii) precise mechanisms vary phylogenetically and operationally; and (iii) epigenetic elements are probabilistic, interactive regulatory factors and not deterministic 'epialleles' with defined genomic locations and effects. We discuss each of these features and offer recommendations for future empirical and theoretical research that implements a unifying inherited gene regulation (IGR) approach to studies of 'non-genetic' inheritance.