RNA nucleation by MSL2 induces selective X chromosome compartmentalization.

Confinement of the X chromosome to a territory for dosage compensation is a prime example of how subnuclear compartmentalization is used to regulate transcription at the megabase scale. In Drosophila melanogaster, two sex-specific non-coding RNAs (roX1 and roX2) are transcribed from the X chromosome. They associate with the male-specific lethal (MSL) complex1, which acetylates histone H4 lysine 16 and thereby induces an approximately twofold increase in expression of male X-linked genes2,3. Current models suggest that X-over-autosome specificity is achieved by the recognition of cis-regulatory DNA high-affinity sites (HAS) by the MSL2 subunit4,5. However, HAS motifs are also found on autosomes, indicating that additional factors must stabilize the association of the MSL complex with the X chromosome. Here we show that the low-complexity C-terminal domain (CTD) of MSL2 renders its recruitment to the X chromosome sensitive to roX non-coding RNAs. roX non-coding RNAs and the MSL2 CTD form a stably condensed state, and functional analyses in Drosophila and mammalian cells show that their interactions are crucial for dosage compensation in vivo. Replacing the CTD of mammalian MSL2 with that from Drosophila and expressing roX in cis is sufficient to nucleate ectopic dosage compensation in mammalian cells. Thus, the condensing nature of roX-MSL2CTD is the primary determinant for specific compartmentalization of the X chromosome in Drosophila.

Temporal expression of MOF acetyltransferase primes transcription factor networks for erythroid fate.

Self-renewal and differentiation of hematopoietic stem cells (HSCs) are orchestrated by the combinatorial action of transcription factors and epigenetic regulators. Here, we have explored the mechanism by which histone H4 lysine 16 acetyltransferase MOF regulates erythropoiesis. Single-cell RNA sequencing and chromatin immunoprecipitation sequencing uncovered that MOF influences erythroid trajectory by dynamic recruitment to chromatin and its haploinsufficiency causes accumulation of a transient HSC population. A regulatory network consisting of MOF, RUNX1, and GFI1B is critical for erythroid fate commitment. GFI1B acts as a Mof activator which is necessary and sufficient for cell type-specific induction of Mof expression. Plasticity of Mof-depleted HSCs can be rescued by expression of a downstream effector, Gata1, or by rebalancing acetylation via a histone deacetylase inhibitor. Accurate timing and dosage of Mof expression act as a rheostat for the feedforward transcription factor network that safeguards progression along the erythroid fate.

Hi-C guided assemblies reveal conserved regulatory topologies on X and autosomes despite extensive genome shuffling.

Genome rearrangements that occur during evolution impose major challenges on regulatory mechanisms that rely on three-dimensional genome architecture. Here, we developed a scaffolding algorithm and generated chromosome-length assemblies from Hi-C data for studying genome topology in three distantly related Drosophila species. We observe extensive genome shuffling between these species with one synteny breakpoint after approximately every six genes. A/B compartments, a set of large gene-dense topologically associating domains (TADs), and spatial contacts between high-affinity sites (HAS) located on the X chromosome are maintained over 40 million years, indicating architectural conservation at various hierarchies. Evolutionary conserved genes cluster in the vicinity of HAS, while HAS locations appear evolutionarily flexible, thus uncoupling functional requirement of dosage compensation from individual positions on the linear X chromosome. Therefore, 3D architecture is preserved even in scenarios of thousands of rearrangements highlighting its relevance for essential processes such as dosage compensation of the X chromosome.

De novo mutations in MSL3 cause an X-linked syndrome marked by impaired histone H4 lysine 16 acetylation.

The etiological spectrum of ultra-rare developmental disorders remains to be fully defined. Chromatin regulatory mechanisms maintain cellular identity and function, where misregulation may lead to developmental defects. Here, we report pathogenic variations in MSL3, which encodes a member of the chromatin-associated male-specific lethal (MSL) complex responsible for bulk histone H4 lysine 16 acetylation (H4K16ac) in flies and mammals. These variants cause an X-linked syndrome affecting both sexes. Clinical features of the syndrome include global developmental delay, progressive gait disturbance, and recognizable facial dysmorphism. MSL3 mutations affect MSL complex assembly and activity, accompanied by a pronounced loss of H4K16ac levels in vivo. Patient-derived cells display global transcriptome alterations of pathways involved in morphogenesis and cell migration. Finally, we use histone deacetylase inhibitors to rebalance acetylation levels, alleviating some of the molecular and cellular phenotypes of patient cells. Taken together, we characterize a syndrome that allowed us to decipher the developmental importance of MSL3 in humans.

Facultative dosage compensation of developmental genes on autosomes in Drosophila and mouse embryonic stem cells.

Haploinsufficiency and aneuploidy are two phenomena, where gene dosage alterations cause severe defects ultimately resulting in developmental failures and disease. One remarkable exception is the X chromosome, where copy number differences between sexes are buffered by dosage compensation systems. In Drosophila, the Male-Specific Lethal complex (MSLc) mediates upregulation of the single male X chromosome. The evolutionary origin and conservation of this process orchestrated by MSL2, the only male-specific protein within the fly MSLc, have remained unclear. Here, we report that MSL2, in addition to regulating the X chromosome, targets autosomal genes involved in patterning and morphogenesis. Precise regulation of these genes by MSL2 is required for proper development. This set of dosage-sensitive genes maintains such regulation during evolution, as MSL2 binds and similarly regulates mouse orthologues via Histone H4 lysine 16 acetylation. We propose that this gene-by-gene dosage compensation mechanism was co-opted during evolution for chromosome-wide regulation of the Drosophila male X.