Summary Dosage compensation in Drosophila is an epigenetic phenomenon utilizing proteins and long noncoding RNAs (lncRNAs) for transcriptional upregulation of the male X chromosome. Here, by using UV crosslinking followed by deep sequencing, we show that two enzymes in the Male-Specific Lethal complex, MLE RNA helicase and MSL2 ubiquitin ligase, bind evolutionarily conserved domains containing tandem stem loops in roX1 and roX2 RNAs in vivo. These domains constitute the minimal RNA unit present in multiple copies in diverse arrangements for nucleation of the MSL complex. MLE binds to these domains with distinct ATP-independent and ATP-dependent behavior. Importantly, we show that different roX RNA domains have overlapping function, since only combinatorial mutations in the tandem stem loops result in severe loss of dosage compensation and consequently male-specific lethality. We propose that repetitive structural motifs in lncRNAs could provide plasticity during multiprotein complex assemblies to ensure efficient targeting in cis or in trans along chromosomes.

Summary Dosage compensation mechanisms provide a paradigm to study the contribution of chromosomal conformation towards targeting and spreading of epigenetic regulators over a specific chromosome. By using Hi-C and 4C analyses we show that high-affinity sites (HAS), landing platforms of the male-specific lethal (MSL) complex, are enriched around topologically associating domain (TAD) boundaries on the X chromosome and harbor more long-range contacts in a sex-independent manner. Ectopically expressed roX1 and roX2 RNA target HAS on the X chromosome in trans and, via spatial proximity, induce spreading of the MSL complex in cis, leading to increased expression of neighboring autosomal genes. We show that the MSL complex regulates nucleosome positioning at HAS, thus acting locally rather than influencing the overall chromosomal architecture. We propose that sex-independent three-dimensional conformation of the X chromosome poises it for exploitation by the MSL complex, thereby facilitating spreading in males.

During development, Drosophila larvae undergo a dramatic increase in body mass wherein nutritional and developmental cues are transduced into growth through the activity of complex signaling pathways. Class I phosphoinositide 3-kinases have an established role in this process. In this study we identify Drosophila phosphatidylinositol 5-phosphate 4-kinase (dPIP4K) as a phosphoinositide kinase that regulates growth during larval development. Loss-of-function mutants in dPIP4K show reduced body weight and prolonged larval development, whereas overexpression of dPIP4K results both in an increase in body weight and shortening of larval development. The growth defect associated with dPIP4K loss of function is accompanied by a reduction in the average cell size of larval endoreplicative tissues. Our findings reveal that these phenotypes are underpinned by changes in the signaling input into the target of rapamycin (TOR) signaling complex and changes in the activity of its direct downstream target p70 S6 kinase. Together, these results define dPIP4K activity as a regulator of cell growth and TOR signaling during larval development.

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.

Endocytic turnover is essential for the regulation of the protein composition and function of the plasma membrane, and thus affects the plasma membrane levels of many receptors. In Drosophila melanogaster photoreceptors, photon absorption by the G-protein-coupled receptor (GPCR) rhodopsin 1 (Rh1; also known as NinaE) triggers its endocytosis through clathrin-mediated endocytosis (CME). We find that CME of Rh1 is regulated by phosphatidylinositol 5 phosphate 4-kinase (PIP4K). Flies lacking PIP4K show mislocalization of Rh1 on expanded endomembranes within the cell body. This mislocalization of Rh1 was dependent on the formation of an expanded Rab5-positive compartment. The Rh1-trafficking defect in PIP4K-depleted cells could be suppressed by downregulating Rab5 function or by selectively reconstituting PIP4K in the PI3P-enriched early endosomal compartment of photoreceptors. We also found that loss of PIP4K was associated with increased CME and an enlarged Rab5-positive compartment in cultured Drosophila cells. Collectively, our findings define PIP4K as a novel regulator of early endosomal homeostasis during CME.