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.

Intergenerationally Maintained Histone H4 Lysine 16 Acetylation Is Instructive for Future Gene Activation.

Before zygotic genome activation (ZGA), the quiescent genome undergoes reprogramming to transition into the transcriptionally active state. However, the mechanisms underlying euchromatin establishment during early embryogenesis remain poorly understood. Here, we show that histone H4 lysine 16 acetylation (H4K16ac) is maintained from oocytes to fertilized embryos in Drosophila and mammals. H4K16ac forms large domains that control nucleosome accessibility of promoters prior to ZGA in flies. Maternal depletion of MOF acetyltransferase leading to H4K16ac loss causes aberrant RNA Pol II recruitment, compromises the 3D organization of the active genomic compartments during ZGA, and causes downregulation of post-zygotically expressed genes. Germline depletion of histone deacetylases revealed that other acetyl marks cannot compensate for H4K16ac loss in the oocyte. Moreover, zygotic re-expression of MOF was neither able to restore embryonic viability nor onset of X chromosome dosage compensation. Thus, maternal H4K16ac provides an instructive function to the offspring, priming future gene activation.

MAPCap allows high-resolution detection and differential expression analysis of transcription start sites.

The position, shape and number of transcription start sites (TSS) are critical determinants of gene regulation. Most methods developed to detect TSSs and study promoter usage are, however, of limited use in studies that demand quantification of expression changes between two or more groups. In this study, we combine high-resolution detection of transcription start sites and differential expression analysis using a simplified TSS quantification protocol, MAPCap (Multiplexed Affinity Purification of Capped RNA) along with the software icetea . Applying MAPCap on developing Drosophila melanogaster embryos and larvae, we detected stage and sex-specific promoter and enhancer activity and quantify the effect of mutants of maleless (MLE) helicase at X-chromosomal promoters. We observe that MLE mutation leads to a median 1.9 fold drop in expression of X-chromosome promoters and affects the expression of several TSSs with a sexually dimorphic expression on autosomes. Our results provide quantitative insights into promoter activity during dosage compensation.

The NSL complex-mediated nucleosome landscape is required to maintain transcription fidelity and suppression of transcription noise.

Nucleosomal organization at gene promoters is critical for transcription, with a nucleosome-depleted region (NDR) at transcription start sites (TSSs) being required for transcription initiation. How NDRs and the precise positioning of the +1 nucleosomes are maintained on active genes remains unclear. Here, we report that the Drosophila nonspecific lethal (NSL) complex is necessary to maintain this stereotypical nucleosomal organization at promoters. Upon NSL1 depletion, nucleosomes invade the NDRs at TSSs of NSL-bound genes. NSL complex member NSL3 binds to TATA-less promoters in a sequence-dependent manner. The NSL complex interacts with the NURF chromatin remodeling complex and is necessary and sufficient to recruit NURF to target promoters. Not only is the NSL complex essential for transcription, but it is required for accurate TSS selection for genes with multiple TSSs. Furthermore, loss of the NSL complex leads to an increase in transcriptional noise. Thus, the NSL complex establishes a canonical nucleosomal organization that enables transcription and determines TSS fidelity.

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.

Multiple microRNAs derived from chemically synthesized precursors regulate thrombospondin 1 expression.

Thrombospondin 1 (THBS1) is a secreted protein with a variety of biological functions, including a potent anti-angiogenic activity and activation of latent transforming growth factor beta (TGF-ß). In many human cancers it is expressed at low levels, although mutations in the THBS1 gene have been rarely reported. Instead, the loss of THBS1 expression has been proposed to be due to transcriptional and post-transcriptional deregulations. In a systematic screen of predicted microRNA (miRNA) binding sites in the THBS1 3' untranslated region (UTR) we employed chemically synthesized pre-miRNAs-a new class of pre-miRNA mimics-to show that several miRNAs (let-7a, miR-18a, miR-29b, miR-194, and miR-221) can modulate THBS1 expression at the post-transcriptional level. Sequence-specific downregulation of THBS1 by let-7a, miR-18a or by a small interfering RNA induced TGF-ß1 and SMAD4 transcript levels. Ectopic expression of latent TGF-ß1 reduced THBS1 protein expression and was associated with increased expression of let-7a, let-7-b, and miR-18a in cells. These data suggest an inverse correlation of THBS1 and latent TGF-ß1 expression levels possibly involving miRNAs.

Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy.

Autophagy is an intracellular trafficking pathway sequestering cytoplasm and delivering excess and damaged cargo to the vacuole for degradation. The Atg1/ULK1 kinase is an essential component of the core autophagy machinery possibly activated by binding to Atg13 upon starvation. Indeed, we found that Atg13 directly binds Atg1, and specific Atg13 mutations abolishing this interaction interfere with Atg1 function in vivo. Surprisingly, Atg13 binding to Atg1 is constitutive and not altered by nutrient conditions or treatment with the Target of rapamycin complex 1 (TORC1)-inhibitor rapamycin. We identify Atg8 as a novel regulator of Atg1/ULK1, which directly binds Atg1/ULK1 in a LC3-interaction region (LIR)-dependent manner. Molecular analysis revealed that Atg13 and Atg8 cooperate at different steps to regulate Atg1 function. Atg8 targets Atg1/ULK1 to autophagosomes, where it may promote autophagosome maturation and/or fusion with vacuoles/lysosomes. Moreover, Atg8 binding triggers vacuolar degradation of the Atg1-Atg13 complex in yeast, thereby coupling Atg1 activity to autophagic flux. Together, these findings define a conserved step in autophagy regulation in yeast and mammals and expand the known functions of LIR-dependent Atg8 targets to include spatial regulation of the Atg1/ULK1 kinase.