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.

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.

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.

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.

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.