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Animal promoters initiate transcription either at precise positions (narrow promoters) or dispersed regions (broad promoters), a distinction referred to as promoter shape. Although highly conserved, the functional properties of promoters with different shapes and the genetic basis of their evolution remain unclear. Here we used natural genetic variation across a panel of 81 Drosophila lines to measure changes in transcriptional start site (TSS) usage, identifying thousands of genetic variants affecting transcript levels (strength) or the distribution of TSSs within a promoter (shape). Our results identify promoter shape as a molecular trait that can evolve independently of promoter strength. Broad promoters typically harbor shape-associated variants, with signatures of adaptive selection. Single-cell measurements demonstrate that variants modulating promoter shape often increase expression noise, whereas heteroallelic interactions with other promoter variants alleviate these effects. These results uncover new functional properties of natural promoters and suggest the minimization of expression noise as an important factor in promoter evolution. Fil: Schor, Ignacio Esteban. European Molecular Biology Laboratory Heidelberg; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Degner, Jacob F.. European Molecular Biology Laboratory; Alemania Fil: Harnett, Dermot. European Molecular Biology Laboratory; Alemania Fil: Cannavò, Enrico. European Molecular Biology Laboratory; Alemania Fil: Casale, Francesco P.. European Bioinformatics Institute; Reino Unido Fil: Shim, Heejung. Purdue University; Estados Unidos Fil: Garfield, David A.. European Molecular Biology Laboratory; Alemania Fil: Birney, Ewan. European Bioinformatics Institute; Reino Unido Fil: Stephens, Matthew. University of Chicago; Estados Unidos Fil: Stegle, Oliver. European Bioinformatics Institute; Reino Unido Fil: Furlong, Eileen E. M.. European Molecular Biology Laboratory; Alemania

Gene expression is regulated by promoters, which initiate transcription, and enhancers, which control their temporal and spatial activity. However, the discovery that mammalian enhancers also initiate transcription questions the inherent differences between enhancers and promoters. Here, we investigate the transcriptional properties of enhancers during Drosophila embryogenesis using characterized developmental enhancers. We show that while the timing of enhancer transcription is generally correlated with enhancer activity, the levels and directionality of transcription are highly varied among active enhancers. To assess how this impacts function, we developed a dual transgenic assay to simultaneously measure enhancer and promoter activities from a single element in the same embryo. Extensive transgenic analysis revealed a relationship between the direction of endogenous transcription and the ability to function as an enhancer or promoter in vivo, although enhancer RNA (eRNA) production and activity are not always strictly coupled. Some enhancers (mainly bidirectional) can act as weak promoters, producing overlapping spatio–temporal expression. Conversely, bidirectional promoters often act as strong enhancers, while unidirectional promoters generally cannot. The balance between enhancer and promoter activity is generally reflected in the levels and directionality of eRNA transcription and is likely an inherent sequence property of the elements themselves. Fil: Mikhaylichenko, Olga. European Molecular Biology Laboratory; Alemania Fil: Bondarenko, Vladyslav. European Molecular Biology Laboratory; Alemania Fil: Harnett, Dermot. European Molecular Biology Laboratory; Alemania Fil: Schor, Ignacio Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Males, Matilda. European Molecular Biology Laboratory; Alemania Fil: Viales, Rebecca R.. European Molecular Biology Laboratory; Alemania Fil: Furlong, Eileen E. M.. European Molecular Biology Laboratory; Alemania

Embryonic development is driven by tightly regulated patterns of gene expression, despite extensive genetic variation among individuals. Studies of expression quantitative trait loci (eQTL) indicate that genetic variation frequently alters gene expression in cell-culture models and differentiated tissues. However, the extent and types of genetic variation impacting embryonic gene expression, and their interactions with developmental programs, remain largely unknown. Here we assessed the effect of genetic variation on transcriptional (expression levels) and post-transcriptional (3' RNA processing) regulation across multiple stages of metazoan development, using 80 inbred Drosophila wild isolates, identifying thousands of developmental-stage-specific and shared QTL. Given the small blocks of linkage disequilibrium in Drosophila, we obtain near base-pair resolution, resolving causal mutations in developmental enhancers, validated transcription-factor-binding sites and RNA motifs. This fine-grain mapping uncovered extensive allelic interactions within enhancers that have opposite effects, thereby buffering their impact on enhancer activity. QTL affecting 3' RNA processing identify new functional motifs leading to transcript isoform diversity and changes in the lengths of 3' untranslated regions. These results highlight how developmental stage influences the effects of genetic variation and uncover multiple mechanisms that regulate and buffer expression variation during embryogenesis.

Efforts to precisely identify tumor human leukocyte antigen (HLA) bound peptides capable of mediating T cell-based tumor rejection still face important challenges. Recent studies suggest that non-canonical tumor-specific HLA peptides derived from annotated non-coding regions could elicit anti-tumor immune responses. However, sensitive and accurate mass spectrometry (MS)-based proteogenomics approaches are required to robustly identify these non-canonical peptides. We present an MS-based analytical approach that characterizes the non-canonical tumor HLA peptide repertoire, by incorporating whole exome sequencing, bulk and single-cell transcriptomics, ribosome profiling, and two MS/MS search tools in combination. This approach results in the accurate identification of hundreds of shared and tumor-specific non-canonical HLA peptides, including an immunogenic peptide derived from an open reading frame downstream of the melanoma stem cell marker gene ABCB5. These findings hold great promise for the discovery of previously unknown tumor antigens for cancer immunotherapy., Non-canonical HLA-bound peptides from presumed non-coding regions are potential targets for cancer immunotherapy, but their discovery remains challenging. Here, the authors integrate exome sequencing, transcriptomics, ribosome profiling, and immunopeptidomics to identify tumor-specific non-canonical HLA-bound peptides.