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Translation modulates the timing and amplification of gene expression after transcription. Brain development requires uniquely complex gene expression patterns, but large-scale measurements of translation directly in the prenatal brain are lacking. We measure the reactants, synthesis, and products of translation spanning mouse neocortex neurogenesis, and discover a transient window of dynamic regulation at mid-gestation. Timed translation upregulation of chromatin binding proteins like Satb2, which is essential for neuronal subtype differentiation, restricts protein expression in neuronal lineages despite broad transcriptional priming in progenitors. In contrast, translation downregulation of ribosomal proteins sharply decreases ribosome number, coinciding with a major shift in protein synthesis dynamics at mid-gestation. Changing levels of eIF4EBP1, a direct inhibitor of ribosomal protein translation, are concurrent with ribosome downregulation and controls Satb2 fate acquisition during neuronal differentiation. Thus, the refinement of transcriptional programs by translation is central to the molecular logic of brain development. Modeling of the developmental neocortex translatome is provided as an open-source searchable resource: https://shiny.mdc-berlin.de/cortexomics/.

Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1’s intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42’s unresponsiveness. Rather, Zfp42’s promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes., We thank the Montpellier Ressources Imagerie facility (BioCampus Montpellier, Centre National de la Recherche Scientifique [CNRS], INSERM, University of Montpellier) and for computer resources from CINECA (ISCRA grant thanks to computer resources from INFN and CINECA [ISCRA Grant HP10C8JWU7]). G.C., Q.S., and F.B. were supported by a grant from the European Research Council (Advanced Grant 3DEpi, 788972) and by the CNRS. This work was funded by EMBO and the Wellcome Trust (ALTF1554-2016 and 206475/Z/17/Z; to M.I.R.) as well as the Deutsche Forschungsgemeinschaft (KR3985/7-3 and MU 880/16-1 to S.M.).

SUMMARYCohesin loop extrusion facilitates precise gene expression by continuously driving promoters to sample all enhancers located within the same topologically-associated domain (TAD). However, many TADs contain multiple genes with divergent expression patterns, thereby indicating additional forces further refine how enhancer activities are utilised. Here, we unravel the mechanisms enabling a new gene, Rex1, to emerge with divergent expression within the ancient Fat1 TAD in placental mammals. We show that such divergent expression is not determined by a strict enhancer-promoter compatibility code, intra-TAD position or nuclear envelope-attachment. Instead, TAD-restructuring in embryonic stem cells (ESCs) separates Rex1 and Fat1 with distinct proximal enhancers that independently drive their expression. By contrast, in later embryonic tissues, DNA methylation renders the inactive Rex1 promoter profoundly unresponsive to Fat1 enhancers within the intact TAD. Combined, these features adapted an ancient regulatory landscape during evolution to support two entirely independent Rex1 and Fat1 expression programs. Thus, rather than operating only as rigid blocks of co-regulated genes, TAD-regulatory landscapes can orchestrate complex divergent expression patterns in evolution.HIGHLIGHTSNew genes can emerge in evolution without taking on the expression pattern of their surrounding pre-existing TAD.Compartmentalisation can restructure seemingly evolutionarily stable TADs to control a promoter’s access to enhancers.Lamina-associated domains neither prevent transcriptional activation nor enhancer-promoter communication.Repression rather than promoter-specificity refines when genes respond to promiscuous enhancer activities in specific tissues.

Ribosome profiling, or Ribo-seq, provides precise information about the position of actively translating ribosomes. It can be used to identify open reading frames (ORFs) that are translated in a given sample. The RiboTaper pipeline, and the ORFquant R package, leverages the periodic distribution of such ribosomes along the ORF to perform a statistically robust test for translation which is insensitive to aperiodic noise and provides a statistically robust measure of translation. In addition to accounting for complex loci with overlapping ORFs, ORFquant is also able to use Ribo-seq as a tool for distinguishing actively translated transcripts from non-translated ones, within a given gene locus.

Ribosome profiling, or Ribo-seq, provides precise information about the position of actively translating ribosomes. It can be used to identify open reading frames (ORFs) that are translated in a given sample. The RiboTaper pipeline, and the ORFquant R package, leverages the periodic distribution of such ribosomes along the ORF to perform a statistically robust test for translation which is insensitive to aperiodic noise and provides a statistically robust measure of translation. In addition to accounting for complex loci with overlapping ORFs, ORFquant is also able to use Ribo-seq as a tool for distinguishing actively translated transcripts from non-translated ones, within a given gene locus.

SUMMARYProtein synthesis must be finely tuned in the nervous system, as it represents an essential feature of neurodevelopmental gene expression, and dominant pathology in neurological disease. However, the architecture of ribosomal complexes in the developing mammalian brain has not been analyzed at high resolution. This study investigates the architecture of ribosomesex vivofrom the embryonic and perinatal mouse neocortex, revealing Ebp1 as a 60S peptide tunnel exit binding factor at near-atomic resolution by multiparticle cryo-electron microscopy. The impact of Ebp1 on the neuronal proteome was analyzed by pSILAC and BONCAT coupled mass spectrometry, implicating Ebp1 in neurite outgrowth proteostasis, within vivoembryonic Ebp1 knockdown resulting in dysregulation of neurite outgrowth. Our findings reveal Ebp1 as a central component of neocortical protein synthesis, and the 60S peptide tunnel exit as a focal point of gene expression control in the molecular specification of neuronal morphology.Graphical abstract

Brain collateral circulation is an essential compensatory mechanism in response to acute brain ischemia. To study the temporal evolution of brain macro and microcollateral recruitment and their reciprocal interactions in response to different ischemic conditions, we applied a combination of complementary techniques (T2-weighted magnetic resonance imaging [MRI], time of flight [TOF] angiography [MRA], cerebral blood flow [CBF] imaging and histology) in two different mouse models. Hypoperfusion was either induced by permanent bilateral common carotid artery stenosis (BCCAS) or 60-min transient unilateral middle cerebral artery occlusion (MCAO). In both models, collateralization is a very dynamic phenomenon with a global effect affecting both hemispheres. Patency of ipsilateral posterior communicating artery (PcomA) represents the main variable survival mechanism and the main determinant of stroke lesion volume and recovery in MCAO, whereas the promptness of external carotid artery retrograde flow recruitment together with PcomA patency, critically influence survival, brain ischemic lesion volume and retinopathy in BCCAS mice. Finally, different ischemic gradients shape microcollateral density and size.

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

Protein synthesis must be finely tuned in the developing nervous system as the final essential step of gene expression. This study investigates the architecture of ribosomes from the neocortex during neurogenesis, revealing Ebp1 as a high-occupancy 60S peptide tunnel exit (TE) factor during protein synthesis at near-atomic resolution by cryoelectron microscopy (cryo-EM). Ribosome profiling demonstrated Ebp1-60S binding is highest during start codon initiation and N-terminal peptide elongation, regulating ribosome occupancy of these codons. Membrane-targeting domains emerging from the 60S tunnel, which recruit SRP/Sec61 to the shared binding site, displace Ebp1. Ebp1 is particularly abundant in the early-born neural stem cell (NSC) lineage and regulates neuronal morphology. Ebp1 especially impacts the synthesis of membrane-targeted cell adhesion molecules (CAMs), measured by pulsed stable isotope labeling by amino acids in cell culture (pSILAC)/bioorthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry (MS). Therefore, Ebp1 is a central component of protein synthesis, and the ribosome TE is a focal point of gene expression control in the molecular specification of neuronal morphology during development.

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.