Mapping the functional impact of non-coding regulatory elements in primary T cells through single-cell CRISPR screens.

BACKGROUND: Drug targets with genetic evidence are expected to increase clinical success by at least twofold. Yet, translating disease-associated genetic variants into functional knowledge remains a fundamental challenge of drug discovery. A key issue is that the vast majority of complex disease associations cannot be cleanly mapped to a gene. Immune disease-associated variants are enriched within regulatory elements found in T-cell-specific open chromatin regions. RESULTS: To identify genes and molecular programs modulated by these regulatory elements, we develop a CRISPRi-based single-cell functional screening approach in primary human T cells. Our pipeline enables the interrogation of transcriptomic changes induced by the perturbation of regulatory elements at scale. We first optimize an efficient CRISPRi protocol in primary CD4+ T cells via CROPseq vectors. Subsequently, we perform a screen targeting 45 non-coding regulatory elements and 35 transcription start sites and profile approximately 250,000 T -cell single-cell transcriptomes. We develop a bespoke analytical pipeline for element-to-gene (E2G) mapping and demonstrate that our method can identify both previously annotated and novel E2G links. Lastly, we integrate genetic association data for immune-related traits and demonstrate how our platform can aid in the identification of effector genes for GWAS loci. CONCLUSIONS: We describe "primary T cell crisprQTL" - a scalable, single-cell functional genomics approach for mapping regulatory elements to genes in primary human T cells. We show how this framework can facilitate the interrogation of immune disease GWAS hits and propose that the combination of experimental and QTL-based techniques is likely to address the variant-to-function problem.

A transcriptional and regulatory map of mouse somite maturation.

The mammalian body plan is shaped by rhythmic segmentation of mesoderm into somites, which are transient embryonic structures that form down each side of the neural tube. We have analyzed the genome-wide transcriptional and chromatin dynamics occurring within nascent somites, from early inception of somitogenesis to the latest stages of body plan establishment. We created matched gene expression and open chromatin maps for the three leading pairs of somites at six time points during mouse embryonic development. We show that the rate of somite differentiation accelerates as development progresses. We identified a conserved maturation program followed by all somites, but somites from more developed embryos concomitantly switch on differentiation programs from derivative cell lineages soon after segmentation. Integrated analysis of the somitic transcriptional and chromatin activities identified opposing regulatory modules controlling the onset of differentiation. Our results provide a powerful, high-resolution view of the molecular genetics underlying somitic development in mammals.

Characterization of a common progenitor pool of the epicardium and myocardium.

The mammalian heart is derived from multiple cell lineages; however, our understanding of when and how the diverse cardiac cell types arise is limited. We mapped the origin of the embryonic mouse heart at single-cell resolution using a combination of transcriptomic, imaging, and genetic lineage labeling approaches. This mapping provided a transcriptional and anatomic definition of cardiac progenitor types. Furthermore, it revealed a cardiac progenitor pool that is anatomically and transcriptionally distinct from currently known cardiac progenitors. Besides contributing to cardiomyocytes, these cells also represent the earliest progenitor of the epicardium, a source of trophic factors and cells during cardiac development and injury. This study provides detailed insights into the formation of early cardiac cell types, with particular relevance to the development of cell-based cardiac regenerative therapies.

Trappc9 deficiency causes parent-of-origin dependent microcephaly and obesity.

Some imprinted genes exhibit parental origin specific expression bias rather than being transcribed exclusively from one copy. The physiological relevance of this remains poorly understood. In an analysis of brain-specific allele-biased expression, we identified that Trappc9, a cellular trafficking factor, was expressed predominantly (~70%) from the maternally inherited allele. Loss-of-function mutations in human TRAPPC9 cause a rare neurodevelopmental syndrome characterized by microcephaly and obesity. By studying Trappc9 null mice we discovered that homozygous mutant mice showed a reduction in brain size, exploratory activity and social memory, as well as a marked increase in body weight. A role for Trappc9 in energy balance was further supported by increased ad libitum food intake in a child with TRAPPC9 deficiency. Strikingly, heterozygous mice lacking the maternal allele (70% reduced expression) had pathology similar to homozygous mutants, whereas mice lacking the paternal allele (30% reduction) were phenotypically normal. Taken together, we conclude that Trappc9 deficient mice recapitulate key pathological features of TRAPPC9 mutations in humans and identify a role for Trappc9 and its imprinting in controlling brain development and metabolism.

Expert curation of the human and mouse olfactory receptor gene repertoires identifies conserved coding regions split across two exons.

BACKGROUND: Olfactory receptor (OR) genes are the largest multi-gene family in the mammalian genome, with 874 in human and 1483 loci in mouse (including pseudogenes). The expansion of the OR gene repertoire has occurred through numerous duplication events followed by diversification, resulting in a large number of highly similar paralogous genes. These characteristics have made the annotation of the complete OR gene repertoire a complex task. Most OR genes have been predicted in silico and are typically annotated as intronless coding sequences. RESULTS: Here we have developed an expert curation pipeline to analyse and annotate every OR gene in the human and mouse reference genomes. By combining evidence from structural features, evolutionary conservation and experimental data, we have unified the annotation of these gene families, and have systematically determined the protein-coding potential of each locus. We have defined the non-coding regions of many OR genes, enabling us to generate full-length transcript models. We found that 13 human and 41 mouse OR loci have coding sequences that are split across two exons. These split OR genes are conserved across mammals, and are expressed at the same level as protein-coding OR genes with an intronless coding region. Our findings challenge the long-standing and widespread notion that the coding region of a vertebrate OR gene is contained within a single exon. CONCLUSIONS: This work provides the most comprehensive curation effort of the human and mouse OR gene repertoires to date. The complete annotation has been integrated into the GENCODE reference gene set, for immediate availability to the research community.

Clustered CTCF binding is an evolutionary mechanism to maintain topologically associating domains.

BACKGROUND: CTCF binding contributes to the establishment of a higher-order genome structure by demarcating the boundaries of large-scale topologically associating domains (TADs). However, despite the importance and conservation of TADs, the role of CTCF binding in their evolution and stability remains elusive. RESULTS: We carry out an experimental and computational study that exploits the natural genetic variation across five closely related species to assess how CTCF binding patterns stably fixed by evolution in each species contribute to the establishment and evolutionary dynamics of TAD boundaries. We perform CTCF ChIP-seq in multiple mouse species to create genome-wide binding profiles and associate them with TAD boundaries. Our analyses reveal that CTCF binding is maintained at TAD boundaries by a balance of selective constraints and dynamic evolutionary processes. Regardless of their conservation across species, CTCF binding sites at TAD boundaries are subject to stronger sequence and functional constraints compared to other CTCF sites. TAD boundaries frequently harbor dynamically evolving clusters containing both evolutionarily old and young CTCF sites as a result of the repeated acquisition of new species-specific sites close to conserved ones. The overwhelming majority of clustered CTCF sites colocalize with cohesin and are significantly closer to gene transcription start sites than nonclustered CTCF sites, suggesting that CTCF clusters particularly contribute to cohesin stabilization and transcriptional regulation. CONCLUSIONS: Dynamic conservation of CTCF site clusters is an apparently important feature of CTCF binding evolution that is critical to the functional stability of a higher-order chromatin structure.

Multi-omics profiling of mouse gastrulation at single-cell resolution.

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes1-5. Global epigenetic reprogramming accompanies these changes6-8, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.

A transcriptomic atlas of mammalian olfactory mucosae reveals an evolutionary influence on food odor detection in humans.

The mammalian olfactory system displays species-specific adaptations to different ecological niches. To investigate the evolutionary dynamics of olfactory sensory neuron (OSN) subtypes across mammalian evolution, we applied RNA sequencing of whole olfactory mucosa samples from mouse, rat, dog, marmoset, macaque, and human. We find that OSN subtypes, representative of all known mouse chemosensory receptor gene families, are present in all analyzed species. Further, we show that OSN subtypes expressing canonical olfactory receptors are distributed across a large dynamic range and that homologous subtypes can be either highly abundant across all species or species/order specific. Highly abundant mouse and human OSN subtypes detect odorants with similar sensory profiles and sense ecologically relevant odorants, such as mouse semiochemicals or human key food odorants. Together, our results allow for a better understanding of the evolution of mammalian olfaction in mammals and provide insights into the possible functions of highly abundant OSN subtypes.

A single-cell molecular map of mouse gastrulation and early organogenesis.

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm. Furthermore, we use single-cell profiling to show that Tal1-/- chimeric embryos display defects in early mesoderm diversification, and we thus demonstrate how combining temporal and transcriptional information can illuminate gene function. Together, this comprehensive delineation of mammalian cell differentiation trajectories in vivo represents a baseline for understanding the effects of gene mutations during development, as well as a roadmap for the optimization of in vitro differentiation protocols for regenerative medicine.

Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci.

We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene structures. Also, 62 new coding loci were added to the reference genome annotation. These genomes identified a large, previously unannotated, gene (Efcab3-like) encoding 5,874 amino acids. Mutant Efcab3-like mice display anomalies in multiple brain regions, suggesting a possible role for this gene in the regulation of brain development.

CTCF maintains regulatory homeostasis of cancer pathways.

BACKGROUND: CTCF binding to DNA helps partition the mammalian genome into discrete structural and regulatory domains. Complete removal of CTCF from mammalian cells causes catastrophic genome dysregulation, likely due to widespread collapse of 3D chromatin looping and alterations to inter- and intra-TAD interactions within the nucleus. In contrast, Ctcf hemizygous mice with lifelong reduction of CTCF expression are viable, albeit with increased cancer incidence. Here, we exploit chronic Ctcf hemizygosity to reveal its homeostatic roles in maintaining genome function and integrity. RESULTS: We find that Ctcf hemizygous cells show modest but robust changes in almost a thousand sites of genomic CTCF occupancy; these are enriched for lower affinity binding events with weaker evolutionary conservation across the mouse lineage. Furthermore, we observe dysregulation of the expression of several hundred genes, which are concentrated in cancer-related pathways, and are caused by changes in transcriptional regulation. Chromatin structure is preserved but some loop interactions are destabilized; these are often found around differentially expressed genes and their enhancers. Importantly, the transcriptional alterations identified in vitro are recapitulated in mouse tumors and also in human cancers. CONCLUSIONS: This multi-dimensional genomic and epigenomic profiling of a Ctcf hemizygous mouse model system shows that chronic depletion of CTCF dysregulates steady-state gene expression by subtly altering transcriptional regulation, changes which can also be observed in primary tumors.

Defining murine organogenesis at single-cell resolution reveals a role for the leukotriene pathway in regulating blood progenitor formation.

During gastrulation, cell types from all three germ layers are specified and the basic body plan is established 1 . However, molecular analysis of this key developmental stage has been hampered by limited cell numbers and a paucity of markers. Single-cell RNA sequencing circumvents these problems, but has so far been limited to specific organ systems 2 . Here, we report single-cell transcriptomic characterization of >20,000 cells immediately following gastrulation at E8.25 of mouse development. We identify 20 major cell types, which frequently contain substructure, including three distinct signatures in early foregut cells. Pseudo-space ordering of somitic progenitor cells identifies dynamic waves of transcription and candidate regulators, which are validated by molecular characterization of spatially resolved regions of the embryo. Within the endothelial population, cells that transition from haemogenic endothelial to erythro-myeloid progenitors specifically express Alox5 and its co-factor Alox5ap, which control leukotriene production. Functional assays using mouse embryonic stem cells demonstrate that leukotrienes promote haematopoietic progenitor cell generation. Thus, this comprehensive single-cell map can be exploited to reveal previously unrecognized pathways that contribute to tissue development.

Revisiting olfactory receptors as putative drivers of cancer.

Background: Olfactory receptors (ORs) recognize odorant molecules and activate a signal transduction pathway that ultimately leads to the perception of smell. This process also modulates the apoptotic cycle of olfactory sensory neurons in an olfactory receptor-specific manner. Recent reports indicate that some olfactory receptors are expressed in tissues other than the olfactory epithelium suggesting that they may have pleiotropic roles. Methods: We investigated the expression of 301 olfactory receptor genes in a comprehensive panel of 968 cancer cell lines. Results: Forty-nine per cent of cell lines show expression of at least one olfactory receptor gene. Some receptors display a broad pattern of expression across tumour types, while others were expressed in cell lines from a particular tissue. Additionally, most of the cancer cell lines expressing olfactory receptors express the effectors necessary for OR-mediated signal transduction. Remarkably, among cancer cell lines, OR2C3 is exclusively expressed in melanoma lines. We also confirmed the expression of OR2C3 in human melanomas, but not in normal melanocytes. Conclusions: The pattern of OR2C3 expression is suggestive of a functional role in the development and/or progression of melanoma. Some olfactory receptors may contribute to tumorigenesis.

Variation in olfactory neuron repertoires is genetically controlled and environmentally modulated.

The mouse olfactory sensory neuron (OSN) repertoire is composed of 10 million cells and each expresses one olfactory receptor (OR) gene from a pool of over 1000. Thus, the nose is sub-stratified into more than a thousand OSN subtypes. Here, we employ and validate an RNA-sequencing-based method to quantify the abundance of all OSN subtypes in parallel, and investigate the genetic and environmental factors that contribute to neuronal diversity. We find that the OSN subtype distribution is stereotyped in genetically identical mice, but varies extensively between different strains. Further, we identify cis-acting genetic variation as the greatest component influencing OSN composition and demonstrate independence from OR function. However, we show that olfactory stimulation with particular odorants results in modulation of dozens of OSN subtypes in a subtle but reproducible, specific and time-dependent manner. Together, these mechanisms generate a highly individualized olfactory sensory system by promoting neuronal diversity.

BCL11A Haploinsufficiency Causes an Intellectual Disability Syndrome and Dysregulates Transcription.

Intellectual disability (ID) is a common condition with considerable genetic heterogeneity. Next-generation sequencing of large cohorts has identified an increasing number of genes implicated in ID, but their roles in neurodevelopment remain largely unexplored. Here we report an ID syndrome caused by de novo heterozygous missense, nonsense, and frameshift mutations in BCL11A, encoding a transcription factor that is a putative member of the BAF swi/snf chromatin-remodeling complex. Using a comprehensive integrated approach to ID disease modeling, involving human cellular analyses coupled to mouse behavioral, neuroanatomical, and molecular phenotyping, we provide multiple lines of functional evidence for phenotypic effects. The etiological missense variants cluster in the amino-terminal region of human BCL11A, and we demonstrate that they all disrupt its localization, dimerization, and transcriptional regulatory activity, consistent with a loss of function. We show that Bcl11a haploinsufficiency in mice causes impaired cognition, abnormal social behavior, and microcephaly in accordance with the human phenotype. Furthermore, we identify shared aberrant transcriptional profiles in the cortex and hippocampus of these mouse models. Thus, our work implicates BCL11A haploinsufficiency in neurodevelopmental disorders and defines additional targets regulated by this gene, with broad relevance for our understanding of ID and related syndromes.

Elevated Cytosolic Cl- Concentrations in Dendritic Knobs of Mouse Vomeronasal Sensory Neurons.

In rodents, the vomeronasal system controls social and sexual behavior. However, several mechanistic aspects of sensory signaling in the vomeronasal organ remain unclear. Here, we investigate the biophysical basis of a recently proposed vomeronasal signal transduction component-a Ca(2+)-activated Cl(-) current. As the physiological role of such a current is a direct function of the Cl(-) equilibrium potential, we determined the intracellular Cl(-) concentration in dendritic knobs of vomeronasal neurons. Quantitative fluorescence lifetime imaging of a Cl(-)-sensitive dye at the apical surface of the intact vomeronasal neuroepithelium revealed increased cytosolic Cl(-) levels in dendritic knobs, a substantially lower Cl(-) concentration in vomeronasal sustentacular cells, and an apparent Cl(-) gradient in vomeronasal neurons along their dendritic apicobasal axis. Together, our data provide a biophysical basis for sensory signal amplification in vomeronasal neuron microvilli by opening Ca(2+)-activated Cl(-) channels.

Detection of pup odors by non-canonical adult vomeronasal neurons expressing an odorant receptor gene is influenced by sex and parenting status.

BACKGROUND: Olfaction is a fundamental sense through which most animals perceive the external world. The olfactory system detects odors via specialized sensory organs such as the main olfactory epithelium and the vomeronasal organ. Sensory neurons in these organs use G-protein coupled receptors to detect chemosensory stimuli. The odorant receptor (OR) family is expressed in sensory neurons of the main olfactory epithelium, while the adult vomeronasal organ is thought to express other types of receptors. RESULTS: Here, we describe Olfr692, a member of the OR gene family identified by next-generation RNA sequencing, which is highly upregulated and non-canonically expressed in the vomeronasal organ. We show that neurons expressing this gene are activated by odors emanating from pups. Surprisingly, activity in Olfr692-positive cells is sexually dimorphic, being very low in females. Our results also show that juvenile odors activate a large number of Olfr692 vomeronasal neurons in virgin males, which is correlated with the display of infanticide behavior. . In contrast, activity substantially decreases in parenting males (fathers), where infanticidal aggressive behavior is not frequently observed. CONCLUSIONS: Our results describe, for the first time, a sensory neural population with a specific molecular identity involved in the detection of pup odors. Moreover, it is one of the first reports of a group of sensory neurons the activity of which is sexually dimorphic and depends on social status. Our data suggest that the Olfr692 population is involved in mediating pup-oriented behaviors in mice.

Pregnancy and estrogen enhance neural progenitor-cell proliferation in the vomeronasal sensory epithelium.

BACKGROUND: The hormonal state during the estrus cycle or pregnancy produces alterations on female olfactory perception that are accompanied by specific maternal behaviors, but it is unclear how sex hormones act on the olfactory system to enable these sensory changes. RESULTS: Herein, we show that the production of neuronal progenitors is stimulated in the vomeronasal organ (VNO) epithelium of female mice during a late phase of pregnancy. Using a wide range of molecular markers that cover the whole VNO cell maturation process in combination with Ca(2+) imaging in early postmitotic neurons, we show that newly generated VNO cells adopt morphological and functional properties of mature sensory neurons. A fraction of these newly generated cells project their axons to the olfactory forebrain, extend dendrites that contact the VNO lumen, and can detect peptides and urinary proteins shown to contain pheromone activity. High-throughput RNA-sequencing reveals concomitant differences in gene expression in the VNO transcriptomes of pregnant females. These include relative increases in expression of 20 vomeronasal receptors, of which 17 belong to the V1R subfamily, and may therefore be considered as candidate receptors for mediating maternal behaviors. We identify the expression of several hormone receptors in the VNO of which estrogen receptor α (Esr1) is directly localized to neural progenitors. Administration of sustained high levels of estrogen, but not progesterone, is sufficient to stimulate vomeronasal progenitor cell proliferation in the VNO epithelium. CONCLUSIONS: Peripheral olfactory neurogenesis driven by estrogen may contribute to modulate sensory perception and adaptive VNO-dependent behaviors during pregnancy and early motherhood.

Hierarchical deconstruction of mouse olfactory sensory neurons: from whole mucosa to single-cell RNA-seq.

The mouse olfactory mucosa is a complex chemosensory tissue composed of multiple cell types, neuronal and non-neuronal. We have here applied RNA-seq hierarchically, in three steps of decreasing cellular heterogeneity: starting with crude tissue samples dissected from the nose, proceeding to flow-cytometrically sorted pools of mature olfactory sensory neurons (OSNs), and finally arriving at single mature OSNs. We show that 98.9% of intact olfactory receptor (OR) genes are expressed in mature OSNs. We uncover a hitherto unknown bipartition among mature OSNs. We find that 19 of 21 single mature OSNs each express a single intact OR gene abundantly, consistent with the one neuron-one receptor rule. For the 9 single OSNs where the two alleles of the abundantly expressed OR gene exhibit single-nucleotide polymorphisms, we demonstrate that monoallelic expression of the abundantly expressed OR gene is extremely tight. The remaining two single mature OSNs lack OR gene expression but express Trpc2 and Gucy1b2. We establish these two cells as a neuronal cell type that is fundamentally distinct from canonical, OR-expressing OSNs and that is defined by the differential, higher expression of 55 genes. We propose this tiered experimental approach as a paradigm to unravel gene expression in other cellularly heterogeneous systems.

Cyclic Regulation of Sensory Perception by a Female Hormone Alters Behavior.

Females may display dramatically different behavior depending on their state of ovulation. This is thought to occur through sex-specific hormones acting on behavioral centers in the brain. Whether incoming sensory activity also differs across the ovulation cycle to alter behavior has not been investigated. Here, we show that female mouse vomeronasal sensory neurons (VSNs) are temporarily and specifically rendered "blind" to a subset of male-emitted pheromone ligands during diestrus yet fully detect and respond to the same ligands during estrus. VSN silencing occurs through the action of the female sex-steroid progesterone. Not all VSNs are targeted for silencing; those detecting cat ligands remain continuously active irrespective of the estrous state. We identify the signaling components that account for the capacity of progesterone to target specific subsets of male-pheromone responsive neurons for inactivation. These findings indicate that internal physiology can selectively and directly modulate sensory input to produce state-specific behavior. PAPERCLIP.