Mutagenesis Sensitivity Mapping of Human Enhancers In Vivo.

Distant-acting enhancers are central to human development. However, our limited understanding of their functional sequence features prevents the interpretation of enhancer mutations in disease. Here, we determined the functional sensitivity to mutagenesis of human developmental enhancers in vivo. Focusing on seven enhancers active in the developing brain, heart, limb and face, we created over 1700 transgenic mice for over 260 mutagenized enhancer alleles. Systematic mutation of 12-basepair blocks collectively altered each sequence feature in each enhancer at least once. We show that 69% of all blocks are required for normal in vivo activity, with mutations more commonly resulting in loss (60%) than in gain (9%) of function. Using predictive modeling, we annotated critical nucleotides at base-pair resolution. The vast majority of motifs predicted by these machine learning models (88%) coincided with changes to in vivo function, and the models showed considerable sensitivity, identifying 59% of all functional blocks. Taken together, our results reveal that human enhancers contain a high density of sequence features required for their normal in vivo function and provide a rich resource for further exploration of human enhancer logic.

Nr2f1 enhancers have distinct functions in controlling Nr2f1 expression during cortical development.

There is evidence that transcription factor (TF) encoding genes, which temporally control development in multiple cell types, can have tens of enhancers that regulate their expression. The NR2F1 TF developmentally promotes caudal and ventral cortical regional fates. Here, we epigenomically compared the activity of Nr2f1's enhancers during mouse cortical development with their activity in a transgenic assay. We identified at least six that are likely to be important in prenatal cortical development, with three harboring de novo mutants identified in ASD individuals. We chose to study the function of two of the most robust enhancers by deleting them singly or together. We found that they have distinct and overlapping functions in driving Nr2f1's regional and laminar expression in the developing cortex. Thus, these two enhancers, probably in combination with the others that we defined epigenetically, precisely tune Nr2f1's regional, cell type, and temporal expression during corticogenesis.

Conserved Cis-Acting Range Extender Element Mediates Extreme Long-Range Enhancer Activity in Mammals.

While most mammalian enhancers regulate their cognate promoters over moderate distances of tens of kilobases (kb), some enhancers act over distances in the megabase range. The sequence features enabling such extreme-distance enhancer-promoter interactions remain elusive. Here, we used in vivo enhancer replacement experiments in mice to show that short- and medium-range enhancers cannot initiate gene expression at extreme-distance range. We uncover a novel conserved cis-acting element, Range EXtender (REX), that confers extreme-distance regulatory activity and is located next to a long-range enhancer of Sall1. The REX element itself has no endogenous enhancer activity. However, addition of the REX to other short- and mid-range enhancers substantially increases their genomic interaction range. In the most extreme example observed, addition of the REX increased the range of an enhancer by an order of magnitude, from its native 71kb to 840kb. The REX element contains highly conserved [C/T]AATTA homeodomain motifs. These motifs are enriched around long-range limb enhancers genome-wide, including the ZRS, a benchmark long-range limb enhancer of Shh. Mutating the [C/T]AATTA motifs within the ZRS does not affect its limb-specific enhancer activity at short range, but selectively abolishes its long-range activity, resulting in severe limb reduction in knock-in mice. In summary, we identify a sequence signature globally associated with long-range enhancer-promoter interactions and describe a prototypical REX element that is necessary and sufficient to confer extreme-distance gene activation by remote enhancers.

Increased enhancer-promoter interactions during developmental enhancer activation in mammals.

Remote enhancers are thought to interact with their target promoters via physical proximity, yet the importance of this proximity for enhancer function remains unclear. Here we investigate the three-dimensional (3D) conformation of enhancers during mammalian development by generating high-resolution tissue-resolved contact maps for nearly a thousand enhancers with characterized in vivo activities in ten murine embryonic tissues. Sixty-one percent of developmental enhancers bypass their neighboring genes, which are often marked by promoter CpG methylation. The majority of enhancers display tissue-specific 3D conformations, and both enhancer-promoter and enhancer-enhancer interactions are moderately but consistently increased upon enhancer activation in vivo. Less than 14% of enhancer-promoter interactions form stably across tissues; however, these invariant interactions form in the absence of the enhancer and are likely mediated by adjacent CTCF binding. Our results highlight the general importance of enhancer-promoter physical proximity for developmental gene activation in mammals.

Dynamic enhancer landscapes in human craniofacial development.

The genetic basis of human facial variation and craniofacial birth defects remains poorly understood. Distant-acting transcriptional enhancers control the fine-tuned spatiotemporal expression of genes during critical stages of craniofacial development. However, a lack of accurate maps of the genomic locations and cell type-resolved activities of craniofacial enhancers prevents their systematic exploration in human genetics studies. Here, we combine histone modification, chromatin accessibility, and gene expression profiling of human craniofacial development with single-cell analyses of the developing mouse face to define the regulatory landscape of facial development at tissue- and single cell-resolution. We provide temporal activity profiles for 14,000 human developmental craniofacial enhancers. We find that 56% of human craniofacial enhancers share chromatin accessibility in the mouse and we provide cell population- and embryonic stage-resolved predictions of their in vivo activity. Taken together, our data provide an expansive resource for genetic and developmental studies of human craniofacial development.

Single-cell, whole-embryo phenotyping of mammalian developmental disorders.

Mouse models are a critical tool for studying human diseases, particularly developmental disorders1. However, conventional approaches for phenotyping may fail to detect subtle defects throughout the developing mouse2. Here we set out to establish single-cell RNA sequencing of the whole embryo as a scalable platform for the systematic phenotyping of mouse genetic models. We applied combinatorial indexing-based single-cell RNA sequencing3 to profile 101 embryos of 22 mutant and 4 wild-type genotypes at embryonic day 13.5, altogether profiling more than 1.6 million nuclei. The 22 mutants represent a range of anticipated phenotypic severities, from established multisystem disorders to deletions of individual regulatory regions4,5. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibit changes in dozens of trajectories whereas others exhibit changes in only a few cell types. We also identify differences between widely used wild-type strains, compare phenotyping of gain- versus loss-of-function mutants and characterize deletions of topological associating domain boundaries. Notably, some changes are shared among mutants, suggesting that developmental pleiotropy might be 'decomposable' through further scaling of this approach. Overall, our findings show how single-cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.

A spatio-temporally constrained gene regulatory network directed by PBX1/2 acquires limb patterning specificity via HAND2.

A lingering question in developmental biology has centered on how transcription factors with widespread distribution in vertebrate embryos can perform tissue-specific functions. Here, using the murine hindlimb as a model, we investigate the elusive mechanisms whereby PBX TALE homeoproteins, viewed primarily as HOX cofactors, attain context-specific developmental roles despite ubiquitous presence in the embryo. We first demonstrate that mesenchymal-specific loss of PBX1/2 or the transcriptional regulator HAND2 generates similar limb phenotypes. By combining tissue-specific and temporally controlled mutagenesis with multi-omics approaches, we reconstruct a gene regulatory network (GRN) at organismal-level resolution that is collaboratively directed by PBX1/2 and HAND2 interactions in subsets of posterior hindlimb mesenchymal cells. Genome-wide profiling of PBX1 binding across multiple embryonic tissues further reveals that HAND2 interacts with subsets of PBX-bound regions to regulate limb-specific GRNs. Our research elucidates fundamental principles by which promiscuous transcription factors cooperate with cofactors that display domain-restricted localization to instruct tissue-specific developmental programs.

Combinatorial transcription factor binding encodes cis-regulatory wiring of forebrain GABAergic neurogenesis.

Transcription factors (TFs) bind combinatorially to genomic cis-regulatory elements (cREs), orchestrating transcription programs. While studies of chromatin state and chromosomal interactions have revealed dynamic neurodevelopmental cRE landscapes, parallel understanding of the underlying TF binding lags. To elucidate the combinatorial TF-cRE interactions driving mouse basal ganglia development, we integrated ChIP-seq for twelve TFs, H3K4me3-associated enhancer-promoter interactions, chromatin and transcriptional state, and transgenic enhancer assays. We identified TF-cREs modules with distinct chromatin features and enhancer activity that have complementary roles driving GABAergic neurogenesis and suppressing other developmental fates. While the majority of distal cREs were bound by one or two TFs, a small proportion were extensively bound, and these enhancers also exhibited exceptional evolutionary conservation, motif density, and complex chromosomal interactions. Our results provide new insights into how modules of combinatorial TF-cRE interactions activate and repress developmental expression programs and demonstrate the value of TF binding data in modeling gene regulatory wiring.

Cell Type- and Tissue-specific Enhancers in Craniofacial Development.

The genetic basis of craniofacial birth defects and general variation in human facial shape remains poorly understood. Distant-acting transcriptional enhancers are a major category of non-coding genome function and have been shown to control the fine-tuned spatiotemporal expression of genes during critical stages of craniofacial development1-3. However, a lack of accurate maps of the genomic location and cell type-specific in vivo activities of all craniofacial enhancers prevents their systematic exploration in human genetics studies. Here, we combined histone modification and chromatin accessibility profiling from different stages of human craniofacial development with single-cell analyses of the developing mouse face to create a comprehensive catalogue of the regulatory landscape of facial development at tissue- and single cell-resolution. In total, we identified approximately 14,000 enhancers across seven developmental stages from weeks 4 through 8 of human embryonic face development. We used transgenic mouse reporter assays to determine the in vivo activity patterns of human face enhancers predicted from these data. Across 16 in vivo validated human enhancers, we observed a rich diversity of craniofacial subregions in which these enhancers are active in vivo. To annotate the cell type specificities of human-mouse conserved enhancers, we performed single-cell RNA-seq and single-nucleus ATAC-seq of mouse craniofacial tissues from embryonic days e11.5 to e15.5. By integrating these data across species, we find that the majority (56%) of human craniofacial enhancers are functionally conserved in mice, providing cell type- and embryonic stage-resolved predictions of their in vivo activity profiles. Using retrospective analysis of known craniofacial enhancers in combination with single cell-resolved transgenic reporter assays, we demonstrate the utility of these data for predicting the in vivo cell type specificity of enhancers. Taken together, our data provide an expansive resource for genetic and developmental studies of human craniofacial development.

Topologically associating domain boundaries are required for normal genome function.

Topologically associating domain (TAD) boundaries partition the genome into distinct regulatory territories. Anecdotal evidence suggests that their disruption may interfere with normal gene expression and cause disease phenotypes1-3, but the overall extent to which this occurs remains unknown. Here we demonstrate that targeted deletions of TAD boundaries cause a range of disruptions to normal in vivo genome function and organismal development. We used CRISPR genome editing in mice to individually delete eight TAD boundaries (11-80 kb in size) from the genome. All deletions examined resulted in detectable molecular or organismal phenotypes, which included altered chromatin interactions or gene expression, reduced viability, and anatomical phenotypes. We observed changes in local 3D chromatin architecture in 7 of 8 (88%) cases, including the merging of TADs and altered contact frequencies within TADs adjacent to the deleted boundary. For 5 of 8 (63%) loci examined, boundary deletions were associated with increased embryonic lethality or other developmental phenotypes. For example, a TAD boundary deletion near Smad3/Smad6 caused complete embryonic lethality, while a deletion near Tbx5/Lhx5 resulted in a severe lung malformation. Our findings demonstrate the importance of TAD boundary sequences for in vivo genome function and reinforce the critical need to carefully consider the potential pathogenicity of noncoding deletions affecting TAD boundaries in clinical genetics screening.