Repression and 3D-restructuring resolves regulatory conflicts in evolutionarily rearranged genomes.

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.

Unblending of Transcriptional Condensates in Human Repeat Expansion Disease.

Expansions of amino acid repeats occur in >20 inherited human disorders, and many occur in intrinsically disordered regions (IDRs) of transcription factors (TFs). Such diseases are associated with protein aggregation, but the contribution of aggregates to pathology has been controversial. Here, we report that alanine repeat expansions in the HOXD13 TF, which cause hereditary synpolydactyly in humans, alter its phase separation capacity and its capacity to co-condense with transcriptional co-activators. HOXD13 repeat expansions perturb the composition of HOXD13-containing condensates in vitro and in vivo and alter the transcriptional program in a cell-specific manner in a mouse model of synpolydactyly. Disease-associated repeat expansions in other TFs (HOXA13, RUNX2, and TBP) were similarly found to alter their phase separation. These results suggest that unblending of transcriptional condensates may underlie human pathologies. We present a molecular classification of TF IDRs, which provides a framework to dissect TF function in diseases associated with transcriptional dysregulation.

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.

The single-cell transcriptional landscape of mammalian organogenesis.

Mammalian organogenesis is a remarkable process. Within a short timeframe, the cells of the three germ layers transform into an embryo that includes most of the major internal and external organs. Here we investigate the transcriptional dynamics of mouse organogenesis at single-cell resolution. Using single-cell combinatorial indexing, we profiled the transcriptomes of around 2 million cells derived from 61 embryos staged between 9.5 and 13.5 days of gestation, in a single experiment. The resulting 'mouse organogenesis cell atlas' (MOCA) provides a global view of developmental processes during this critical window. We use Monocle 3 to identify hundreds of cell types and 56 trajectories, many of which are detected only because of the depth of cellular coverage, and collectively define thousands of corresponding marker genes. We explore the dynamics of gene expression within cell types and trajectories over time, including focused analyses of the apical ectodermal ridge, limb mesenchyme and skeletal muscle.

Formation of new chromatin domains determines pathogenicity of genomic duplications.

Chromosome conformation capture methods have identified subchromosomal structures of higher-order chromatin interactions called topologically associated domains (TADs) that are separated from each other by boundary regions. By subdividing the genome into discrete regulatory units, TADs restrict the contacts that enhancers establish with their target genes. However, the mechanisms that underlie partitioning of the genome into TADs remain poorly understood. Here we show by chromosome conformation capture (capture Hi-C and 4C-seq methods) that genomic duplications in patient cells and genetically modified mice can result in the formation of new chromatin domains (neo-TADs) and that this process determines their molecular pathology. Duplications of non-coding DNA within the mouse Sox9 TAD (intra-TAD) that cause female to male sex reversal in humans, showed increased contact of the duplicated regions within the TAD, but no change in the overall TAD structure. In contrast, overlapping duplications that extended over the next boundary into the neighbouring TAD (inter-TAD), resulted in the formation of a new chromatin domain (neo-TAD) that was isolated from the rest of the genome. As a consequence of this insulation, inter-TAD duplications had no phenotypic effect. However, incorporation of the next flanking gene, Kcnj2, in the neo-TAD resulted in ectopic contacts of Kcnj2 with the duplicated part of the Sox9 regulatory region, consecutive misexpression of Kcnj2, and a limb malformation phenotype. Our findings provide evidence that TADs are genomic regulatory units with a high degree of internal stability that can be sculptured by structural genomic variations. This process is important for the interpretation of copy number variations, as these variations are routinely detected in diagnostic tests for genetic disease and cancer. This finding also has relevance in an evolutionary setting because copy-number differences are thought to have a crucial role in the evolution of genome complexity.

Mutation in LBX1/Lbx1 precludes transcription factor cooperativity and causes congenital hypoventilation in humans and mice.

The respiratory rhythm is generated by the preBötzinger complex in the medulla oblongata, and is modulated by neurons in the retrotrapezoid nucleus (RTN), which are essential for accelerating respiration in response to high CO2 Here we identify a LBX1 frameshift (LBX1FS ) mutation in patients with congenital central hypoventilation. The mutation alters the C-terminal but not the DNA-binding domain of LBX1 Mice with the analogous mutation recapitulate the breathing deficits found in humans. Furthermore, the mutation only interferes with a small subset of Lbx1 functions, and in particular with development of RTN neurons that coexpress Lbx1 and Phox2b. Genome-wide analyses in a cell culture model show that Lbx1FS and wild-type Lbx1 proteins are mostly bound to similar sites, but that Lbx1FS is unable to cooperate with Phox2b. Thus, our analyses on Lbx1FS (dys)function reveals an unusual pathomechanism; that is, a mutation that selectively interferes with the ability of Lbx1 to cooperate with Phox2b, and thus impairs the development of a small subpopulation of neurons essential for respiratory control.

Characterization of hundreds of regulatory landscapes in developing limbs reveals two regimes of chromatin folding.

Complex regulatory landscapes control the pleiotropic transcriptional activities of developmental genes. For most genes, the number, location, and dynamics of their associated regulatory elements are unknown. In this work, we characterized the three-dimensional chromatin microarchitecture and regulatory landscape of 446 limb-associated gene loci in mouse using Capture-C, ChIP-seq, and RNA-seq in forelimb, hindlimb at three developmental stages, and midbrain. The fine mapping of chromatin interactions revealed a strong preference for functional genomic regions such as repressed or active domains. By combining chromatin marks and interaction peaks, we annotated more than 1000 putative limb enhancers and their associated genes. Moreover, the analysis of chromatin interactions revealed two regimes of chromatin folding, one producing interactions stable across tissues and stages and another one associated with tissue and/or stage-specific interactions. Whereas stable interactions associate strongly with CTCF/RAD21 binding, the intensity of variable interactions correlates with changes in underlying chromatin modifications, specifically at the viewpoint and at the interaction site. In conclusion, this comprehensive data set provides a resource for the characterization of hundreds of limb-associated regulatory landscapes and a framework to interpret the chromatin folding dynamics observed during embryogenesis.

Engaging with Children Using Augmented Reality on Clothing to Prevent Them from Smoking.

Smoking is a harmful habit, causing a range of severe consequences which could lead to premature death. This habit is still prevalent amongst young people. In order to protect children, effective early interventions supported by public instances need to be set in place. Raising awareness and educating the youth is crucial to change their mindset about the severity of smoking. Emerging technologies, such as augmented reality (AR) on mobile devices, have been shown to be useful in providing engaging experiences and educating children about a range of issues, including health and anatomy. This chapter presents a research which explores the use of AR as an exciting and engaging medium to effectively help educating children from 5 to 13 years about the effects of smoking. A mobile application, called SmokAR, was developed. This app includes AR visualization amongst other functionalities, whereby children are presented a realistic model of the human lungs of a healthy person and of a smoker. The aim of this research is to propose a transformative experience in order to put children off this dangerous habit whilst they gain knowledge about the effect of smoking on their organs. The anatomical accuracy of the 3D models and animations proposed by the app has been verified by an expert anatomist. A group of children (n = 17) also took part in usability and knowledge acquisition testing at the Glasgow Science Centre. Findings showed a significant high usability suggesting a user-friendly app design. Moreover, results also suggested that participants gained knowledge to a certain extent and felt discouraged from smoking after seeing the model of the smoker's lungs. Although there were several limitations to the study, the potential of the app to support learning and raising awareness is encouragingly positive. In addition, user testing in a more controlled environment, such as a classroom, can help gain further insights into the effectiveness and usability of the app. In the future, this simple but engaging approach to raise public awareness and support education could be used to further communicate with children about negative health effects of other harmful habits such as alcohol or drug consumption.

Genome-Wide Binding of Posterior HOXA/D Transcription Factors Reveals Subgrouping and Association with CTCF.

Homeotic genes code for key transcription factors (HOX-TFs) that pattern the animal body plan. During embryonic development, Hox genes are expressed in overlapping patterns and function in a partially redundant manner. In vitro biochemical screens probing the HOX-TF sequence specificity revealed largely overlapping sequence preferences, indicating that co-factors might modulate the biological function of HOX-TFs. However, due to their overlapping expression pattern, high protein homology, and insufficiently specific antibodies, little is known about their genome-wide binding preferences. In order to overcome this problem, we virally expressed tagged versions of limb-expressed posterior HOX genes (HOXA9-13, and HOXD9-13) in primary chicken mesenchymal limb progenitor cells (micromass). We determined the effect of each HOX-TF on cellular differentiation (chondrogenesis) and gene expression and found that groups of HOX-TFs induce distinct regulatory programs. We used ChIP-seq to determine their individual genome-wide binding profiles and identified between 12,721 and 28,572 binding sites for each of the nine HOX-TFs. Principal Component Analysis (PCA) of binding profiles revealed that the HOX-TFs are clustered in two subgroups (Group 1: HOXA/D9, HOXA/D10, HOXD12, and HOXA13 and Group 2: HOXA/D11 and HOXD13), which are characterized by differences in their sequence specificity and by the presence of cofactor motifs. Specifically, we identified CTCF binding sites in Group 1, indicating that this subgroup of HOX-proteins cooperates with CTCF. We confirmed this interaction by an independent biological assay (Proximity Ligation Assay) and demonstrated that CTCF is a novel HOX cofactor that specifically associates with Group 1 HOX-TFs, pointing towards a possible interplay between HOX-TFs and chromatin architecture.

Saturation analysis of ChIP-seq data for reproducible identification of binding peaks.

Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) is a powerful technology to identify the genome-wide locations of transcription factors and other DNA binding proteins. Computational ChIP-seq peak calling infers the location of protein-DNA interactions based on various measures of enrichment of sequence reads. In this work, we introduce an algorithm, Q, that uses an assessment of the quadratic enrichment of reads to center candidate peaks followed by statistical analysis of saturation of candidate peaks by 5' ends of reads. We show that our method not only is substantially faster than several competing methods but also demonstrates statistically significant advantages with respect to reproducibility of results and in its ability to identify peaks with reproducible binding site motifs. We show that Q has superior performance in the delineation of double RNAPII and H3K4me3 peaks surrounding transcription start sites related to a better ability to resolve individual peaks. The method is implemented in C++ and is freely available under an open source license.

Distinct global shifts in genomic binding profiles of limb malformation-associated HOXD13 mutations.

Gene regulation by transcription factors (TFs) determines developmental programs and cell identity. Consequently, mutations in TFs can lead to dramatic phenotypes in humans by disrupting gene regulation. To date, the molecular mechanisms that actually cause these phenotypes have been difficult to address experimentally. ChIP-seq, which couples chromatin immunoprecipitation with high-throughput sequencing, allows TF function to be investigated on a genome-wide scale, enabling new approaches for the investigation of gene regulation. Here, we present the application of ChIP-seq to explore the effect of missense mutations in TFs on their genome-wide binding profile. Using a retroviral expression system in chicken mesenchymal stem cells, we elucidated the mechanism underlying a novel missense mutation in HOXD13 (Q317K) associated with a complex hand and foot malformation phenotype. The mutated glutamine (Q) is conserved in most homeodomains, a notable exception being bicoid-type homeodomains that have lysine (K) at this position. Our results show that the mutation results in a shift in the binding profile of the mutant toward a bicoid/PITX1 motif. Gene expression analysis and functional assays using in vivo overexpression studies confirm that the mutation results in a partial conversion of HOXD13 into a TF with bicoid/PITX1 properties. A similar shift was not observed with another mutation, Q317R, which is associated with brachysyndactyly, suggesting that the bicoid/PITX1-shift observed for Q317K might be related to the severe clinical phenotype. The methodology described can be used to investigate a wide spectrum of TFs and mutations that have not previously been amenable to ChIP-seq experiments.

A homozygous HOXD13 missense mutation causes a severe form of synpolydactyly with metacarpal to carpal transformation.

Synpolydactyly (SPD) is a rare congenital limb disorder characterized by syndactyly between the third and fourth fingers and an additional digit in the syndactylous web. In most cases SPD is caused by heterozygous mutations in HOXD13 resulting in the expansion of a N-terminal polyalanine tract. If homozygous, the mutation results in severe shortening of all metacarpals and phalanges with a morphological transformation of metacarpals to carpals. Here, we describe a novel homozygous missense mutation in a family with unaffected consanguineous parents and severe brachydactyly and metacarpal-to-carpal transformation in the affected child. We performed whole exome sequencing on the index patient, followed by Sanger sequencing of parents and patient to investigate cosegregation. The DNA-binding ability of the mutant protein was tested with electrophoretic mobility shift assays. We demonstrate that the c.938C>G (p.313T>R) mutation in the DNA-binding domain of HOXD13 prevents binding to DNA in vitro. Our results show to our knowledge for the first time that a missense mutation in HOXD13 underlies severe brachydactyly with metacarpal-to-carpal transformation. The mutation is non-penetrant in heterozygous carriers. In conjunction with the literature we propose the possibility that the metacarpal-to-carpal transformation results from a homozygous loss of functional HOXD13 protein in humans in combination with an accumulation of non-functional HOXD13 that might be able to interact with other transcription factors in the developing limb.

Regulation of cell polarity in the cartilage growth plate and perichondrium of metacarpal elements by HOXD13 and WNT5A.

The morphology of bones is genetically determined, but the molecular mechanisms that control shape, size and the overall gestalt of bones remain unclear. We previously showed that metacarpals in the synpolydactyly homolog (spdh) mouse, which carries a mutation in Hoxd13 similar to the human condition synpolydactyly (SPD), were transformed to carpal-like bones with cuboid shape that lack cortical bone and a perichondrium and are surrounded by a joint surface. Here we provide evidence that spdh metacarpal growth plates have a defect in cell polarization with a random instead of linear orientation. In parallel prospective perichondral cells failed to adopt the characteristic flattened cell shape. We observed a similar cell polarity defect in metacarpals of Wnt5a(-/-) mice. Wnt5a and the closely related Wnt5b were downregulated in spdh handplates, and HOXD13 induced expression of both genes in vitro. Concomitant we observed mislocalization of core planar cell polarity (PCP) components DVL2 and PRICKLE1 in spdh metacarpals indicating a defect in the WNT/PCP pathway. Conversely the WNT/ß-CATENIN pathway, a hallmark of joint cells lining carpal bones, was upregulated in the perichondral region. Finally, providing spdh limb explant cultures with cells expressing either HOXD13 or WNT5A led to a non-cell autonomous partial rescue of cell polarity the perichondral region and restored the expression of perichondral markers. This study provides a so far unrecognized link between HOX proteins and cell polarity in the perichondrium and the growth plate, a failure of which leads to transformation of metacarpals to carpal-like structures.

Homeotic arm-to-leg transformation associated with genomic rearrangements at the PITX1 locus.

The study of homeotic-transformation mutants in model organisms such as Drosophila revolutionized the field of developmental biology, but how these mutants relate to human developmental defects remains to be elucidated. Here, we show that Liebenberg syndrome, an autosomal-dominant upper-limb malformation, shows features of a homeotic limb transformation in which the arms have acquired morphological characteristics of a leg. Using high-resolution array comparative genomic hybridization and paired-end whole-genome sequencing, we identified two deletions and a translocation 5' of PITX1. The structural changes are likely to remove active PITX1 forelimb suppressor and/or insulator elements and thereby move active enhancer elements in the vicinity of the PITX1 regulatory landscape. We generated transgenic mice in which PITX1 was misexpressed under the control of a nearby enhancer and were able to recapitulate the Liebenberg phenotype.

When 3D genome changes cause disease: the impact of structural variations in congenital disease and cancer.

Large structural variations (SV) are a class of mutations that have long been known to cause a wide range of genetic diseases, from rare congenital disease to cancer. Many of these SVs do not directly disrupt disease-related genes and determining causal genotype-phenotype relationships has been challenging to disentangle in the past. This has started to change with our increased understanding of the 3D genome folding. The pathophysiologies of the different types of genetic diseases influence the type of SVs observed and their genetic consequences, and how these are connected to 3D genome folding. We propose guiding principles for interpreting disease-associated SVs based on our current understanding of 3D chromatin architecture and the gene-regulatory and physiological mechanisms disrupted in disease.

The role of 3D chromatin domains in gene regulation: a multi-facetted view on genome organization.

The causal relationship between 3D chromatin domains and gene regulation has been of considerable debate in recent years. Initial Hi-C studies profiling the 3D chromatin structure of the genome described evolutionarily conserved Topologically Associating Domains (TADs) that correlated with gene expression. Subsequent evidence from mouse models and human disease directly linked TADs to gene regulation. However, a number of focused genetic and genome-wide studies questioned the relevance of 3D chromatin domains for orchestrating gene expression, ultimately yielding a more multi-layered view of 3D chromatin structure and gene regulation. We review the evidence for and against the importance of 3D chromatin structure for gene regulation and argue for a more comprehensive classification of regulatory chromatin domains that integrates 3D chromatin structure with genomic, functional, and evolutionary conservation.

Three-dimensional chromatin in disease: What holds us together and what drives us apart?

Recent advances in understanding spatial genome organization inside the nucleus have shown that chromatin is compartmentalized into megabase-scale units known as topologically associating domains (TADs). In further studies, TADs were linked to differing transcriptional activity, suggesting that they might provide a scaffold for gene regulation by promoting enhancer-promoter interaction and by insulating regulatory activities. One strong argument for this hypothesis was provided by the effects of disease-causing structural variations in congenital disease and cancer. By rearranging TADs, these mutations result in a rewiring of enhancer-promoter contacts, consecutive gene misexpression, and ultimately disease. However, not all rearrangements are equally effective in creating these effects. Here, we review several recent studies aiming to understand the mechanisms by which disease-causing mutations achieve gene misregulation. We will discuss which regulatory effects are to be expected by different disease mutations and how this new knowledge can be used for diagnostics in the clinic.

Biomechanical evaluation of an independent acromioclavicular ligament repair for acromioclavicular joint reconstruction.

BACKGROUND: A number of surgical techniques for the treatment of acromioclavicular joint separations have been described; however, few have been able to create a strong intra-operative construct that provides minimal joint translation. A biomechanical study was conducted to examine joint translation in an independent acromioclavicular ligament repair. METHODS: Three variations of a novel independent acromioclavicular ligament repair technique underwent testing using a Sawbones model. The technique involves threading sutures through two acromial bone tunnels in a suture-bridge configuration and anchoring them into the distal clavicle. Three groups of eight specimens underwent reconstruction; group 1 using FiberTape, group 2 using FiberWire and group 3 using FiberTape in a modified (under-over) suture-bridge configuration. Superior, anterior and posterior translation was tested at loads of 10, 20 and 30 N. RESULTS: Group 3 repair yielded the least translation in both anterior-posterior and superior-inferior planes, with a two-fold decrease in superior translation compared to groups 1 and 2 (P < .05). Both groups 1 and 3 using FiberTape resulted in significantly less anterior and posterior translation compared to the FiberWire group (P < .05). DISCUSSION: The independent acromioclavicular ligament repair, without repair of the coracoclavicular ligament, demonstrated significant translational stability in the anterior-posterior and superior-inferior planes.

Functional dissection of the Sox9-Kcnj2 locus identifies nonessential and instructive roles of TAD architecture.

The genome is organized in three-dimensional units called topologically associating domains (TADs), through a process dependent on the cooperative action of cohesin and the DNA-binding factor CTCF. Genomic rearrangements of TADs have been shown to cause gene misexpression and disease, but genome-wide depletion of CTCF has no drastic effects on transcription. Here, we investigate TAD function in vivo in mouse limb buds at the Sox9-Kcnj2 locus. We show that the removal of all major CTCF sites at the boundary and within the TAD resulted in a fusion of neighboring TADs, without major effects on gene expression. Gene misexpression and disease phenotypes, however, were achieved by redirecting regulatory activity through inversions and/or the repositioning of boundaries. Thus, TAD structures provide robustness and precision but are not essential for developmental gene regulation. Aberrant disease-related gene activation is not induced by a mere loss of insulation but requires CTCF-dependent redirection of enhancer-promoter contacts.