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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.

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.

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., This study was supported by grants from the Deutsche Forschungsgemeinschaft (KR3985/7-3 and MU 880/16-1 to S.M.).

The genome is organized in megabase-sized three-dimensional units, called Topologically Associated Domains (TADs), that are separated by boundaries. TADs bring distant cis-regulatory elements into proximity, a process dependent on the cooperative action of cohesin and the DNA binding factor CTCF. Surprisingly, genome-wide depletion of CTCF has little effect on transcription, yet structural variations affecting TADs have been shown to cause gene misexpression and congenital disease. Here, we investigate TAD functionin vivoin mice by systematically editing components of TAD organization at theSox9/Kcnjlocus. We find that TADs are formed by a redundant system of CTCF sites requiring the removal of all major sites within the TAD and at the boundary for two neighboring TADs to fuse. TAD fusion resulted in leakage of regulatory activity from theSox9to theKcnjTAD, but no major changes in gene expression. This indicates that TAD structures provide robustness and precision, but are not essential for developmental gene regulation. Gene misexpression and resulting disease phenotypes, however, were attained by re-directing regulatory activity through inversions and/or the re-positioning of boundaries. Thus, efficient re-wiring of enhancer promoter interaction and aberrant disease causing gene activation is not induced by a mere loss of insulation but requires the re-direction of contacts.

Rare diseases and their underlying molecular causes are often poorly studied, posing challenges for patient diagnosis and prognosis. The development of next-generation sequencing and its decreasing costs promises to alleviate such issues by supplying personal genomic information at a moderate price. Here, we used crowdfunding as an alternative funding source to sequence the genome of Lil BUB, a celebrity cat affected by rare disease phenotypes characterized by supernumerary digits, osteopetrosis and dwarfism, all phenotypic traits that also occur in human patients. We discovered that Lil BUB is affected by two distinct mutations: a heterozygous mutation in the limb enhancer of the Sonic hedgehog gene, previously associated with polydactyly in Hemingway cats; and a novel homozygous frameshift deletion affecting the TNFRSF11A (RANK) gene, which has been linked to osteopetrosis in humans. We communicated the progress of this project to a large online audience, detailing the ‘inner workings’ of personalized whole genome sequencing with the aim of improving genetic literacy. Our results highlight the importance of genomic analysis in the identification of disease-causing mutations and support crowdfunding as a means to fund low-budget projects and as a platform for scientific communication.

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.

Significance Maintaining low CO2 levels in our bodies is critical for life and depends on neurons that generate the respiratory rhythm and monitor tissue gas levels. Inadequate response to increasing levels of CO2 is common in congenital hypoventilation diseases. Here, we identified a mutation in LBX1, a homeodomain transcription factor, that causes congenital hypoventilation in humans. The mutation alters the C terminus of the protein without disturbing its DNA-binding domain. Mouse models carrying an analogous mutation recapitulate the disease. The mutation spares most Lbx1 functions, but selectively affects development of a small group of neurons central in respiration. Our work reveals a very unusual pathomechanism, a mutation that hampers a small subset of functions carried out by a transcription factor., 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.

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. Data from single-cell combinatorial-indexing RNA-sequencing analysis of 2 million cells from mouse embryos between embryonic days 9.5 and 13.5 are compiled in a cell atlas of mouse organogenesis, which provides a global view of developmental processes occurring during this critical period.

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.

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+l+ and is freely available under an open source license.

Zusammenfassung Transkriptionsfaktoren sind entscheidende Regulatoren der Embryonalentwicklung, da sie die Genexpression in jeder Zelle kontrollieren. Mutationen in Transkriptionsfaktoren liegen häufig angeborenen Entwicklungsdefekten zugrunde, jedoch ist die funktionelle Einschätzung der Pathogenität einzelner Transkriptionsfaktorvarianten anspruchsvoll, da die molekulare Funktionsweise von Transkriptionsfaktoren nicht vollkommen verstanden ist. Besonders Gain-of-Function-Mutationen führen häufig zu neuen, unerwarteten Phänotypen, deren funktionelle Charakterisierung eine Herausforderung darstellt. Die im letzten Jahrzehnt entwickelte ChIP-seq-Technologie ermöglicht es, die molekularen Mechanismen zu unterscheiden, welche Transkriptionsfaktor-assoziierten Krankheiten zugrunde liegen. Dieser Artikel fasst die molekularen Pathomechanismen diverser Transkriptionsfaktormutationen zusammen und versucht einen molekularbiologischen Rahmen für die Bewertung neuer Transkriptionsfaktormutationen zu geben.

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., Author Summary Hox genes encode transcription factors that determine the vertebrate body plan and pattern structures and organs in the developing embryo. Despite decades of effort and research on Hox genes, little is known about the HOX-DNA binding properties in vivo. This lack of knowledge is mainly due to the absence of appropriate antibodies to distinguish between different HOX transcription factors. Here, we adapt a cell culture system that allows us to investigate HOX-DNA binding on a genome-wide scale. With this approach, we define and directly compare the genome-wide binding sites of nine posterior HOXA and HOXD transcription factors. We report that the in vivo HOX binding specificity differs from the in vitro specificity and find that HOX-TFs largely rely on co-factor binding and not only on direct HOX-DNA binding. Finally, we identify a novel HOX co-factor, a genome architecture protein, CTCF suggesting a possible interplay between HOX-TF function and chromatin architecture.

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.

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 regions1, 2. By subdividing the genome into discrete regulatory units, TADs restrict the contacts that enhancers establish with their target genes3, 4, 5. 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 humans6, 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.

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.

SummaryStructural variations (SVs) contribute to the variability of our genome and are often associated with disease. Their study in model systems was hampered until now by labor-intensive genetic targeting procedures and multiple mouse crossing steps. Here we present the use of CRISPR/Cas for the fast (10 weeks) and efficient generation of SVs in mice. We specifically produced deletions, inversions, and also duplications at six different genomic loci ranging from 1.1 kb to 1.6 Mb with efficiencies up to 42%. After PCR-based selection, clones were successfully used to create mice via aggregation. To test the practicability of the method, we reproduced a human 500 kb disease-associated deletion and were able to recapitulate the human phenotype in mice. Furthermore, we evaluated the regulatory potential of a large genomic interval by deleting a 1.5 Mb fragment. The method presented permits rapid in vivo modeling of genomic rearrangements.

Signaling between cells in the anterior (A) and posterior (P) compartments directs Drosophila wing disc development and is dependent on expression of the homeodomain transcription factor Engrailed (En) in P cells. Downstream of en, posteriorly expressed Hedgehog (Hh) protein signals across the A/P border to establish a developmental organizer that directs pattern formation and growth throughout the wing primordium. Here we extend investigations of the processes downstream of en by using expression array analysis to compare A and P cells. A total of 102 candidate genes were identified that express differentially in the A and P compartments; four were characterized: Stubble (Sb) expression is restricted to A cells due to repression by en. CG15905, CG16884; CG10200/hase und igel (hui) are expressed in A cells downstream of Hh signaling; and RNA interference for hui, Stubble, and CG16884 revealed that each is essential to wing development.

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/β-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.

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.