Pre-hypertrophic chondrogenic enhancer landscape of limb and axial skeleton development.

Chondrocyte differentiation controls skeleton development and stature. Here we provide a comprehensive map of chondrocyte-specific enhancers and show that they provide a mechanistic framework through which non-coding genetic variants can influence skeletal development and human stature. Working with fetal chondrocytes isolated from mice bearing a Col2a1 fluorescent regulatory sensor, we identify 780 genes and 2'704 putative enhancers specifically active in chondrocytes using a combination of RNA-seq, ATAC-seq and H3K27ac ChIP-seq. Most of these enhancers (74%) show pan-chondrogenic activity, with smaller populations being restricted to limb (18%) or trunk (8%) chondrocytes only. Notably, genetic variations overlapping these enhancers better explain height differences than those overlapping non-chondrogenic enhancers. Finally, targeted deletions of identified enhancers at the Fgfr3, Col2a1, Hhip and, Nkx3-2 loci confirm their role in regulating cognate genes. This enhancer map provides a framework for understanding how genes and non-coding variations influence bone development and diseases.

Inhibitory SMAD6 interferes with BMP-dependent generation of muscle progenitor cells and perturbs proximodistal pattern of murine limb muscles.

The mechanism of pattern formation during limb muscle development remains poorly understood. The canonical view holds that naïve limb muscle progenitor cells (MPCs) invade a pre-established pattern of muscle connective tissue, thereby forming individual muscles. Here, we show that early murine embryonic limb MPCs highly accumulate pSMAD1/5/9, demonstrating active signaling of bone morphogenetic proteins (BMP) in these cells. Overexpression of inhibitory human SMAD6 (huSMAD6) in limb MPCs abrogated BMP signaling, impaired their migration and proliferation, and accelerated myogenic lineage progression. Fewer primary myofibers developed, causing an aberrant proximodistal muscle pattern. Patterning was not disturbed when huSMAD6 was overexpressed in differentiated muscle, implying that the proximodistal muscle pattern depends on BMP-mediated expansion of MPCs before their differentiation. We show that limb MPCs differentially express Hox genes, and Hox-expressing MPCs displayed active BMP signaling. huSMAD6 overexpression caused loss of HOXA11 in early limb MPCs. In conclusion, our data show that BMP signaling controls expansion of embryonic limb MPCs as a prerequisite for establishing the proximodistal muscle pattern, a process that involves expression of Hox genes.

Genetic cold cases: lessons from solving complex congenital limb disorders.

Congenital genetic disorders affecting limb morphology in humans and other mammals are particularly well described, due to both their rather high frequencies of occurrence and the ease of their detection when expressed as severe forms. In most cases, their molecular and cellular etiology remained unknown long after their initial description, often for several decades, and sometimes close to a century. Over the past 20 yr, however, experimental and conceptual advances in our understanding of gene regulation, in particular over large genomic distances, have allowed these cold cases to be reopened and, eventually, for some of them to be solved. These investigations led not only to the isolation of the culprit genes and mechanisms, but also to the understanding of the often complex regulatory processes that are disturbed in such mutant genetic configurations. Here, we present several cases in which dormant regulatory mutations have been retrieved from the archives, starting from a historical perspective up to their molecular explanations. While some cases remain open, waiting for new tools and/or concepts to bring their investigations to an end, the solutions to others have contributed to our understanding of particular features often found in the regulation of developmental genes and hence can be used as benchmarks to address the impact of noncoding variants in the future.

Context-dependent enhancer function revealed by targeted inter-TAD relocation.

The expression of some genes depends on large, adjacent regions of the genome that contain multiple enhancers. These regulatory landscapes frequently align with Topologically Associating Domains (TADs), where they integrate the function of multiple similar enhancers to produce a global, TAD-specific regulation. We asked if an individual enhancer could overcome the influence of one of these landscapes, to drive gene transcription. To test this, we transferred an enhancer from its native location, into a nearby TAD with a related yet different functional specificity. We used the biphasic regulation of Hoxd genes during limb development as a paradigm. These genes are first activated in proximal limb cells by enhancers located in one TAD, which is then silenced when the neighboring TAD activates its enhancers in distal limb cells. We transferred a distal limb enhancer into the proximal limb TAD and found that its new context suppresses its normal distal specificity, even though it is bound by HOX13 transcription factors, which are responsible for the distal activity. This activity can be rescued only when a large portion of the surrounding environment is removed. These results indicate that, at least in some cases, the functioning of enhancer elements is subordinated to the host chromatin context, which can exert a dominant control over its activity.

Cell-specific alterations in Pitx1 regulatory landscape activation caused by the loss of a single enhancer.

Developmental genes are frequently controlled by multiple enhancers sharing similar specificities. As a result, deletions of such regulatory elements have often failed to reveal their full function. Here, we use the Pitx1 testbed locus to characterize in detail the regulatory and cellular identity alterations following the deletion of one of its enhancers (Pen). By combining single cell transcriptomics and an in-embryo cell tracing approach, we observe an increased fraction of Pitx1 non/low-expressing cells and a decreased fraction of Pitx1 high-expressing cells. We find that the over-representation of Pitx1 non/low-expressing cells originates from a failure of the Pitx1 locus to coordinate enhancer activities and 3D chromatin changes. This locus mis-activation induces a localized heterochrony and a concurrent loss of irregular connective tissue, eventually leading to a clubfoot phenotype. This data suggests that, in some cases, redundant enhancers may be used to locally enforce a robust activation of their host regulatory landscapes.

Non-coding deletions identify Maenli lncRNA as a limb-specific En1 regulator.

Long non-coding RNAs (lncRNAs) can be important components in gene-regulatory networks1, but the exact nature and extent of their involvement in human Mendelian disease is largely unknown. Here we show that genetic ablation of a lncRNA locus on human chromosome 2 causes a severe congenital limb malformation. We identified homozygous 27-63-kilobase deletions located 300 kilobases upstream of the engrailed-1 gene (EN1) in patients with a complex limb malformation featuring mesomelic shortening, syndactyly and ventral nails (dorsal dimelia). Re-engineering of the human deletions in mice resulted in a complete loss of En1 expression in the limb and a double dorsal-limb phenotype that recapitulates the human disease phenotype. Genome-wide transcriptome analysis in the developing mouse limb revealed a four-exon-long non-coding transcript within the deleted region, which we named Maenli. Functional dissection of the Maenli locus showed that its transcriptional activity is required for limb-specific En1 activation in cis, thereby fine-tuning the gene-regulatory networks controlling dorso-ventral polarity in the developing limb bud. Its loss results in the En1-related dorsal ventral limb phenotype, a subset of the full En1-associated phenotype. Our findings demonstrate that mutations involving lncRNA loci can result in human Mendelian disease.

Modeling Single-Molecule Conformations of the HoxD Region in Mouse Embryonic Stem and Cortical Neuronal Cells.

Complex architectural rearrangements are associated to the control of the HoxD genes in different cell types; yet, how they are implemented in single cells remains unknown. By use of polymer models, we dissect the locus 3D structure at the single DNA molecule level in mouse embryonic stem and cortical neuronal cells, as the HoxD cluster changes from a poised to a silent state. Our model describes published Hi-C, 3-way 4C, and FISH data with high accuracy and is validated against independent 4C data on the Nsi-SB 0.5-Mb duplication and on triple contacts. It reveals the mode of action of compartmentalization on the regulation of the HoxD genes that have gene- and cell-type-specific multi-way interactions with their regulatory elements and high cell-to-cell variability. It shows that TADs and higher-order 3D structures, such as metaTADs, associate with distinct combinations of epigenetic factors, including but not limited to CCCTC-binding factor (CTCF) and histone marks.

Preformed chromatin topology assists transcriptional robustness of Shh during limb development.

Long-range gene regulation involves physical proximity between enhancers and promoters to generate precise patterns of gene expression in space and time. However, in some cases, proximity coincides with gene activation, whereas, in others, preformed topologies already exist before activation. In this study, we investigate the preformed configuration underlying the regulation of the Shh gene by its unique limb enhancer, the ZRS, in vivo during mouse development. Abrogating the constitutive transcription covering the ZRS region led to a shift within the Shh-ZRS contacts and a moderate reduction in Shh transcription. Deletion of the CTCF binding sites around the ZRS resulted in the loss of the Shh-ZRS preformed interaction and a 50% decrease in Shh expression but no phenotype, suggesting an additional, CTCF-independent mechanism of promoter-enhancer communication. This residual activity, however, was diminished by combining the loss of CTCF binding with a hypomorphic ZRS allele, resulting in severe Shh loss of function and digit agenesis. Our results indicate that the preformed chromatin structure of the Shh locus is sustained by multiple components and acts to reinforce enhancer-promoter communication for robust transcription.

Chromatin topology in development and disease.

The discovery of domains of preferential interaction or Topologically Associating Domains (TADs) has provided a framework to understand the relation between enhancers and promoters within intricate regulatory landscapes. It has also enabled the conceptualization of the effect of non-coding structural variants on TADs structure and insulation and reveal new patho-mechanisms leading to disease. Here, we will review current knowledge on enhancer-promoter communication in relation to TAD structure. In particular, we will discuss how enhancer-promoter interaction dynamics is established within or outside of TADs. We will further provide an overview of how mutations affect the normal organization of the genome and how it impacts the normal ability of enhancers to induce transcription at their cognate promoters in disease. Finally, we will discuss the future directions to be explored to understand the mutual influences between 3D chromatin topology and gene regulation.

Serial genomic inversions induce tissue-specific architectural stripes, gene misexpression and congenital malformations.

Balanced chromosomal rearrangements such as inversions and translocations can cause congenital disease or cancer by inappropriately rewiring promoter-enhancer contacts1,2. To study the potentially pathogenic consequences of balanced chromosomal rearrangements, we generated a series of genomic inversions by placing an active limb enhancer cluster from the Epha4 regulatory domain at different positions within a neighbouring gene-dense region and investigated their effects on gene regulation in vivo in mice. Expression studies and high-throughput chromosome conformation capture from embryonic limb buds showed that the enhancer cluster activated several genes downstream that are located within asymmetric regions of contact, the so-called architectural stripes3. The ectopic activation of genes led to a limb phenotype that could be rescued by deleting the CCCTC-binding factor (CTCF) anchor of the stripe. Architectural stripes appear to be driven by enhancer activity, because they do not form in mouse embryonic stem cells. Furthermore, we show that architectural stripes are a frequent feature of developmental three-dimensional genome architecture often associated with active enhancers. Therefore, balanced chromosomal rearrangements can induce ectopic gene expression and the formation of asymmetric chromatin contact patterns that are dependent on CTCF anchors and enhancer activity.

Similarities and differences in the regulation of HoxD genes during chick and mouse limb development.

In all tetrapods examined thus far, the development and patterning of limbs require the activation of gene members of the HoxD cluster. In mammals, they are regulated by a complex bimodal process that controls first the proximal patterning and then the distal structure. During the shift from the former to the latter regulation, this bimodal regulatory mechanism allows the production of a domain with low Hoxd gene expression, at which both telomeric (T-DOM) and centromeric regulatory domains (C-DOM) are silent. These cells generate the future wrist and ankle articulations. We analyzed the implementation of this regulatory mechanism in chicken, i.e., in an animal for which large morphological differences exist between fore- and hindlimbs. We report that although this bimodal regulation is globally conserved between the mouse and the chick, some important modifications evolved at least between these two model systems, in particular regarding the activity of specific enhancers, the width of the TAD boundary separating the two regulations, and the comparison between the forelimb versus hindlimb regulatory controls. At least one aspect of these regulations seems to be more conserved between chick and bats than with mouse, which may relate to the extent to which forelimbs and hindlimbs of these various animals differ in their morphologies.

Dynamic 3D chromatin architecture contributes to enhancer specificity and limb morphogenesis.

The regulatory specificity of enhancers and their interaction with gene promoters is thought to be controlled by their sequence and the binding of transcription factors. By studying Pitx1, a regulator of hindlimb development, we show that dynamic changes in chromatin conformation can restrict the activity of enhancers. Inconsistent with its hindlimb-restricted expression, Pitx1 is controlled by an enhancer (Pen) that shows activity in forelimbs and hindlimbs. By Capture Hi-C and three-dimensional modeling of the locus, we demonstrate that forelimbs and hindlimbs have fundamentally different chromatin configurations, whereby Pen and Pitx1 interact in hindlimbs and are physically separated in forelimbs. Structural variants can convert the inactive into the active conformation, thereby inducing Pitx1 misexpression in forelimbs, causing partial arm-to-leg transformation in mice and humans. Thus, tissue-specific three-dimensional chromatin conformation can contribute to enhancer activity and specificity in vivo and its disturbance can result in gene misexpression and disease.

Polymer physics predicts the effects of structural variants on chromatin architecture.

Structural variants (SVs) can result in changes in gene expression due to abnormal chromatin folding and cause disease. However, the prediction of such effects remains a challenge. Here we present a polymer-physics-based approach (PRISMR) to model 3D chromatin folding and to predict enhancer-promoter contacts. PRISMR predicts higher-order chromatin structure from genome-wide chromosome conformation capture (Hi-C) data. Using the EPHA4 locus as a model, the effects of pathogenic SVs are predicted in silico and compared to Hi-C data generated from mouse limb buds and patient-derived fibroblasts. PRISMR deconvolves the folding complexity of the EPHA4 locus and identifies SV-induced ectopic contacts and alterations of 3D genome organization in homozygous or heterozygous states. We show that SVs can reconfigure topologically associating domains, thereby producing extensive rewiring of regulatory interactions and causing disease by gene misexpression. PRISMR can be used to predict interactions in silico, thereby providing a tool for analyzing the disease-causing potential of SVs.

The three-dimensional genome: regulating gene expression during pluripotency and development.

The precise expression of genes in time and space during embryogenesis is largely influenced by communication between enhancers and promoters, which is propagated and governed by the physical proximity of these elements in the nucleus. Here, we review how chromatin domains organize the genome by guiding enhancers to their target genes thereby preventing non-specific interactions with other neighboring regions. We also discuss the dynamics of chromatin interactions between enhancers and promoters, as well as the consequent changes in gene expression, that occur in pluripotent cells and during development. Finally, we evaluate how genomic changes such as deletions, inversions and duplications affect 3D chromatin configuration overall and lead to ectopic enhancer-promoter contacts, and thus gene misexpression, which can contribute to abnormal development and disease.

Integration of Shh and Fgf signaling in controlling Hox gene expression in cultured limb cells.

During embryonic development, fields of progenitor cells form complex structures through dynamic interactions with external signaling molecules. How complex signaling inputs are integrated to yield appropriate gene expression responses is poorly understood. In the early limb bud, for instance, Sonic hedgehog (Shh) is expressed in the distal posterior mesenchyme, where it acts as a mediator of anterior to posterior (AP) patterning, whereas fibroblast growth factor 8 (Fgf8) is produced by the apical ectodermal ridge (AER) at the distal tip of the limb bud to direct outgrowth along the proximal to distal (PD) axis. Here we use cultured limb mesenchyme cells to assess the response of the target Hoxd genes to these two factors. We find that they act synergistically and that both factors are required to activate Hoxd13 in limb mesenchymal cells. However, the analysis of the enhancer landscapes flanking the HoxD cluster reveals that the bimodal regulatory switch observed in vivo is only partially achieved under these in vitro conditions, suggesting an additional requirement for other factors.

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.

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.

CRISPR/Cas9 Genome Editing in Embryonic Stem Cells.

Targeted mutagenesis is required to evaluate the function of DNA segments across the genome. In recent years the CRISPR/Cas9 technology has been widely used for functional genome studies and is partially replacing classical homologous recombination methods in different aspects. CRISPR/Cas9-derived tools indeed allow the production of a wide-range of engineered mutations: from point mutations to large chromosomal rearrangements such as deletions, duplications and inversions. Here we present a protocol to engineer Embryonic Stem Cells (ESC) with desired mutations using transfection of custom-made CRISPR/Cas9 vectors. These methods allow the in vivo modeling of congenital mutations and the functional interrogation of DNA sequences.