Guillaume Andrey

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.

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.

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.

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. Summary: This Review summarizes the role of 3D chromatin architecture in organizing the regulatory genome and evaluates how its misfolding can lead to 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 authors present a polymer-physics-based approach (PRISMR) to model 3D chromatin folding and to predict enhancer–promoter contacts. PRISMR correctly predicts ectopic contacts induced by pathogenic SVs at the mouse Epha4 locus.

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.

CONSERVATION AND VARIATIONS OF BIMODAL HoxD GENE REGULATION DURING TETRAPOD 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 controlled by a complex bimodal regulation, which controls first the proximal patterning, then the distal structure, allowing at the same time the formation of the wrist and ankle articulations. We analyzed the implementation of this regulatory mechanism in chicken, i.e. in an animal where large morphological differences exist between fore-and hindlimbs. We report that while this bimodal regulation is globally conserved between mammals and avian, 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. Some aspects of these regulations seem to be more conserved between chick and bats than with the mouse situation, which may relate to the extent to which forelimbs and hindlimbs of these various animals differ in their functions. AUTHOR SUMMARY The morphologies of limbs largely vary either amongst tetrapod species, or even between the fore-and hindlimbs of the same animal species. In order to try and evaluate whether variations in the complex regulation of Hoxd genes during limb development may contribute to these differences, we compared their transcriptional controls during both fore-and hindlimb buds development in either the mouse, or the chicken embryos. We combined transcriptome analyses with 3D genome conformation, histone modification profiles and mouse genetics and found that the regulatory mechanism underlying Hoxd gene expression was highly conserved in all contexts, though with some clear differences. For instance, we observed a variation in the TAD boundary interval between the mouse and the chick, as well as differences in the activity of a conserved enhancer element (CS93) situated within the T-DOM regulatory landscape. In contrast to the mouse, the chicken enhancer indeed displayed a stronger activity in fore-than in hindlimb buds, coinciding with the observed striking differences in the mRNA levels. Altogether, differences in both the timing and duration of TAD activities and in the width of their boundary may parallel the important decrease in Hoxd gene transcription in chick hindlimb versus forelimb buds. These differences may also account for the slightly distinct regulatory strategies implemented by mammals and birds at this locus, potentially leading to substantial morphological variations.

Large genomic insertion at the Shh locus results in hammer toes through enhancer adoption

Enhancers are cis -regulatory elements which control the expression of genes in a defined spatiotemporal pattern, enabling the normal morphogenesis of organs and structures during embryogenesis. Enhancers control their target genes independently of their orientation or distance through chromosomal looping and are thought to evolve through various mutational mechanisms (1). A critical biological process that leads to the acquisition of new gene expression domains as well as pathological outcomes is termed “enhancer adoption,” whereby a gene is regulated by an enhancer that is not normally its own (2). Enhancer adoption can derive from the insertion of transposable elements (TEs) with regulatory capacities or genomic structural variants (SVs). In particular, many studies have demonstrated the importance of SVs, including deletions, inversions, duplications, and translocations, with regard to 3D genome organization, gene regulation, and disease (3⇓–5). Recent advances in genome engineering and in sequencing technologies have contributed to a deeper understanding of the molecular mechanisms behind genomic rearrangements and their role in evolution and pathology. Therefore, researchers have seized the opportunity to reinterpret old mouse alleles obtained by spontaneous mutations like X-irradiation-induced phocomelia, Hemimelic extra toes ( Hx ), Ulnaless , and so on, in the light of modern molecular tools (6⇓–8). In PNAS, Mouri et al. (10) set out to reanalyze a mouse mutant with syndactyly and interdigital webbing, known as Hammer toe ( Hm ), first described in 1964 (9). The authors found that a genomic insertion of an interdigital regulatory region in the vicinity of the gene Sonic hedgehog ( Shh ) results in its ectopic expression and the Hm phenotype. The authors use chromatin technologies and CRISPR/Cas9 genetic engineering to dissect the regulatory function of … [↵][1]1To whom correspondence should be addressed. Email: andrey{at}molgen.mpg.de. [1]: #xref-corresp-1-1

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.A Pitx1 enhancer shows activity in forelimbs and hindlimbs but only interacts with Pitx1 in hindlimbs because of its three-dimensional configuration. Structural variants that affect three-dimensional conformation induce Pitx1 expression in forelimbs and cause partial arm-to-leg transformation in mice and humans.