Project description:During limb development, Hoxd genes are transcribed in two waves: Early on, when the arm and forearm are specified and subsequently, when digits form. While the latter phase is controlled by enhancers centromeric to the HoxD cluster, we show here that the early phase requires enhancers located in the opposite telomeric gene desert. The transition between the two types of regulations involves a functional switch between two distinct topological domains, as reflected by a subset of genes mapping centrally into the cluster, which initially interact with the telomeric domain and subsequently shift to establish new contacts on the opposite side. This transition between two regulatory landscapes generates an intermediate area of low Hox dose developing into the wrist, the transition between our arms and our hands. This intriguing correspondence between genomic and morphological boundaries illustrates the mechanism underlying collinear Hox gene regulation in our developing appendages. Chromatin ImmunoPrecipitation and Sequencing (ChIP-seq) of H3K27A in developing proximal and distal limbs at E9.5, E10.5 and E12.5
Project description:During limb development, Hoxd genes are transcribed in two waves: Early on, when the arm and forearm are specified and subsequently, when digits form. While the latter phase is controlled by enhancers centromeric to the HoxD cluster, we show here that the early phase requires enhancers located in the opposite telomeric gene desert. The transition between the two types of regulations involves a functional switch between two distinct topological domains, as reflected by a subset of genes mapping centrally into the cluster, which initially interact with the telomeric domain and subsequently shift to establish new contacts on the opposite side. This transition between two regulatory landscapes generates an intermediate area of low Hox dose developing into the wrist, the transition between our arms and our hands. This intriguing correspondence between genomic and morphological boundaries illustrates the mechanism underlying collinear Hox gene regulation in our developing appendages. Circular Chromosome Conformation Capture (4C seq) at the HoxD locus in developing proximal and distal limbs at E9.5 and E12.5
Project description:During limb development, Hoxd genes are transcribed in two waves: Early on, when the arm and forearm are specified and subsequently, when digits form. While the latter phase is controlled by enhancers centromeric to the HoxD cluster, we show here that the early phase requires enhancers located in the opposite telomeric gene desert. The transition between the two types of regulations involves a functional switch between two distinct topological domains, as reflected by a subset of genes mapping centrally into the cluster, which initially interact with the telomeric domain and subsequently shift to establish new contacts on the opposite side. This transition between two regulatory landscapes generates an intermediate area of low Hox dose developing into the wrist, the transition between our arms and our hands. This intriguing correspondence between genomic and morphological boundaries illustrates the mechanism underlying collinear Hox gene regulation in our developing appendages. Chromatin ImmoPrecipitation on chip (Tiling array): Distribution of H3K4me3 and H3K27me3 in early limb buds at E9.5, E10.5 and proximal late limbs E12.5. Distribution of H3K27me3 in del(8-13) and del(8-13)/del(attP-TpSB3) E10.5 limb buds. Distribution of H3K27me3 in WT and homozygote del(Nsi-Atf2) (Montavon et al., 2011) forelimb autopods.
Project description:HoxA genes exhibit central roles during development and causal mutations have been found in several human syndromes including limb malformation. Despite their importance, information on how these genes are regulated is lacking. Here, we report on the first identification of bona fide transcriptional enhancers controlling HoxA genes in developing limbs and show that these enhancers are grouped into distinct topological domains at the sub-megabase scale (sub-TADs). We provide evidence that target genes and regulatory elements physically interact with each other through contacts between sub-TADs rather than by the formation of discreet "DNA loops". Interestingly, there is no obvious relationship between the functional domains of the enhancers within the limb and how they are partitioned among the topological domains, suggesting that sub-TAD formation does not rely on enhancer activity. Moreover, we show that suppressing the transcriptional activity of enhancers does not abrogate their contacts with HoxA genes. Based on these data, we propose a model whereby chromatin architecture defines the functional landscapes of enhancers. From an evolutionary standpoint, our data points to the convergent evolution of HoxA and HoxD regulation in the fin-to-limb transition, one of the major morphological innovations in Vertebrates. The chromatin binding of the RNA polymerase II and the sub-unit Med12 of the Mediator complexe were analysed by ChIP-sequencing mouse distal E11.5 forelimb. This approach allowed us to identify the position of limb-specific enhancers.
Project description:The evolution of tetrapod limbs from fish fins enabled the conquest of land by vertebrates and thus represents a key step in evolution. Despite the use of comparative gene expression analyses, critical aspects of this transformation remain controversial, in particularly the origin of digits. Hoxa and Hoxd genes are essential for the specification of the different limb segments and their functional abrogation leads to large truncations of the appendages. Here we show that the selective transcription of mouse Hoxa genes in proximal and distal limbs is related to a bimodal higher order chromatin structure, similar to that reported for Hoxd genes, thus revealing a generic regulatory strategy implemented by both gene clusters during limb development. We found the same bimodal chromatin architecture in fish embryos, indicating that the regulatory strategy used to pattern tetrapod limbs predates the divergence between fish and tetrapods. However, when assessed in mice, both fish regulatory domains triggered transcription in proximal, rather than distal limb territories, supporting an evolutionary scenario whereby digits arose as true tetrapod novelties through genetic retrofitting of a preexisting bimodal chromatin framework. We discuss the possibility to consider regulatory circuitries, rather than expression patterns, as essential parameters to define evolutionary synapomorphies. Circular Chromosome Conformation Capture (4C seq) at the mouse HoxA and HoxD loci in proximal and distal forelimbs and forebrain at E12.5 and at the zebrafish HoxAa, HoxAb and HoxDa loci in 5 dpf whole embryos.
Project description:HoxA genes exhibit central roles during development and causal mutations have been found in several human syndromes including limb malformation. Despite their importance, information on how these genes are regulated is lacking. Here, we report on the first identification of bona fide transcriptional enhancers controlling HoxA genes in developing limbs and show that these enhancers are grouped into distinct topological domains at the sub-megabase scale (sub-TADs). We provide evidence that target genes and regulatory elements physically interact with each other through contacts between sub-TADs rather than by the formation of discreet "DNA loops". Interestingly, there is no obvious relationship between the functional domains of the enhancers within the limb and how they are partitioned among the topological domains, suggesting that sub-TAD formation does not rely on enhancer activity. Moreover, we show that suppressing the transcriptional activity of enhancers does not abrogate their contacts with HoxA genes. Based on these data, we propose a model whereby chromatin architecture defines the functional landscapes of enhancers. From an evolutionary standpoint, our data points to the convergent evolution of HoxA and HoxD regulation in the fin-to-limb transition, one of the major morphological innovations in Vertebrates.
Project description:During limb development, Hoxd genes are transcribed in two waves: Early on, when the arm and forearm are specified and subsequently, when digits form. While the latter phase is controlled by enhancers centromeric to the HoxD cluster, we show here that the early phase requires enhancers located in the opposite telomeric gene desert. The transition between the two types of regulations involves a functional switch between two distinct topological domains, as reflected by a subset of genes mapping centrally into the cluster, which initially interact with the telomeric domain and subsequently shift to establish new contacts on the opposite side. This transition between two regulatory landscapes generates an intermediate area of low Hox dose developing into the wrist, the transition between our arms and our hands. This intriguing correspondence between genomic and morphological boundaries illustrates the mechanism underlying collinear Hox gene regulation in our developing appendages.