Tschopp P, Fraudeau N, Bena F, Duboule D
National Research Centre Frontiers in Genetics, Department of Genetics and Evolution, University of Geneva, Sciences III, 1211 Geneva 4, Switzerland.
The emergence of Vertebrata was accompanied by two rounds of whole-genome duplications. This enabled paralogous genes to acquire novel functions with high evolutionary potential, a process suggested to occur mostly by changes in gene regulation, rather than in protein sequences. In the case of Hox gene clusters, such duplications favored the appearance of distinct global regulations. To assess the impact of such "regulatory evolution" upon neo-functionalization, we developed PANTHERE (PAN-genomic Translocation for Heterologous Enhancer RE-shuffling) to bring the entire megabase-scale HoxD regulatory landscape in front of the HoxC gene cluster via a targeted translocation in vivo. At this chimeric locus, Hoxc genes could both interpret this foreign regulation and functionally substitute for their Hoxd counterparts. Our results emphasize the importance of evolving regulatory modules rather than their target genes in the process of neo-functionalization and offer a genetic tool to study the complexity of the vertebrate regulatory genome.
Schorderet P, Duboule D
School of Life Sciences, Federal Institute of Technology (EPFL), Lausanne, Switzerland.
Long non-coding RNAs regulate various biological processes such as dosage compensation, imprinting, and chromatin organization. HOTAIR, a paradigm of this new class of RNAs, is localized within the human HOXC gene cluster and was shown, in human cells, to regulate HOXD genes in trans via the recruitment of Polycomb Repressive Complex 2 (PRC2), followed by the trimethylation of lysine 27 of histone H3. We looked for the presence of Hotair in mice to assess whether this in trans mechanism was conserved, in particular at the developmental stages, when Hoxd genes must be tightly regulated. We show that the cognate mouse Hotair is poorly conserved in sequence; and its absence, along with the deletion of the HoxC cluster, has surprisingly little effect in vivo, neither on the expression pattern or transcription efficiency, nor on the amount of K27me3 coverage of different Hoxd target genes. We conclude that Hotair may have rapidly evolved within mammals and acquired a functional importance in humans that is not easily revealed in mice. Alternatively, redundant or compensatory mechanisms may mask its function when studied under physiological conditions.
Tschopp P, Duboule D
National Research Centre 'Frontiers in Genetics', Department of Zoology and Animal Biology, University of Geneva, Sciences III, Quai Ernest-Ansermet 30, 1211, Geneva 4, Lausanne, Switzerland.
Faithful expression of Hox genes in both time and space is essential for proper patterning of the primary body axis. Transgenic approaches in vertebrates have suggested that this collinear activation process is regulated in a largely gene cluster-autonomous manner. In contrast, more recently co-opted expression specificities, required in other embryonic structures, depend upon long-range enhancer sequences acting from outside the gene clusters. This regulatory dichotomy was recently questioned, since gene activation along the trunk seems to be partially regulated by signals located outside of the cluster. We investigated these alternative regulatory strategies by engineering a large inversion that precisely separates the murine HoxD complex from its centromeric neighborhood. Mutant animals displayed posterior transformations along with subtle deregulations of Hoxd genes, indicating an impact of the centromeric landscape on the fine-tuning of Hoxd gene expression. Proximal limbs were also affected, suggesting that this 'landscape effect' is generic and impacts upon regulatory mechanisms of various qualities and evolutionary origins.
Soshnikova N, Montavon T, Leleu M, Galjart N, Duboule D
Department of Zoology and Animal Biology, University of Geneva, Sciences III, 1211 Geneva 4, Switzerland.
CCCTC-binding factor (CTCF) is a nuclear zinc-finger protein that displays insulating activity in a variety of biological assays. For example, CTCF-binding sites have been suggested to isolate Hox gene clusters from neighboring transcriptional interference. We investigated this issue during limb development, where Hoxd genes must remain isolated from long-range effects to allow essential regulation within independent sub-groups. We used conditional Ctcf inactivation in incipient forelimbs and show that the overall pattern of Hoxd gene expression remains unchanged. Transcriptome analysis using tiling arrays covering chromosomes 2 and X confirmed the weak effect of CTCF depletion on global gene regulation. However, Ctcf deletion caused massive apoptosis, leading to a nearly complete loss of limb structure at a later stage. We conclude that, at least in this physiological context, rather than being an insulator, CTCF is required for cell survival via the direct transcriptional regulation of target genes critical for cellular homeostasis.
Duboule D
University of Geneva, Ecole Polytechnique Federale, Lausanne, Switzerland. denis.duboule@unige.ch
Villavicencio-Lorini P, Kuss P, Friedrich J, Haupt J, Farooq M, Turkmen S, Duboule D, Hecht J, Mundlos S
Max Planck Institute for Molecular Genetics, Berlin, Germany.
The molecular mechanisms that govern bone and joint formation are complex, involving an integrated network of signaling pathways and gene regulators. We investigated the role of Hox genes, which are known to specify individual segments of the skeleton, in the formation of autopod limb bones (i.e., the hands and feet) using the mouse mutant synpolydactyly homolog (spdh), which encodes a polyalanine expansion in Hoxd13. We found that no cortical bone was formed in the autopod in spdh/spdh mice; instead, these bones underwent trabecular ossification after birth. Spdh/spdh metacarpals acquired an ovoid shape and developed ectopic joints, indicating a loss of long bone characteristics and thus a transformation of metacarpals into carpal bones. The perichondrium of spdh/spdh mice showed abnormal morphology and decreased expression of Runt-related transcription factor 2 (Runx2), which was identified as a direct Hoxd13 transcriptional target. Hoxd11-/-Hoxd12-/-Hoxd13-/- triple-knockout mice and Hoxd13-/-Hoxa13+/- mice exhibited similar but less severe defects, suggesting that these Hox genes have similar and complementary functions and that the spdh allele acts as a dominant negative. This effect was shown to be due to sequestration of other polyalanine-containing transcription factors by the mutant Hoxd13 in the cytoplasm, leading to their degradation. These data indicate that Hox genes not only regulate patterning but also directly influence bone formation and the ossification pattern of bones, in part via Runx2.
Di-Poi N, Koch U, Radtke F, Duboule D
National Research Center Frontiers in Genetics, Department of Zoology and Animal Biology, University of Geneva, Sciences III, 1211 Geneva 4, Switzerland.
Hox genes encode transcription factors that play a central role in the specification of regional identities along the anterior to posterior body axis. In the developing mouse embryo, Hox genes from all four genomic clusters are involved in range of developmental processes, including the patterning of skeletal structures and the formation of several organs. However, the functional redundancy observed either between paralogous genes, or among neighboring genes from the same cluster, has hampered functional analyses, in particular when synergistic, cluster-specific functions are considered. Here, we report that mutant mice lacking the entire HoxA cluster in mesodermal lineages display the expected spectrum of postnatal respiratory, cardiac and urogenital defects, previously reported for single gene mutations. Likewise, mild phenotypes are observed in both appendicular and axial skeleton. However, a striking effect was uncovered in the hematopoietic system, much stronger than that seen for Hoxa9 inactivation alone, which involves stem cells (HSCs) as well as the erythroid lineage, indicating that several Hoxa genes are necessary for normal hematopoiesis to occur. Finally, the combined deletions of Hoxa and Hoxd genes reveal abnormalities in axial elongation as well as skin morphogenesis that are likely the results of defects in epithelial-mesenchymal interactions.
Woltering J M, Duboule D
National Research Centre Frontiers in Genetics, Department of Zoology and Animal Biology, University of Geneva, Sciences III, Quai Ernest-Ansermet 30, 1211 Geneva 4, Switzerland.
In the emerging discipline of Evo-Devo, the analysis of gene expression patterns can be deceptive without a clear understanding of the underlying regulatory strategies. Here, we use the paradigm of hand and foot evolution to argue that the consideration of the regulatory mechanisms controlling developmental gene expression is essential to resolve comparative conundrums. In this context, we discuss the adaptive relevance of evolving stepwise, distinct developmental regulatory mechanisms to build an arm, i.e., a composite structure with functional coherence.
Di-Poi N, Montoya-Burgos J I, Miller H, Pourquie O, Milinkovitch M C, Duboule D
National Research Center Frontiers in Genetics, Department of Zoology and Animal Biology, University of Geneva, Sciences III, 1211 Geneva 4, Switzerland.
Hox genes are central to the specification of structures along the anterior-posterior body axis, and modifications in their expression have paralleled the emergence of diversity in vertebrate body plans. Here we describe the genomic organization of Hox clusters in different reptiles and show that squamates have accumulated unusually large numbers of transposable elements at these loci, reflecting extensive genomic rearrangements of coding and non-coding regulatory regions. Comparative expression analyses between two species showing different axial skeletons, the corn snake and the whiptail lizard, revealed major alterations in Hox13 and Hox10 expression features during snake somitogenesis, in line with the expansion of both caudal and thoracic regions. Variations in both protein sequences and regulatory modalities of posterior Hox genes suggest how this genetic system has dealt with its intrinsic collinear constraint to accompany the substantial morphological radiation observed in this group.
Friedli M, Barde I, Arcangeli M, Verp S, Quazzola A, Zakany J, Lin-Marq N, Robyr D, Attanasio C, Spitz F, Duboule D, Trono D, Antonarakis S E
Department of Genetic Medicine and Development, University of Geneva Medical School and University Hospitals of Geneva, Geneva, Switzerland.
Finding sequences that control expression of genes is central to understanding genome function. Previous studies have used evolutionary conservation as an indicator of regulatory potential. Here, we present a method for the unbiased in vivo screen of putative enhancers in large DNA regions, using the mouse as a model. We cloned a library of 142 overlapping fragments from a 200 kb-long murine BAC in a lentiviral vector expressing LacZ from a minimal promoter, and used the resulting vectors to infect fertilized murine oocytes. LacZ staining of E11 embryos obtained by first using the vectors in pools and then testing individual candidates led to the identification of 3 enhancers, only one of which shows significant evolutionary conservation. In situ hybridization and 3C/4C experiments suggest that this enhancer, which is active in the neural tube and posterior diencephalon, influences the expression of the Olig1 and/or Olig2 genes. This work provides a new approach for the large-scale in vivo screening of transcriptional regulatory sequences, and further demonstrates that evolutionary conservation alone seems too limiting a criterion for the identification of enhancers.
Soshnikova N, Duboule D
National Research Centre Frontiers in Genetics, Department of Zoology and Animal Biology, University of Geneva, Sciences III, Geneva, Switzerland.
Temporal and spatial control of Hox gene expression is essential for correct patterning of many animals. In both Drosophila and vertebrates, Polycomb and Trithorax group complexes control the maintenance of Hox gene expression in appropriate domains. In vertebrates, dynamic changes in chromatin modifications are also observed during the sequential activation of Hox genes in the embryo, suggesting that progressive epigenetic modifications could regulate collinear gene activation.
Soshnikova N, Duboule D
National Research Centre Frontiers in Genetics, Department of Zoology and Animal Biology, University of Geneva, Sciences III, Quai Ernest-Ansermet 30, 1211 Geneva 4, Switzerland.
During vertebrate development, the temporal control of Hox gene transcriptional activation follows the genomic order of the genes within the Hox clusters. Although it is recognized that this "Hox clock" serves to coordinate body patterning, the underlying mechanism remains elusive. We have shown that successive Hox gene activation in the mouse embryo is closely associated with a directional transition in chromatin status, as judged by the dynamic progression of transcription-competent modifications: Increases in activation marks correspond to decreases in repressive marks. Furthermore, using a mouse in which a Hox cluster was split into two pieces, we document the necessity to maintain a clustered organization to properly implement this process. These results suggest that chromatin modifications are important parameters in the temporal regulation of this gene family.