1,721,179 research outputs found
Convergent evolution of complex regulatory landscapes and pleiotropy at Hox loci
No full textHox genes are required during the morphogenesis of both vertebrate digits and external genitals. We investigated whether transcription in such distinct contexts involves a shared enhancer-containing landscape. We show that the same regulatory topology is used, yet with some tissue-specific enhancer-promoter interactions, suggesting the hijacking of a regulatory backbone from one context to the other. In addition, comparable organizations are observed at both HoxA and HoxD clusters, which separated through genome duplication in an ancestral invertebrate animal. We propose that this convergent regulatory evolution was triggered by the preexistence of some chromatin architecture, thus facilitating the subsequent recruitment of the appropriate transcription factors. Such regulatory topologies may have both favored and constrained the evolution of pleiotropic developmental loci in vertebrates.UPDUBLonfat, Nicolas Montavon, Thomas Darbellay, Fabrice Gitto, Sandra Duboule, Denis eng Research Support, Non-U.S. Gov't New York, N.Y. 2014/11/22 06:00 Science. 2014 Nov 21;346(6212):1004-6. doi: 10.1126/science.1257493
Coordinated Hoxd genes expression during embryogenesis is dependent on a strict disposition of genes and polarized regulators within the HoxD cluster
Full text linkHOX transcription factors determine the identity of body regions along the rostro-caudal axis during bilaterian embryogenesis. In vertebrates Hox genes distinctively lie organized in dense clusters, each typically composed of a dozen paralogous transcription units spread over 100 kb of genomic DNA. In every Hox cluster the Hox genes are arranged following a complete 5'-3' transcriptional polarization. Even though Hox are found across the whole animal kingdom, this ordered physical arrangement distinctly characterizes vertebrate Hox clusters. This organization, and the attendant collinearity phenomena have been proposed to be a crucial feature to Hox genes' co-option in several structures organized along secondary body axes, as exemplified by the patterning of various tetrapod-specific structures (e.g. limbs, dig-its and external genitalia). The aim of this thesis is to investigate to what extent Hox transcriptional polarization results from constraints imposed by their close linear proximity when arranged in clusters. Therefore, we challenged the murine HoxD cluster by engineering inversions inside the cluster using CRISPR/Cas9 mutagenesis approaches in vivo. We produced two novel alleles bearing inversions of either Hoxd11 or Hoxd12. Complementarily, we also reanalyzed extensively a larger targeted inversion encompassing Hoxd11-d12 loci in a variety of embryonic organs and tissues. Together, the comparative analysis of these alleles by a combination of approaches (WISH, RNAseq, ChIPseq and 4Cseq) illuminated different types of outcomes resulting from the disruption of HoxD transcriptional polarity and the inversion of non-coding regulatory elements. From our work, we developed the view that engineered inversions of particular Hox genes are tolerated by this genetic system and does not result systematically in major functional disruption of the affected cluster. This result therefore demonstrates that Hox transcriptional polarity is not a feature arising from an absolute developmental constraint. This conclusion is nevertheless nuanced by two instances where intra-HoxD inversion disrupts the regulation and function of the HoxD cluster. In one case, the inversion of Hoxd11 affects the transcriptional output of its posterior neighbor Hoxd12. This perturbation is likely implying interference mechanism related to the transcriptional leakage emitted onto Hoxd12 by the inverted Hoxd11 locus. In the second case, the inversion of the Hoxd11-d12 loci in a Hoxd13lacZ background results in the specific gain-of-expression of this reporter gene in a variety of embryonic structures. Of interest, we present here diverse pieces of evidence that this dysregulation may not be directly related to the disruption of Hox gene transcriptional polarity but instead caused by the translocation and inversion of a CTCF binding site. This observation supports the hypothesis that CTCF dependent topological organization of the chromatin plays a role in the regulatory insulation of Hoxd13. It appears therefore that spontaneous inversions within polarized Hox clusters might affect the function and regulation of the Hox patterning mechanism in different functional contexts. This conclusion leads us to conjecture that once disposed in dense clusters, the developmental expression of Hox genes is extremely sensitive to genetic perturbations, due notably to the presence of several conserved polarized regulatory elements interspersed between the individual Hox genes iUPDU
Analysis of long-range gene regulation at the HoxD locus
No full textHoxd genes are essential for the development of the various body axes in vertebrates and hence the underlying regulatory mechanisms are of paramount importance. Among these various mechanisms are long-range acting enhancers, which are located in the two adjacent regulatory landscapes. Analyses of chromatin architecture at this gene cluster has revealed the existence of two topologically associating domains (TADs) flanking the cluster and encompassing these regulatory landscapes. However, the dynamics of such regulatory regions as well as the stability and functional contribution of specific enhancer-promoter interactions during development remains to be established.
In this work, we analysed the 3D chromatin organization and transcription profile at the HoxD locus, at different time points during genital tubercle (GT) development, and observe that the 3D conformation of this regulatory region predates the embryonic emergence of the GT. Along with this tissue development, we observe a reduction in transcript levels correlating with a decrease in enhancer-promoter chromatin loops within the adjacent gene desert. This decrease occurs while maintaining a subset of CTCF/Cohesin associated contacts, which are preserved independently from the transcriptional status of the gene cluster.
To further explore the functional contribution of this regulatory landscape, we used CRISPR-Cas9 technology to generate mice carrying partial deletions of this region, as well as targeted deletions of both transient (enhancer associated) and constitutive (CTCF/Cohesin associated) contacts. We observe that single deletions of both transient and constitutive contacts displayed little if any effect on Hoxd genes expression in the GT. On the contrary, the single deletion of a previous characterized Hoxd enhancer, the Prox element, or deletions comprising several enhancers, result in the reduction of Hoxd genes expression levels. Overall our results suggest that not all enhancer elements within a complex regulatory landscape have the same functional strength, and highlight the existence of a dynamic yet robust system to tightly regulate Hoxd genes expression.UPDU
Regulatory Switches Underlie Hoxd Gene Activation in Tetrapod Limbs
No full textAccurate spatio-temporal control of Hox cluster genes expression is critical for the proper organization of body structures during vertebrate embryogenesis. In the limbs, Hoxd genes are critical in setting up the proximodistal and anteroposterior axes. Several studies have shown that long-range global regulatory elements are required to establish Hoxd genes expression territories in this tissue, via the co-regulation of several genes at once. Limb buds take their origin from the lateral plate mesoderm where the HoxD cluster is controlled by cues from the main body axis. During limb outgrowth Hoxd genes are then subjected to two subsequent activation waves: an early phase patterning proximal limb structures, arms and forearms and a late one instructing digits. As the late activation of Hoxd genes is known to rely upon a large regulatory archipelago located centromeric to the gene cluster, the control of the early phase remains poorly described (Montavon et al., 2011). In this work we show that the early activation domain requires a different, telomeric-located, regulatory landscape. This one megabase (Mb) large domain holds at least three regulatory regions contributing to Hoxd gene activation in early forelimb and hindlimb buds. To our surprise the abrogation of most of the early telomeric enhancers induced a dramatic decrease of central Hoxd transcription in forelimbs but only removed 50% of the transcripts in hindlimbs. This discrepancy most likely arises from the difference in the epigenetic status of the HoxD cluster in anterior and posterior limb fields where forelimb and hindlimb respectively take their origin. As the cluster does not possess the same fraction of active genes in these progenitors it most likely does not respond to early cues in the same way. In the second part of this work we noticed an unexpected decrease of the late expression phase in all mutants lacking early enhancers. This observation shows that digit progenitors have to implement the early phase in order to gain competence toward the late centromeric regulation. This transition between early and late regulations involves a set of central Hoxd genes, i.e. Hoxd11 to Hoxd9. To implement both regulations, these loci switch between two topological domains containing the centromeric and telomeric regulatory landscapes respectively (Dixon et al., 2012). The ability to switch from one domain to another depends on the relative position of genes towards the boundary between both domains and correlates with their respective expression. Moreover, the switch between both topological domains mechanically induces an intermediate, Hoxd-free zone, in-between the two expression phases that will later produce the mesopodium, the articulation between our arms and our hands.UPDU
Polycomb Recruitment and Targeting in Mammals
No full textPolycomb- (PcG) and trithorax (trxG) group proteins are essential components in the regulation of multiple developmental regulators. In particular, during early stages, they act as chromatin modifiers in such a way as to set and maintain the transcriptional status of Hox genes. Little is known, in vertebrates, concerning the modalities of PcG binding to their target sites. One possibility is that they are recruited, in a synergistic manner, through their binding to specific DNA regulatory elements to control spatial and temporal restriction of Hox genes by trimethylating lysine 27 on histone 3 (H3K27me3). Whereas these DNA sequences, referred to as polycomb response elements (PREs), have been identified in Drosophila, their existence in mammals remains to be clearly demonstrated. In this work, we aim to decipher the means by which polycomb repressive complexes (PRCs) are recruited to PREs during early mouse development and focus on the HoxD cluster, a genomic segment that is amongst the most heavily covered by H3K27me3 marks in embryonic stem (ES) cells and where one of the few vertebrate PRE has been previously reporter. We describe relatively small DNA sequences lying within the HoxD locus that are necessary and sufficient for the tethering of polycomb to ectopic loci. These elements work synergistically to form a fully functional repressive domain. Moreover, we show that a sequence with high GC content, although not necessary for the initial recruitment of PRC per se, may stabilize the link between the complex and the underlying DNA. Finally, we propose a model for the evolution of polycomb targets and discuss the emergence of diverging recruitment mechanisms amongst Drosophila, mice and humans.UPDU
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Evolution of Hoxd gene regulation
No full textThe phenotypic diversity found in vertebrates is closely related to modifications in the transcription patterns of developmental genes. Developmental genes often participate in several patterning processes and are thus very well conserved. In contrast, their associated cis-regulatory elements (CREs) function in a mostly time and tissue specific manner. Genetic modifications within CREs may thus alter the pattern of expression of their target developmental gene in a spatiotemporal limited manner. Consequently, they may lead to morphological changes with less probability of associated lethality than mutations affecting coding sequences. For this reason, understanding the mechanisms of CREs evolution is key to gain insight into vertebrate diversification.La diversité phénotypique des vertébrés est fortement liée à des variations dans les patterns de transcription des gènes du développement. Ces gènes sont généralement impliqués dans plusieurs processus d'organogenèse et ont par conséquent été fortement conservés au cours de l'évolution. En revanche, leurs séquences régulatrices, appelées cis-regulatory elements (CREs), ont une activité majoritairement spécifique aux tissus et aux stades de développement. Ainsi, certaines modifications de ces séquences de régulation affectent le pattern d'expression de leur gène cible de manière limitée dans le temps et dans l'espace. De ce fait, elles sont plus susceptibles de provoquer des altérations morphologiques non létales que des modifications des séquences codantes. Une compréhension accrue des mécanismes entraînant l'évolution des séquences régulatrices permettrait donc d'améliorer nos déductions quant aux raisons de l'incroyable diversité biologique des vertébrés
Hoxd gene regulation in the developing caecum
Full text link[...] In this project, we investigated the qualitative and quantitative profile of expression of the HoxD complex in the developing caecum of wild-type animals. [... click on "Download fulltext" for full abstract]SS
Mechanisms of Hoxd gene regulation during the development and evolution of the intestinal caecum
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