Biology subjects

Mascrez, B.

Publications and source records attributed to Mascrez, B..

10 recordsLinked to original sources

bioRxiv2024

CTCF-DEPENDENT INSULATION OF Hoxb13 AND THE HETEROCHRONIC CONTROL OF TAIL LENGTH

In mammals, tail length is controlled by several genetic determinants, amongst which Hox13 genes located at the posterior extremities of Hox clusters, whose main function are to terminate the extension of the body axis. In this view, the precise timing in the transcriptional activation of these genes may impact upon body length. Unlike other Hox clusters, HoxB lacks all posterior genes between Hoxb9 and Hoxb13, two genes separated by a ca. 70 kb large DNA segment containing an unusually high number of CTCF sites, suggesting it isolates Hoxb13 from the rest of the cluster, thereby delaying its negative impact on trunk extension. We deleted the spacer DNA to induce a potential heterochronic gain of function of Hoxb13 at physiological concentration and observed a shortening of the tail as well as other abnormal phenotypes, which were all rescued by inactivating Hoxb13 in-cis with the deletion. A comparable gain of function was observed in mutant ES cells grown as pseudo-embryos in vitro, which allowed us to examine in details the importance of both the number and the orientation of CTCF sites in the insulating activity of the DNA spacer. A short cassette containing all the CTCF sites was sufficient to insulate Hoxb13 from the rest of HoxB and additional modifications of this CTCF cassette showed that two CTCF sites in convergent orientations are already capable of importantly delaying Hoxb13 activation in these conditions. We discuss the relative importance of genomic distance versus number and orientation of CTCF sites in preventing Hoxb13 to be activated too early during trunk extension and hence to modulate tail length.

developmental biology↗

bioRxiv2024

EVOLUTIONARY CO-OPTION OF AN ANCESTRAL CLOACAL REGULATORY LANDSCAPE DURING THE EMERGENCE OF DIGITS AND GENITALS

The transition from fins to limbs has been a rich source of discussion for more than a century. One open and important issue is understanding how the mechanisms that pattern digits arose during vertebrate evolution. In this context, the analysis of Hox gene expression and functions to infer evolutionary scenarios has been a productive approach to explain the changes in organ formation, particularly in limbs. In tetrapods, the transcription of Hoxd genes in developing digits depends on a well-characterized set of enhancers forming a large regulatory landscape1,2. This control system has a syntenic counterpart in zebrafish, even though they lack bona fide digits, suggestive of deep homology3 between distal fin and limb developmental mechanisms. We tested the global function of this landscape to assess ancestry and source of limb and fin variation. In contrast to results in mice, we show here that the deletion of the homologous control region in zebrafish has a limited effect on the transcription of hoxd genes during fin development. However, it fully abrogates hoxd expression within the developing cloaca, an ancestral structure related to the mammalian urogenital sinus. We show that similar to the limb, Hoxd gene function in the urogenital sinus of the mouse also depends on enhancers located in this same genomic domain. Thus, we conclude that the current regulation underlying Hoxd gene expression in distal limbs was co-opted in tetrapods from a preexisting cloacal program. The orthologous chromatin domain in fishes may illustrate a rudimentary or partial step in this evolutionary co-option.

evolutionary biology↗

bioRxiv2022

Sequential And Directional Insulation By Conserved CTCF Sites Underlies The Hox Timer In Pseudo-Embryos

During development, Hox genes are activated in a time sequence following their relative positions on their clusters, leading to the proper identities of structures along the rostral to caudal axis. To understand the mechanism operating this Hox timer, we used ES-cells derived stembryos and show that the core of the process involves the start of transcription at the 3 part of the cluster, following Wnt signaling, and the concomitant loading of cohesin complexes on the transcribed DNA segments, i.e., with an asymmetric distribution along the gene cluster. Chromatin extrusion then occurs with successively more posterior CTCF sites acting as transient insulators, thus generating a progressive time-delay in the activation of more 5-located genes due to long-range contacts with a flanking TAD. Mutant stembryos support this model and reveal that the iterated presence of evolutionary conserved and regularly spaced intergenic CTCF sites control the precision and the pace of this temporal mechanism.

developmental biology↗

bioRxiv2022

CONTEXT-DEPENDENT ENHANCER FUNCTION REVEALED BYTARGETED INTER-TAD RELOCATION

The expression of genes with a key function during development is frequently controlled by large regulatory landscapes containing multiple enhancer elements. These landscapes often match Topologically Associating Domains (TADs) and sometimes integrate range of similar enhancers, thus leading to TADs having a global regulatory specificity. To assess the relative functional importance of enhancer sequences versus the regulatory domain they are included in, we set out to transfer one particular enhancer sequence from its native domain into a TAD with a closely related, yet different functional specificity. We used Hoxd genes and their biphasic regulation during limb development as a paradigm, since they are first activated in proximal limb cells by enhancers located in one TAD, which is then silenced at the time when the neighboring TAD starts to activate its enhancers in distal limb cells. We introduced a strong distal limb enhancer into the proximal limb TAD and found that its new context strongly suppresses its distal specificity, even though it continues to be bound by HOX13 transcription factors, which normally are responsible for this activity. Using local genetic alterations and chromatin conformation measurements, we see that the enhancer is capable of interacting with target genes, with a pattern comparable to its adoptive neighborhood of enhancers. Its activity in distal limb cells 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 local chromatin context, which can exert a dominant control over its activity.

developmental biology↗

bioRxiv2021

Sequential in-cis mutagenesis in vivo reveals various functions for CTCF sites at the mouse HoxD cluster

Mammalian Hox gene clusters contain a range of CTCF binding sites. In addition to their importance in organizing a TAD border, which isolates the most posterior genes from the rest of the cluster, the positions and orientations of these sites suggest that CTCF may be instrumental in the selection of various subsets of contiguous genes, which are targets of distinct remote enhancers located in the flanking regulatory landscapes. We examined this possibility by producing an allelic series of cumulative in-cis mutations in these sites, up to the abrogation of CTCF binding in the five sites located on one side of the TAD border. In the most impactful alleles, the global chromatin architecture of the locus was modified, yet not drastically, illustrating that CTCF sites located on one side of a strong TAD border are sufficient to organize at least part of this insulation. Spatial colinearity in the expression of these genes along the major body axis was nevertheless maintained, despite abnormal expression boundaries. In contrast, strong effects were scored in the selection of target genes responding to particular enhancers, leading to the mis-regulation of Hoxd genes in specific structures. Altogether, while most enhancer-promoter interactions can occur in the absence of this series of CTCF sites, it seems that the binding of CTCF in the Hox cluster is required to properly transform a rather unprecise process into a highly discriminative mechanism of interactions, which is translated into various patterns of transcription accompanied by the distinctive chromatin topology found at this locus. Our allelic series also allowed us to reveal the distinct functional contributions for CTCF sites within this Hox cluster, some acting as insulator elements, others being necessary to anchor or stabilize enhancer-promoter interactions and some doing both, whereas all together contribute to the formation of a TAD border. This variety of tasks may explain the amazing evolutionary conservation in the distribution of these sites amongst paralogous Hox clusters or between various vertebrates.

developmental biology↗

bioRxiv2021

MESOMELIC DYSPLASIAS ASSOCIATED WITH THE HOXD LOCUS ARE CAUSED BY REGULATORY REALLOCATIONS

Some human families display severe shortening and bending of the radius and ulna, a condition referred to as mesomelic dysplasia. Many of these families contain chromosomal rearrangements at 2q31, where the human HOXD locus maps. In mice, the dominant X-ray-induced Ulnaless inversion of the HoxD gene cluster produces a similar phenotype suggesting that the same mechanism is responsible for this pathology in humans and mice. Amongst the proposed explanations, the various alterations to the genomic structure of HOXD could expose Hoxd13 to proximal limb enhancers, leading to its deleterious gain-of-expression in the embryonic forelimb. To assess this hypothesis, we used an engineered 1Mb large inversion including the HoxD gene cluster, in order to position Hoxd13 within a chromatin domain rich in proximal limb enhancers. We show that these enhancers contact and activate Hoxd13 in proximal cells, concomitant to the formation of a mesomelic dysplasia phenotype. A secondary mutation in the coding frame of the HOXD13 protein in-cis with the inversion completely rescued the limb alterations, demonstrating that ectopic HOXD13 is indeed the unique cause of this bone anomaly. Single cell expression analysis and evaluation of HOXD13 binding sites in cells from this ectopic expression domain suggests that the phenotype arises primarily by acting through genes normally controlled by HOXD13 in distal limb cells. Altogether, these results provide a conceptual and mechanistic framework to understand and unify the molecular origins of human mesomelic dysplasia associated with 2q31.

developmental biology↗

bioRxiv2020

HOX13-MEDIATED DBX2 REGULATION IN LIMBS SUGGESTS INTER-TAD SHARING OF ENHANCERS

BackgroundDuring tetrapod limb development, the HOXA13 and HOXD13 transcription factors are critical for the emergence and organization of the autopod, the most distal aspect where digits will develop. Since previous work had suggested that the Dbx2 gene is a target of these factors, we set up to analyze in detail this potential regulatory interaction. ResultsWe show that HOX13 proteins bind to eutherian-specific sequences at the vicinity of the Dbx2 locus that have enhancer activity in developing digits. However, the functional inactivation of the DBX2 protein did not elicit any particular phenotype related to Hox genes inactivation in digits, suggesting either redundant or compensatory mechanisms. We report that the neighboring Nell2 and Ano6 genes are also expressed in distal limb buds and are, in part, controlled by the same Dbx2 enhancers despite being localized into two different topologically associating domains (TADs) flanking the Dbx2 locus. ConclusionsWe conclude that Hoxa13 and Hoxd genes cooperatively activate Dbx2 expression in developing digits through binding to eutherian specific regulators elements in the Dbx2 neighborhood. Furthermore, these enhancers can overcome TAD boundaries in either direction to co-regulate a set of genes located in distinct chromatin domains. Bullet pointsO_LIHoxa13 and Hoxd genes cooperatively regulate Dbx2 expression in developing digits via eutherian specific enhancers. C_LIO_LIDbx2 is expressed in different digit joint precursors but its function there is not essential. C_LIO_LIDbx2 enhancers also control the expression of the Nell2 and Ano6 genes, which are located in different TADs, thus overcoming the boundary effect. C_LIO_LIDbx2 chromatin architecture and enhancers evolved in the mammalian lineage. C_LI Grant Sponsor and NumberSwiss National Research Foundation #310030B_138662. European Research Council grants RegulHox #588029

developmental biology↗

bioRxiv2019

A Complex Regulatory Landscape Involved In The Development Of External Genitals

In vertebrates, developmental genes are often controlled by large regulatory landscapes matching the dimensions of topologically associating domains (TADs). In various ontogenic contexts, the associated constitutive chromatin backbone is modified by fine-tuned specific variations in enhancer-enhancer and enhancer-promoter interaction profiles. In this work, we take one of the TADs flanking the HoxD gene cluster as a paradigm to address the question of how these complex regulatory architectures are formed and how they are de-constructed once their function has been achieved. We suggest that this TAD can be considered as a coherent functional unit in itself, with several regulatory sequences acting together to elicit a transcriptional response. With one notable exception, the deletion of each of these sequences in isolation did not produce any substantial modification in the global transcriptional outcome of the system, a result at odds with a conventional view of long-range enhancer function. Likewise, both the deletion and inversion of a supposedly critical CTCF site located in a region rich in such sequences did not affect transcription of the target gene. In the latter case, however, slight modifications were observed in interaction profiles in vivo in agreement with the loop extrusion model, despite no apparent functional consequences. We discuss these unexpected results by considering both conventional explanations and an alternative possibility whereby a rather unspecific accumulation of particular factors within the TAD backbone may have a global impact upon transcription.

developmental biology↗

bioRxiv2019

Constrained Transcriptional Polarity In The Organization Of Mammalian Hox Gene Clusters

In many animal species with a bilateral symmetry, Hox genes are clustered either at one or at several genomic loci. This organization has a functional relevance, as the transcriptional control applied to each gene depends upon its relative position within the gene cluster. It was previously noted that vertebrate Hox clusters display a much higher level of genomic organization than their invertebrate counterparts. The former are always more compact than the latter, they are generally devoid of repeats and of interspersed genes, and all genes are transcribed by the same DNA strand, suggesting that particular factors constrained these clusters towards a tighter structure during the evolution of the vertebrate lineage. Here we investigate the importance of uniform transcriptional orientation by engineering several alleles within the HoxD cluster such as to invert one or several transcription unit(s), with or without a neighboring CTCF site. We observe that the association between the tight structure of mammalian Hox clusters and their regulation makes inversions likely detrimental to the proper implementation of this complex genetic system. We propose that the consolidation of Hox clusters in vertebrates, including transcriptional polarity, evolved in conjunction with the emergence of global gene regulation via the flanking regulatory landscapes, to optimize a coordinated response of selected subsets of target genes in cis.

developmental biology↗

bioRxiv2018

Single-cell mRNA profiling reveals heterogeneous combinatorial expression of Hoxd genes during limb development

ABSTRACT (177 words)A global analysis of gene expression during development reveals specific transcription patterns associated with the emergence of various cell types, tissues and organs. These heterogeneous patterns are instrumental to ensure the proper formation of the different parts of our body, as shown by the phenotypic effects generated by functional genetic approaches. However, variations at the cellular level can be observed within each structure or organ. In the developing mammalian limbs, expression of Hoxd genes is differentially controlled in space and time in cells that will pattern the digits and the arms. Here we analyze single-cell transcriptomes of limb bud cells and show that Hox genes are expressed in specific combinations that match particular cell types. In the presumptive digits, we find that the expression of Hoxd gene is unbalanced, despite their common genomic proximity to known global enhancers, often expressing only a subset of the five genes transcribed in these cells. We also report that combinatorial expression follows a pseudo-time sequence, suggesting that a progression in combinatorial expression may be associated with cellular diversity in developing digits.\n\nHIGHLIGHTSO_LICollinear expression of Hox genes is only weaved at the tissue scale\nC_LIO_LIEnhancer-sharing to specific target genes is reduced at the single-cell level\nC_LIO_LIHoxd gene combinatorial expression is linked to distinct transcriptional signatures\nC_LIO_LIIn presumptive digits, Hoxd combinations follow a pseudotime trajectory\nC_LI

developmental biology↗