Biology subjects

Bolt, C. C.

Publications and source records attributed to Bolt, C. C..

8 recordsLinked to original sources

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

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

DEVELOPMENTAL AND EVOLUTIONARY COMPARATIVE ANALYSIS OF A HOXD REGULATORY LANDSCAPE IN MAMMALS AND BIRDS

Modifications in gene regulation during development are considered to be a driving force in the evolution of organisms. Part of these changes involve rapidly evolving cis-regulatory elements (CREs), which interact with their target genes through higher-order 3D chromatin structures. However, how such 3D architectures and variations in CREs contribute to transcriptional evolvability remains elusive. During vertebrate evolution, Hox genes were redeployed in different organs in a class-specific manner, while maintaining the same basic function in organizing the primary body axis. Since a large part of the relevant enhancers are located in a conserved regulatory landscape, this gene cluster represents an interesting paradigm to study the emergence of regulatory innovations. Here, we analysed Hoxd gene regulation in both murine vibrissae and chicken feather primordia, two mammalian- and avian-specific skin appendages which express different subsets of Hoxd genes, and compared their regulatory modalities with the regulations at work during the elongation of the posterior trunk, a mechanism highly conserved in amniotes. We show that in the former two structures, distinct subsets of Hoxd genes are contacted by different lineage-specific enhancers, likely as a result of using an ancestral chromatin topology as an evolutionary playground, whereas the regulations implemented in the mouse and chicken embryonic trunk partially rely on conserved CREs. Nevertheless, a high proportion of these non-coding sequences active in the trunk appear to have functionally diverged between the two species, suggesting that transcriptional robustness is maintained despite a considerable divergence in CREs sequence, an observation supported by a genome-wide comparative approach.

developmental biology↗

bioRxiv2021

CONTEXT-INDEPENDENT FUNCTION OF A CHROMATIN BOUNDARY IN VIVO

Mammalian genomes are partitioned into sub-megabase to megabase-sized units of preferential interactions called topologically associating domains or TADs, which are likely important for the proper implementation of gene regulatory processes. These domains provide structural scaffolds for distant cis regulatory elements to interact with their target genes within the three-dimensional nuclear space and architectural proteins such as CTCF as well as the cohesin complex participate in the formation of the boundaries between them. However, the importance of the genomic context in providing a given DNA sequence the capacity to act as a boundary element remains to be fully investigated. To address this question, we randomly relocated a topological boundary functionally associated with the mouse HoxD gene cluster and show that it can indeed act similarly outside its initial genomic context. In particular, the relocated DNA segment recruited the required architectural proteins and induced a significant depletion of contacts between genomic regions located across the integration site. The host chromatin landscape was re-organized, with the splitting of the TAD wherein the boundary had integrated. These results provide evidence that topological boundaries can function independently of their site of origin, under physiological conditions during mouse development. AUTHOR SUMMARYDuring development, enhancer sequences tightly regulate the spatio-temporal expression of target genes often located hundreds of kilobases away. This complex process is made possible by the folding of chromatin into domains, which are separated from one another by specific genomic regions referred to as boundaries. In order to understand whether such boundary sequences require their particular genomic contexts to achieve their isolating effect, we analyzed the impact of introducing one such boundary, taken from the HoxD gene cluster, into a distinct topological domain. We show that this ectopic boundary splits the host domain into two sub-domains and affects the expression levels of a neighboring gene. We conclude that this sequence can work independently from its genomic context and thus carries all the information necessary to act as a boundary element.

genetics↗

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

SPECIFIC ECTODERMAL ENHANCERS CONTROL THE EXPRESSION OF HoxC GENES IN DEVELOPING MAMMALIAN INTEGUMENTS

Vertebrate Hox genes are key players in the establishment of structures during the development of the main body axis. Subsequently, they play important roles either in organizing secondary axial structures such as the appendages, or during homeostasis in postnatal stages and adulthood. Here we set up to analyze their elusive function in the ectodermal compartment, using the mouse limb bud as a model. We report that the HoxC gene cluster was globally co-opted to be transcribed in the distal limb ectoderm, where it is activated following the rule of temporal colinearity. These ectodermal cells subsequently produce various keratinized organs such as nails or claws. Accordingly, deletion of the HoxC cluster led to mice lacking nails (anonychia) and also hairs (alopecia), a condition stronger than the previously reported loss of function of Hoxc13, which is the causative gene of the ectodermal dysplasia 9 (ECTD9) in human patients. We further identified two ectodermal, mammalian-specific enhancers located upstream of the HoxC gene cluster, which act synergistically to regulate Hoxc gene expression in the hair and nail ectodermal organs. Deletion of these regulatory elements alone or in combination revealed a strong quantitative component in the regulation of Hoxc genes in the ectoderm, suggesting that these two enhancers may have evolved along with mammals to provide the level of HOXC proteins necessary for the full development of hairs and nails. Significance StatementIn this study, we report a unique and necessary function for the HoxC gene cluster in the development of some ectodermal organs, as illustrated both by the hair and nail phenotype displayed by mice lacking the Hoxc13 function and by the congenital anonychia (absence of nails) in full HoxC cluster mutants. We show that Hoxc genes are activated in a colinear manner in the embryonic limb ectoderm and are subsequently transcribed in developing nails and hairs. We identify two mammalian-specific enhancers located upstream of the HoxC cluster with and exclusive ectodermal specificity. Individual or combined enhancer deletions suggest that they act in combination to raise the transcription level of several Hoxc genes during hairs and nails development.

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↗

bioRxiv2018

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.\n\nAUTHOR SUMMARYThe 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.

developmental biology↗