staff

Sandra Gitto

Research assistant in Physical principles of regeneration

  • T: +41 22 379 30 53
  • office 4077a (Sciences III)

  • EVOLUTIONARY CO-OPTION OF AN ANCESTRAL CLOACAL REGULATORY LANDSCAPE DURING THE EMERGENCE OF DIGITS AND GENITALS. bioRxiv 2024 Mar;():. PMC10996561. 10.1101/2024.03.24.586442. 2024.03.24.586442.

    abstract

    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 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 genes in developing digits depends on a well-characterized set of enhancers forming a large regulatory landscape. This control system has a syntenic counterpart in zebrafish, even though they lack digits, suggestive of deep homology 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 genes during fin development. However, it fully abrogates expression within the developing cloaca, an ancestral structure related to the mammalian urogenital sinus. We show that similar to the limb, 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 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.

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  • Developmental and evolutionary comparative analysis of a regulatory landscape in mouse and chicken. Development 2022 Jun;149(12):. 275867. 10.1242/dev.200594.

    abstract

    Modifications in gene regulation are driving forces in the evolution of organisms. Part of these changes involve cis-regulatory elements (CREs), which contact their target genes through higher-order chromatin structures. However, how such architectures and variations in CREs contribute to transcriptional evolvability remains elusive. We use Hoxd genes as a paradigm for the emergence of regulatory innovations, as many relevant enhancers are located in a regulatory landscape highly conserved in amniotes. Here, we analysed their regulation in murine vibrissae and chicken feather primordia, two skin appendages expressing different Hoxd gene subsets, and compared the regulation of these genes in these appendages with that in the elongation of the posterior trunk. In the two former structures, distinct subsets of Hoxd genes are contacted by different lineage-specific enhancers, probably as a result of using an ancestral chromatin topology as an evolutionary playground, whereas the gene regulation that occurs in the mouse and chicken embryonic trunk partially relies on conserved CREs. A high proportion of these non-coding sequences active in the trunk have functionally diverged between species, suggesting that transcriptional robustness is maintained, despite considerable divergence in enhancer sequences.

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  • Sequential in mutagenesis in vivo reveals various functions for CTCF sites at the mouse cluster. Genes Dev 2021 Oct;():. gad.348934.121. 10.1101/gad.348934.121.

    abstract

    Mammalian 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 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 misregulation of genes in specific structures. Altogether, while most enhancer-promoter interactions can occur in the absence of this series of CTCF sites, the binding of CTCF in the 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 cluster, some acting as insulator elements, others being necessary to anchor or stabilize enhancer-promoter interactions, and some doing both, whereas they 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 among paralogous clusters or between various vertebrates.

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  • Hox13-Mediated Dbx2 Regulation In Limbs Suggests Inter-Tad Sharing Of Enhancers. Dev Dyn 2021 Jan;():. 10.1002/dvdy.303.

    abstract

    During 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.

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  • Chromatin topology and the timing of enhancer function at the locus. Proc Natl Acad Sci U S A 2020 Nov;():. 2015083117. 10.1073/pnas.2015083117.

    abstract

    The gene cluster is critical for proper limb formation in tetrapods. In the emerging limb buds, different subgroups of genes respond first to a proximal regulatory signal, then to a distal signal that organizes digits. These two regulations are exclusive from one another and emanate from two distinct topologically associating domains (TADs) flanking , both containing a range of appropriate enhancer sequences. The telomeric TAD (T-DOM) contains several enhancers active in presumptive forearm cells and is divided into two sub-TADs separated by a CTCF-rich boundary, which defines two regulatory submodules. To understand the importance of this particular regulatory topology to control gene transcription in time and space, we either deleted or inverted this sub-TAD boundary, eliminated the CTCF binding sites, or inverted the entire T-DOM to exchange the respective positions of the two sub-TADs. The effects of such perturbations on the transcriptional regulation of genes illustrate the requirement of this regulatory topology for the precise timing of gene activation. However, the spatial distribution of transcripts was eventually resumed, showing that the presence of enhancer sequences, rather than either their exact topology or a particular chromatin architecture, is the key factor. We also show that the affinity of enhancers to find their natural target genes can overcome the presence of both a strong TAD border and an unfavorable orientation of CTCF sites.

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  • Reorganisation of Hoxd regulatory landscapes during the evolution of a snake-like body plan. Elife 2016 ;5():. 10.7554/eLife.16087. PMC4969037.

    abstract

    Within land vertebrate species, snakes display extreme variations in their body plan, characterized by the absence of limbs and an elongated morphology. Such a particular interpretation of the basic vertebrate body architecture has often been associated with changes in the function or regulation of Hox genes. Here, we use an interspecies comparative approach to investigate different regulatory aspects at the snake HoxD locus. We report that, unlike in other vertebrates, snake mesoderm-specific enhancers are mostly located within the HoxD cluster itself rather than outside. In addition, despite both the absence of limbs and an altered Hoxd gene regulation in external genitalia, the limb-associated bimodal HoxD chromatin structure is maintained at the snake locus. Finally, we show that snake and mouse orthologous enhancer sequences can display distinct expression specificities. These results show that vertebrate morphological evolution likely involved extensive reorganisation at Hox loci, yet within a generally conserved regulatory framework.

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  • Convergent evolution of complex regulatory landscapes and pleiotropy at Hox loci. Science 2014 Nov;346(6212):1004-6. 346/6212/1004. 10.1126/science.1257493.

    abstract

    Hox 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.

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  • A genetic approach to the recruitment of PRC2 at the HoxD locus. PLoS Genet. 2013 Nov;9(11):e1003951. 10.1371/journal.pgen.1003951. PGENETICS-D-13-01888. PMC3820793.

    abstract

    Polycomb group (PcG) proteins are essential for the repression of key factors during early development. In Drosophila, the polycomb repressive complexes (PRC) associate with defined polycomb response DNA elements (PREs). In mammals, however, the mechanisms underlying polycomb recruitment at targeted loci are poorly understood. We have used an in vivo approach to identify DNA sequences of importance for the proper recruitment of polycomb proteins at the HoxD locus. We report that various genomic re-arrangements of the gene cluster do not strongly affect PRC2 recruitment and that relatively small polycomb interacting sequences appear necessary and sufficient to confer polycomb recognition and targeting to ectopic loci. In addition, a high GC content, while not sufficient to recruit PRC2, may help its local spreading. We discuss the importance of PRC2 recruitment over Hox gene clusters in embryonic stem cells, for their subsequent coordinated transcriptional activation during development.

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