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Summary-Heading
In 2008, Françoise Gofflot joined The Institute of Life Science, now LIBST, with a "Mandat d’impulsion Ulysse (FNRS)" to establish the TeamGofflot in close partnership with colleagues of what is now the AMCB group. Building upon a strong background in developmental biology, mainly centered on neurodevelopment, her research interest is focused on deciphering the functions of HOX transcription factors in late aspects of the central nervous system development, and the impact of their deregulation on neurological disorders.
To tackle these questions, the team currently focus on HOXA5. Five years ago, they were the first to demonstrate that the HOXA5 protein is present and functional in the mouse brain during the first postnatal weeks, where it regulates key players involved in synapse function. They are currently pursuing the functional characterization of HOXA5 in post-mitotic neurons, using in vivo and in vitro models, but they are also addressing the question of HOXA5 modes of action in post-mitotic neurons.
The PhD students currently working at the lab are Amandine Vandenberghe (FRIA fellow) and Hadrien Glibert (Teaching assistant, UCLouvain). We benefit from the expertise of Laure Bridoux, post-doctoral fellow (“Chargée de recherches”, FRS-FNRS ), “shared” with my colleague René Rezsohazy.
Marie-Thérèse Ahn and Coralie Piget, technicians, members of the AMCB and ANCA staff are also active members of the team.
In 2022-2023, we welcome two master students in the team, Victoria Lievens and Lucile Wittock.
And all of our research are in close collaboration with the other members of the AMCB group
Research interests
Deciphering new Hox gene functions in the postnatal and adult brain
Hox genes define a subset of the homeobox gene family coding for homeodomain transcription factors involved in the embryogenesis, morphogenesis and organogenesis of bilaterian embryos. Although their expression and functions within the embryo are well established, it has appeared more recently that Hox genes are expressed at later stages of development as well as in several adult tissues. At the origin of this project, we established a quantitative and qualitative atlas of the 39 Hox genes expression in the postnatal mouse brain. We confirmed and extended previous data by showing that expression of several Hox genes is still present in the mouse brain at postnatal stages and is maintained until adulthood. However, the functional importance of HOX presence after birth remained to be determined. In this context, we selected Hoxa5 and developed projects addressing its functions in the postnatal brain and its potential involvement in diseases.
Five years ago, we could demonstrate that the HOXA5 protein is indeed present and functional in the brain during the first postnatal weeks, where it regulates key players involved in synapse function (Figure 1). Building upon the Hoxa5 expression profile in the brainstem, we hypothesized that HOXA5 regulates critical steps of the formation and maturation of the pre-cerebellar circuits that project to the cerebellum and thus contributes to the function of this important brain structure. Moreover, HOXA5 could be an important regulator of pathways altered in synaptopathies (synapse dysfunctions) such as autism spectrum disorders (ASD). We were able to extend our observations to other HOX proteins, and suggest that several of these transcription integrate into gene regulatory networks that are involved in late processes of central nervous system development, including the assembly and maturation of synapses.
Figure1 - Schematic representations of a glutamatergic synapse allowing to visualize the localization HOXA5 targets (blue and green circles) identified by transcriptome analysis of P21 brainstem. Adapted from KEGG pathway database.
Current research projects: HOXA5 functions and modes of action in neurons
To test these hypotheses, we are exploring the function of HOXA5 in the postnatal brain using an original mouse model inactivated for Hoxa5 just after birth (Hoxa5 conditional knock-out or cKO)(Figure 2A). Our recent data have shown that these Hoxa5 cKO mice do not show deficits in brain function related to the Hoxa5 expression territory. In contrast, these mutant mice show deficits in autism-related behaviors, such as impaired social interaction and increased repetitive and stereotyped behaviors, reinforcing the hypothesis of a correlation between HOXA5 and ASD.
To further decipher the role of HOXA5 in neuronal circuit formation and synaptogenesis we also explore HOXA5 impact on synapse formation, maturation and function using in vitro assays. Indeed, culture of neurons enables the conversion of complex three-dimensional brain tissue, difficult to study in vivo, into a two-dimensional monolayer of cells, allowing easier access and visualization of individual neurons and synapses. Two assays, involving either primary culture of neurons or culture of cell lines differentiated into neurons are currently being validated in the lab (Figure 2B). Using these models, we aim to evaluate synaptogenesis, and to analyze the morphological, molecular and physiological characteristics of synapses in presence and absence of HOXA5. We also aimed at improving our knowledge of the molecular networks controlled by HOXA5 in synaptogenesis.
Figure2 – A. In vivo model of Hoxa5-induced postnatal loss-of-function. Using the Tamoxifen-inducible CreERT2 – LoxP system, Hoxa5 is inactivated at early postnatal stages. B. In vitro model: Hindbrain-derived primary neurons (identified by MAP2 expression in green) express HOXA5 (in red).
To fully apprehend HOXA5 functions in the late processes of central nervous system development, it is also necessary to address the question of its regulation and modes of action, and to consider the protein, its stability and its molecular interactions. Through a Y2H screening and database mining, we identified 12 HOXA5 candidate interactants, which are under investigation. Notably, we identified a nuclear import receptor (importin) as an HOXA5 interactant that could serve its nuclear import, and we are currently characterizing their interaction domains and the importin impact on HOXA5 cellular localization. Preliminary data also support that HOXA5 has a rather long half-life ant that its stability may be controlled by interacting ubiquitin ligases. Using two methods, coprecipitation (CoP) and Bimolecular Fluorescence Complementation (BIFC), our preliminary data reveal interactions between HOXA5 and some Ubiquitin Conjugating Enzymes and a member of the E3 ubiquitin ligase complex. This can be correlated with other assays showing that HOXA5 is indeed ubiquitinated.
We are also investigating the impact of other partners on HOXA5 activity at the chromatin as transcription factor. Among our 12 candidates, three transcription factors hold the attention from the SMAD, SOX and TSHZ family. The interaction between HOXA5 and these transcription factors was next confirmed in our laboratory, by CoP and BIFC. We also confirmed that the three candidates are expressed in cells and brain regions expressing Hoxa5, strengthening their physiological relevance. To further analysed these interactions at the chromatin, we first need to identify regulatory sites (enhancers, silencers and promoters) common to HOXA5 and these proteins. As these data are not available, we aim to identify and characterize HOXA5 DNA-binding sites by chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-Seq), in a neuronal cellular context.
Publications from the lab
1. Bridoux, Laure, Gofflot, Françoise, & Rezsohazy, René (2021). HOX Protein Activity Regulation by Cellular Localization. Journal of Developmental Biology, 9, 56. doi :10.3390/jdb9040056
2. Gofflot, Françoise, Jeannotte Lucie, & Rezsohazy, René (2018). A scientific journey in the garden of the Hox genes: an interview with Jacqueline Deschamps. . The International Journal of Developmental Biology, 62, 665-671. doi :10.1387/ijdb.180305rr
3. Gofflot, Françoise, Jeannotte Lucie, & Rezsohazy, René (2018). Hox genes: past, present and future of master regulator genes. Four decades of Hox gene investigation and many more to go. The International Journal of Developmental Biology, 62, 653-657. doi :10.1387/ijdb.180332fg
4. Gofflot, Françoise, & Lizen Benoît (2018). Emerging roles for Hox proteins in synaptogenesis. The International Journal of Developmental Biology, 62, 807-818. doi :10.1387.ijdb.180299fg
6. Lizen, Benoît, Moens, Charlotte, Mouheiche, Jinane, Sacré, Thomas, Ahn, Marie-Thérèse, Jeannotte, Lucie, Salti, Ahmad, & Gofflot, Françoise (2017). Conditional loss of Hoxa5 function early after birth impacts on expression of genes with synaptic function. Frontiers in Molecular Neuroscience, 10, 1-15. doi :10.3389/ fnmol.2017.00369
7. Lizen, Benoît, Hutlet, Bertrand, Bissen, Diane, Sauvegarde, Deborah, Hermant, Maryse, Ahn, Marie- Thérèse, & Gofflot, Françoise (2017). HOXA5 localization in postnatal and adult mouse brain is suggestive of regulatory roles in postmitotic neurons. The Journal of Comparative Neurology, 525(5), 1155-1175. doi :10.1002/cne.24123
8. Hutlet, Bertrand, Theys, Nicolas, Coste, Cécile, Ahn, Marie-Thérèse, Doshishti-Agolli, Konstantin, Lizen, Benoît, & Gofflot, Françoise (2016). Systematic expression analysis of Hox genes at adulthood reveals novel patterns in the central nervous system. Brain Structure and Function (Print Edition), 221, 1223-1243. doi :10.1007/s00429-014-0965-8
9. Lizen, Benoît, Claus, Mélissa, Jeannotte, Lucie, Rijli , F.M., & Gofflot, Françoise (2015). Perinatal induction of Cre recombination with tamoxifen. Transgenic Research, 24, 1065-1077. doi :10.1007/ s11248-015-9905-5