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Topological organization of vertebrate regulatory landscapes: The Hox genes paradigm

Periodic Reporting for period 4 - RegulHox (Topological organization of vertebrate regulatory landscapes: The Hox genes paradigm)

Reporting period: 2020-03-01 to 2021-02-28

Hox genes code for an important family of transcription factors, which organise structures during animal development. In mammals, they are grouped into four different genomic clusters and these genes are active in many different structures, at various times. For example, they are necessary to organise the vertebral column early on, but also the intestin and the uro-genital apparatus -for example- subsequently.

How is this possible? Which are the regulatory mechanisms that can control these genes faithfully, yet in different contexts and at different times? And how did these mechanisms evolve from our invertebrate ancestors? These are the main question that this grant application wants to answer, i.e. what is the precise nature of the regulatory choreography that determine when and where which Hox gene(s) is (are) either active or silent.

Besides the importance of this work as a paradigm for gene regulation, it also impacts the society in two different aspects. First, more and more genetic diseases turn out not to be due to problems in the genes themselves but instead in their regulatory sequences. This is the case with Hox genes and many syndromes affecting their regulations have been described lately. Secondly, the precise understanding of these complex regulatory circuits will shed light on the multiple effects that such diseases often elicit ('syndromes'). This is of course due to the intrication of their regulations and hence it is important to understand how this works and consequently, how this was built over evolution.
We decided to take a global approach to characterise all the enhancers regulating one particular subset of Hox genes (the HoxD cluster), in multiple tissues at different times. We then tried to associate these regulatory sequences to particular topological structures in the chromatin (i.e. how can the supra structure of the chromatin influence the activity of these enhancers?) and several publications have been produced in this context.

We have shown that the global structure of the chromatin does not change much from one tissue to another, but that small internal modifications were scored, as if various regulations are implemented in the frame of the same structure (a regulatory landscape). Also, we reported that systems have evolved to prevent particular regulations to act over some Hox genes as they would be detrimental, a case reported in some human syndromes.

To understand in details these complex chains of regulations, we have started an important programme of genome editing, to remove all these enhancers in isolation by using the CRISPR/cas9 technology. Several mouse lines have been obtained and have been analysed. We have also used the same approach to remove several of the 'blocking sequences' to see their effect upon gene regulation and these mice have also been characterised.

To try and facilitate these approches, we have started to produce gastruloids, i.e. artificial mouse 'embryos', a novel model system that should in principle speed up the processes and allow to carry out biochemical analyses in a more efficient way than by using the mouse embryo. The ERC funding has allowed us to fully develop this system for our own needs and we have almost totally given up using living mice to address these questions. This system looked so promising that it has changed the composition of my laboratory, which is now fully engaged into this 3R-compliant technology.
In this first part of the ERC granting period, we have been able to progress much faster and to produce most of the mutant stocks we can now analyse. The regulatory landscape of the HoxD cluster has been modified to an extent that has no equivalent in the literature and the next two years will be devoted to the analyses of these various genomic situations, which should give us critical informations as to how complex regulatory circuits are implemented during mammalian development.

In the final part of this project, we were able to analyse all these stocks and reach conclusions on almost all the sub-projects we had started. More than 20 publications were produced, amongst which some that truly terminate very long-term projects, such as for example the mechanistic explanation to the mesomelic dysplasia syndromes, a genetic disease affecting human families and involving regulatory re-allocations at the HoxD gene cluster.
Expression of a Hox gene in two embryos and corresponding pseudo-embryos in culture