Les récepteurs nucléaires, intégrateurs de signaux endogènes et environnementaux

Theme 1: Mechanisms of gene regulation by nuclear receptors

P Germain, A le Maire, W Bourguet

The overall goal of this theme is to use a combination of biochemical, structural, cellular, genomic and in vivo approaches to gain mechanistic insights into the general principles governing nuclear receptor action and regulation.

The precise regulation of gene expression requires activation and repression of DNA transcription and both activities appear critical for the biological functions of a number of receptors including the retinoic acid receptors (RARα, β, γ) that form functional heterodimers with retinoid X receptors (RXRα, β, γ). We previously discovered that the unliganded RARα and γ repress gene transcription via formation of a very specific interface with corepressors [le Maire, Nat Struct Mol Biol, 2010].

 

Team5 Fig1

Association and dissociation of coregulators from RAR upon ligand binding (CoA, coactivator ; CoR, corepressor).

More recently, we combined a large set of biophysical and computational methods to show that while the NR interaction domain (NID) of corepressors is mainly disordered, it presents transient but robust intramolecular contacts upon interaction with the heterodimer, indicating that disorder-to-order transitions of both receptors and corepressors are key events in the regulation of RXR–RAR heterodimers [Cordeiro, Structure, 2019] (coll. Team 2). A similar approach is currently being used to investigate the ligand-dependent interaction between the RAR–RXR heterodimer and the NID of the coactivator TIF2 [Senicourt, submitted, bioarchiv]. The fine understanding of these molecular processes at the atomic scale allowed us to insert a mutation into the RARα and γ sequences that specifically disrupts corepressor binding without affecting any other function of the receptors. RARAI396E and RARGI387E were subsequently introduced in mouse to probe the role of RAR-mediated gene silencing in vivo (coll. M Mark/N Ghyselinck). We are currently running genome-wide analyses of genes repressed by RARs through ChIP-Seq and RNA-Seq methods. The interaction sites of the corepressor SMRT in RAR target genes promoters in the absence of ligand or in the presence of various classes of retinoids were identified, as well as the associated epigenetic modifications of chromatin (ongoing work).

We are also interested by the structural and functional characterization of pathological forms of RAR. In particular, acute promyelocytic leukemias (APLs) arise from different chromosomal translocations that create x-RARα fusion proteins, including PML-RARα. Unlike RARα, these x-RARα proteins fail to release properly corepressors in response to ATRA and function as dominant-negative inhibitors of target gene expression and of myeloid cell differentiation. Despite important insights on PML-RARα (and to a lesser extent on other fusion proteins) functioning, the question still remains as to what are the crucial determinants for x-RARα proteins oncogenic activity. Using a set of structural methods, together with cell-based and biophysical assays, we expect to bring new insights into the molecular mechanisms of gene repression mediated by RAR in pathological situations.

Theme 1 also comprises the study of secondary coactivators that are part of coregulatory complexes but not directly bound to receptors. In particular, we studied the human mixed lineage leukemia 5 (MLL5), identified in a multi-subunit complex associated with RARα and thought to play a key role in hematopoiesis, spermatogenesis and cell cycle progression. Having provided compelling evidence that MLL5 is not a functioning enzyme despite its first identification as a member of the histone methyltransferases (HMT) family, we proposed that MLL5 functions as a mediator of gene regulation, not through an HMT activity, but as a structural component ensuring multi-protein complex integrity [Mas-y-Mas, PloS One, 2016].

We are also interested in the evolution of ligand binding activity/specificity in retinoid receptors [Handberg-Thorsager, Sci Adv, 2018 ; Gutierrez-Mazariegos, R Soc Open Sci, 2016 ; Gutierrez-Mazariegos, Endocrinology, 2014] (coll. V Laudet) and in the characterization of selective retinoids that can be used as investigation tools [le Maire, Cells, 2019]. Particularly, many studies have provided conflicting information on the role of RARβ in cancer development so that the availability of RARβ-selective ligands would allow dissecting the specific activities exerted by this receptor. By combining medicinal chemistry, cell biology and X-ray crystallography, we discovered the first full RARβ-selective agonist [Nadendla, PloS One, 2015], whose unexpected binding mode may, in addition, provide a comprehensive set of guidelines for the rational design of RARβ-selective antagonists.

Main collaborators: G Benoit (ENS Lyon), N Ghyselinck/M Mark (IGBMC Illkirch), P Veber (LBBE Lyon), V Laudet (OOB Banyuls/mer), Team 2 (CBS), H de Thé (Collège de France)

 

Connexion