DS0401 -

The Mediator complex : from a protein-protein interaction network to gene regulation in vivo – MEDNET

Submission summary

Transcription initiation by RNA polymerase II (PolII) requires many protein-protein interactions, whose roles in vivo have been poorly investigated. One key element in this process is Mediator (MED), a conserved multisubunit complex serving as a physical as well as functional interface between DNA-bound transcription factors (TFs) and the PolII machinery.

Despite a general role of Mediator in controlling transcriptional activation or repression, some MED subunits (SUs) display strikingly-specific functions, as evidenced by human diseases associated with their dysfunctions. For example, Med13 point mutations have been associated with intellectual disabilities, while Med19 is over-expressed in a majority of cancers. Even though MED SU specificity comes in part from their ability to interact with dedicated TFs (Boube et al., 2014; Verger et al., 2013), their molecular specificity and their functional in vivo roles in tissue-specific gene regulation, remains poorly understood.

Another black-box in the transcription field lies in the regulation of MED assembly, notably of “CKM” module anchoring. This separable regulatory MED subcomplex, composed of Med12, Med13 and a Cdk8-CycC pair, whose association with core Mediator induces gene repression, has been more recently involved in gene activation as well. Determining the molecular links between cMED and CKM is thus crucial to better understand its regulatory role.
Importantly, the Med19 SU, located at the core MED tip, is thought to anchor CKM via direct interaction with Med13. In addition, Drosophila Med19 acts as a functional partner of two GATA TFs through direct interaction with their zinc-finger domain, as well as of HOX TFs, directly binding their DNA-binding homeodomain. Thus Med19 seems unique in its ability to serve as a molecular hub within Mediator for specific TFs on the one hand and for CKM on the other. However, direct partnerships remain to be molecularly characterized and their functional relevance to be tested in vivo.

The present project proposes to combine Bourbon’s (Partner#1) and Villeret‘s (Partner#2) interests for the MED complex and to join their long-standing respective expertise in developmental genetics and structural biology to unravel its mechanisms of action. We propose to perform an in depth structure–function analysis of one key MED SU, Med19, starting from the characterization of structural determinants involved in specific partnerships to a detailed analysis of their functional relevance in a living animal.

The project will address three issues:

(1) Deciphering Med19 structural determinants for direct interaction with GATA TFs and CKM, using in vitro (Partner#2) and in-vivo (Partner#1) assays, as well as structural analyses at the atomic resolution (Crystallography or NMR – Partner#2).
(2) Dissecting the regulatory roles of Med19 molecular partnerships in gene expression in vivo. Taking advantage of the newly-developed CRISPR/Cas9 technique in Drosophila to specifically mutate molecular determinants involved in Med19 and Med13 partnerships, we will analyze their functional consequences, through phenotypic, transcriptomic and chromatin immunoprecipitation analyses (Partner#1).
(3) Exploring the conservation of Med19 partnerships and their relevance to human diseases. To this end, we will (i) examine conserved Med19 interactions with GATA TFs and CKM and when appropriate, infer from Drosophila the 3D structures of human complexes (Partner#2), (ii) shed to light new Med19-TF partnerships in both species (Partner#1), and (iii) check the potential involvement of these partnerships in human pathologies.

The originality of our project results from multi-disciplinary experimental approaches combining structural studies at the atomic level with in vivo genome-wide analyses. This project should provide huge knowledge regarding the functional specificity of particularly relevant MED SUs and will help to understand their links with human diseases.

Project coordination

Muriel Boube (Centre National de la Recherche Scientifique/Centre de Biologie du Développement)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRS/UMR5547 Centre National de la Recherche Scientifique/Centre de Biologie du Développement
CNRS/UMR8576 Centre National de la Recherche Scientifique/Unité de Glycobiologie structurale et fonctionnelle

Help of the ANR 457,585 euros
Beginning and duration of the scientific project: January 2017 - 48 Months

Useful links

Explorez notre base de projets financés

 

 

ANR makes available its datasets on funded projects, click here to find more.

Sign up for the latest news:
Subscribe to our newsletter