Défense de thèse de doctorat en sciences biologiques "RNA Polymerase II"

RNA Polymerase II Carboxy-Terminal Domain Serine 2 phosphorylation: a role in Nucleosome Depleted Regions establishment on specific genes

Jury

Karl EKWALL (Karolinska Institute, Suède), Karine VAN DONINCK (UNamur),  Eric DEPIEREUX, président (UNamur), Xavier DE BOLLE (UNamur), Damien HERMAND, promoteur (UNamur)

Résumé

 

The RNA Polymerase II is a macromolecular complex composed of 12 subunits. Its biggest subunit, Rpb1, exhibits a peculiar domain on its carboxy-terminal region named CTD. It is composed of the highly conserved repeat of the heptapeptid Y₁S₂P₃T₄S₅P₆S₇ repeated 26, 29 and 52 times in S. cerevisiae, S. pombe and mammals respectively. The CTD follows a pattern of phosphorylation-dephosphorylation in the course of gene transcription. The serine in position 5 (of every heptapeptid repetition) is phosphorylated (S5P) close to the promoter region while the serine in position 2 is phosphorylated (S2P) next to the 3’ of transcribed regions. This phosphorylation combination both reinforces and restricts the recruitment of proteins required for mRNA maturation. Indeed, under certain states of phosphorylation, the CTD serves as a docking platform for capping and splicing enzymes or poly-adenylation factors. It was also later shown that the phosphorylated CTD recruits chromatin modifiers.

Transcription occurs through nucleosomes, the base unit of chromatin. One nucleosome is formed of a heterodimer of histones H2A and H2B and a tetramer of histones H3 and H4 surrounded by 147 base pairs of DNA. The histones can be moved, replaced, evicted or modified in a covalent way. Many enzymes are involved in such active processes. The nucleosome occupancy is not homogeneous across the genome. Some regions are depleted in nucleosomes and hence called Nucleosome Depleted Region (NDR). Their formation is an ATP-consuming active process and requires chromatin remodelers. Those NDRs are often located in genes promoter regions and make the DNA sequence accessible to initiate transcription. Their presence/absence is thus an important layer of transcriptional regulation.

Surprisingly, in S. pombe, a subset of genes shows an unexpected pattern of CTD phosphorylation: S2P reaches high level in 5' regions where it is usually low. Strikingly, the complete absence of S2P has only a minor impact on genome expression. However, S2P is critical for sexual differentiation in fission yeast. Indeed, in a S2A mutant (where every serine in position 2 of the CTD are replaced by alanine) or in a S2 kinase mutant, the ste11 expression (master regulator of sexual differentiation) is severely impaired making those strains sterile. This lowered expression correlates with a severe drop in Pol II occupancy along the ste11 locus.

Here we show by a genome-wide nucleosome mapping study that S2P is required in S. pombe for the proper establishment of NDR in promoter region in a subset of genes with early S2P. We investigated the mechanical of this process through two model genes in S. pombe: ste11 and inv1 (model gene for sugar metabolism). We also broadened the scope of this work with another model gene in a different model organism: IME1 (key regulator of meiosis) in S. cerevisiae.

We propose the following model of a new kind of transcriptional regulation: S2P prevents the binding of histone deacetylases in the promoter region of genes with early S2P by interfering with S5P. It results in hyperacetylated histones in the promoter region allowing the recruitment of histone remodeling complexes promoting NDR formation whence potentiating chromatin for gene expression.

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