Structural basis of nucleosome binding by PRC2 and its regulation by histone modifications

Finogenova, Ksenia (2021): Structural basis of nucleosome binding by PRC2 and its regulation by histone modifications. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Many histone modifications have chromatin modulating and gene regulating functions. Typically, the underlying mechanism involves 'writing' and/or 'reading' of the histone marks by chromatin regulating proteins. Alternatively, the histone marks interfere with these processes. These events ultimately result in an altered chromatin state. Polycomb Repressive Complex 2 (PRC2) is a histone methyltransferase that mono-, di- and trimethylates histone H3 at lysine 27 (H3K27). Among the different methylation states, H3K27me3 marks the chromatin of Polycomb-regulated genes for transcriptional repression by another complex, called PRC1. The correct regulation of H3K27me3 deposition has emerged as a critical step in both animals and plants as defects in this process are directly linked to developmental abnormalities or diseases such as cancer. The methyltransfer is catalyzed by the PRC2 subunit EZH2 at its SET domain, the stereochemical properties of which limit the rate of di- to trimethylation. As a consequence, to generate high levels of H3K27me3 at its target genes PRC2 requires positive regulation. Such regulation can be allostery through recognition of H3K27me3 by the PRC2 subunit EED, recruitment mechanisms or association with different accessory subunits. In contrast, mono- and dimethylation marks seem to be deposited by PRC2 in a more serendipitous manner. Examples of negative PRC2 regulation are histone modifications such H3K4me3 and H3K36me2/3, which are catalyzed by different Trithorax/COMPASS complexes. These marks are characteristic for active genes and inhibit PRC2 in cis but not in trans. However, the molecular details of how PRC2 interpretes these pre-existing H3 tail modifications on the context of a nucleosome remain unknown. This thesis employs single-particle cryo-electron microscopy for structural characterization of PRC2-PHF1 interaction with a heterodimeric dinucleosome, which contains one substrate nucleosome and one allosteric/H3K27me3-containing nucleosome. The obtained structure reveals that the CXC domain of PRC2 subunit EZH2 establishes binding to the nucleosomal DNA of the substrate nucleosome. A combined interface of EZH2 SBD/SANT1 domains and EED recognizes the nucleosomal DNA on the allosteric nucleosome. Mutational analysis of these binding sites suggest that these interactions establish the subsequent productive recognition of the respective H3 tails by the active site of EZH2 and the β-propeller of EED. Furthermore, signal subtraction and focused 3D refinement procedures applied during cryo-EM data processing allow to extend previous structural descriptions to a model of the H3 tail stretching from the substrate nucleosome into the EZH2 active site. Unmodified H3K36 is found sandwiched in between EZH2 and nucleosomal DNA in close proximity to the EZH2 CXC residues. Its ε-amino group is seemingly engaged in long range electrostatic interactions with the phosphate backbone of the nucleosomal DNA and in polar interactions with the carbonyl group of CXC residue Q570. Biochemical analyses showed that substitutions of H3K36 to either a shorter apolar alanine or a bulkier arginine side chain reduce the activity of PRC2, while binding of PRC2 to H3K36me3-nucleosomes is not affected. Taken together, results presented in this thesis suggest a model in which, within the time frame of PRC2 binding and reaction cycle, H3K36me2/3 hinders the subsequent optimal alignment of lysine 27 in the EZH2 active site. The bulkier quaternary ε-ammonium of H3K36me3, the positive charge of which is dispersed into the three additional methyl groups, thereby impedes but not entirely blocks PRC2 catalysis. Furthermore, it doesn’t prevent PRC2 from binding to the nucleosome. This model of allosteric inhibition allows for a targeted and tuned activity regulation of PRC2 in dependence of the surrounding chromatin and the presence/absence of other activity regulating mechanisms.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Polycomb, PRC2
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 8. März 2021
• 1. Bericht­erstatter:in: Conti, Elena