Publication

Dynamic protein-chromatin interactions in heterochromatin and during the DNA damage response on the single molecule level

Andreas Linus Bachmann
2018
Thèse EPFL
Résumé

Dynamic regulation of chromatin, a structure consisting of DNA and histone proteins, is mediated through histone post-translational modifications (PTMs) and effector proteins. HP1a and PRC2 are recruited to repressive PTMs (H3K9me3, H3K27me3) and enable heterochromatin formation, which leads to gene silencing. During the DNA damage response, tight spatial and temporal control of the chromatin compaction state is required and novel PTMs are installed to guide proteins through dynamic signalling pathways. In the RNF168-mediated signalling cascade, appropriate DNA repair mechanisms are selected through ubiquitination (H2AK15ub) of chromatin. Improper regulation of these processes leads to cancer and neurodegenerative diseases. Here, we present a method to study chromatin-protein interactions employing total internal reflection fluorescence microscopy (TIRFM) and in vitro chromatin assembly of chemically defined, modified histones. Colocalization experiments of fluorescently labelled chromatin fibres and proteins allowed us to observe dynamic chromatin-protein interactions on the single-molecule level. We tested different binding models of HP1a, and we measured a highly dynamic behaviour of HP1a towards chromatin in dependence of the available binding sites or HP1a concentration. HP1a dimerization allows multivalent binding, increased chromatin association and decreased dissociation and thus leads to a more stable mode of chromatin binding. Further, we studied PRC2 recruitment to chromatin and DNA in dependence of the accessory factor PHF1. We measured increased residence times of PRC2 when PHF1 was included. Structure prediction and mutagenesis studies identified a novel winged-helix (WH) motif of PHF1, which was responsible for the enhanced binding effect in PRC2-PHF1 measurements. Moreover, binding of PRC2 towards chromatin was prolonged in the presence of H3K27me3, H3K27M or H3K36me3 histone PTMs. Next, we characterized the chromatin writer RNF168. We show altered ubiquitination activities dependent on accessibility of the nucleosome acidic patch, which was tested by addition of a competitor (RAPTA-C) or by introducing H4K16 acetylation, which leads to an open chromatin conformation. RNF168 is a multivalent reader of ubiquitin marks and we show binding specificity of ubiquitin binding module 1 (UDM1) towards native and synthetic K63-linked ubiquitin chains, while no interaction with K48-linked ubiquitin chains was detected. In contrast, we could not determine a clear contribution of the second binding module (UDM2), which recognizes H2A.XK15ub. In summary, we gained insight into different binding modes of HP1a, PRC2 and RNF168. Understanding basal protein binding mechanisms and chromatin regulation is essential to find new drug targets.

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Concepts associés (33)
Remodelage de la chromatine
Le remodelage de la chromatine est l'un des trois mécanismes de modification de la structure de la chromatine. Intervenant d'abord au cours de l'étape de maturation de la chromatine, il permet l'obtention d'un certain état final de sa structure. Plusieurs protéines sont impliquées dans ce processus de remodelage (familles SW1/SNF, ISW1, INO, CHD). Ces protéines, appelées facteurs de remodelage de la chromatine, forment des complexes multi-protéiques et utilisent l'énergie libérée par l'hydrolyse de l'ATP pour induire des changements conformationnels au niveau du nucléosome et des domaines de la chromatine.
Chromatine
alt=Compaction de l'ADN dans la chromatine|vignette|upright=1.9|Compaction de l'ADN au sein de la chromatine. De gauche à droite : l'ADN, le nucléosome, le nucléofilament, la fibre de 30 nm et le chromosome métaphasique. La chromatine est la structure au sein de laquelle l'ADN se trouve empaqueté et compacté dans le volume limité du noyau des cellules eucaryotes. La chromatine est constituée d'une association d'ADN, d'ARN et de protéines de deux types : histones et non-histones. C'est le constituant principal des chromosomes eucaryotes.
Ubiquitine
L'ubiquitine est une protéine de 76 acides aminés servant, elle-même, de marqueur de protéines à éliminer. Elle est ainsi appelée parce qu'elle est localisée dans tous les compartiments subcellulaires de toutes les cellules des organismes, elle est dite ubiquitaire. L'ubiquitination désigne la fixation (covalente, ATP dépendante grâce à une cascade d'enzymes E1, E2, E3) spécifique et régulée d'une ou plusieurs ubiquitines sur une protéine cible (il faut quatre ubiquitines pour qu'une protéine soit dégradée).
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