Recognition of nucleosomes by protein factors that regulate chromatin structure and dynamics

• Main Author: Lee, Phoi Leng
• Other Authors: Curtis Alexander Davey
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/142395
• Institution: Nanyang Technological University

One of the key players in genomic structure and dynamics is chromatin modification enzymes, which regulate epigenetic marks and alter chromatin activity. The first part of this thesis is focused on poly(ADP-ribose) polymerase 1 (PARP1) and sirtuin-6 (SIRT6), two chromatin modification enzymes that modulate distinct cellular pathways. Both proteins are involved in the DNA damage response and are crucial for upholding genome stability. PARP1 is a multipartite polymerase that modulates transcription, DNA damage repair and cell-death signaling and is an important cancer drug target since it plays a key role in maintaining genomic stability. The enzyme specifically recognizes single- and double-stranded DNA breaks, resulting in activation of the poly[ADP-ribos(e)]ylation (PARylation) activity towards self (PARP1) and other protein substrates. This automodification and PAR attachment to other nuclear factors evokes a cascade cellular response that leads to recruitment of additional DNA repair factors and chromatin remodeling machinery to the site of DNA damage. PARP1 is of further interest in chromatin studies because it is known to bind more strongly to nucleosomal substrates as compared to naked DNA. The SIRT6 protein is also involved in multiple signalling pathways, including DNA repair, telomere maintenance, glycolysis and inflammation. It is a histone deacetylase that is specific for K9 and K56 of H3. The first part of this thesis work was aimed at structural and functional characterization of PARP-nucleosome and SIRT6-nucleosome recognition. The original objective was to determine the structure of a PARP1-nucleosome complex by X-ray crystallography or electron microscopy (EM) and to solve the structure of a SIRT6-nucleosome core particle complex. Full length PARP1 and SIRT6 proteins were overexpressed and purified. The enzymes were subsequently used to generate homogenous assemblies with a variety of different nucleosomal constructs, which were subjected to both crystallographic screening and EM analysis. In SIRT6 co-crystallizations with nucleosome core particle, crystals were obtained under several different conditions, but these did not contain SIRT6 incorporated into the lattice. Additionally a number of different preparative approaches were taken to try to optimize EM grids and material integrity. Alternative nucleosome constructs and conditions were also explored in an attempt to improve both crystallization behavior and EM imaging resolution. Nonetheless, a recent publication indicates that PARP1 binding to the nucleosome in fact induces (partial) unfolding of the nucleosome core. This would mean that PARP1-nucleosome assemblies are likely highly dynamic and varied in structural nature, and as such would constitute highly challenging candidates for study by X-ray and EM approaches. Condensing nucleosomes into highly compact states coincides with an epigenetic mechanism in chromatin, which is required for gene regulation, maintaining genomic integrity and generating mitotic chromosomes for cell division. Chromatin compaction is facilitated by nucleosome association of linker histones, which are essential proteins in higher eukaryotes. However, in spite of its importance for genomic function, the biological relevance of different types of condensed nucleosomal structures observed in vitro is unclear, and we lack a unified understanding of how linker histones bind to nucleosomes and promote compaction in the cell. Chromatin’s structural characterization is complicated by its heterogeneous composition in the cell and the conformational disorder of nucleosome assemblies in vitro. One approach is to explore methods that would allow one to elucidate crystal structures of assemblies with differing lengths of linker DNA connecting the nucleosome core regions. Yet, as with the nucleosome core particle, the challenge is finding constructs that yield well diffracting crystals. To overcome this obstacle, we developed an approach that promotes ordered lattice formation, which appears to have general utility for chromatin structural studies. In the second part of this thesis, X-ray structures of novel nucleosome-linker histone assemblies based on engineered nucleosomal constructs are described. The structures reveal different nucleosome packing configurations and linker histone binding modes and demonstrate that a slight alteration in the linker DNA length can result in a dramatically distinct configuration. We propose that different nucleosome packing configurations in crystals obtained by varying linker DNA length could serve as a platform for both rationalizing and understanding chromatin structural heterogeneity in the cell.