Towards X-ray crystallographic characterization of nucleosome interactions with linker histones, FOXA proteins and high mobility group N factors

• Main Author: Ravi, Sailatha
• Other Authors: Curtis Alexander Davey
• Format: Theses and Dissertations
• Language: English
• Published: 2018
• Subjects: DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/74380
• Institution: Nanyang Technological University

In the eukaryotic cell nucleus, genetic material is packaged into chromatin, consisting of arrays of nucleosomes that can be densely compacted. The three-dimensional organization of chromatin imparts a sophisticated temporally and spatially controlled regulation of accessibility to the genes. Globally, the chromatin landscape is compartmentalized into chromatin territories and further into topologically active domains. Locally, gene regulation events can be triggered by various factors including interactions of enhancer/silencer with promoter regions, role of chromatin architectural proteins that enhances gene accessibility. Histone and non-histone chromatin architectural proteins can recognize structural features of nucleosomes, which are the fundamental units of chromatin. In general, the binding of linker histone proteins to the linker DNA between nucleosomes facilitates the condensation of chromatin into higher order structures. Other chromatin architectural proteins, such as those in the FOXA and High Mobility Group N [HMGN] families, can interact with nucleosomes and help to maintain open and active chromatin states by competing with linker histone activities. Studying such interactions could provide a new perspective for understanding chromatin structure and activity, as well as shed light on the bases of developmental defects and cancer-related disorders. X-ray crystallography has provided a powerful platform to study biological macromolecules in great atomic detail. In this dissertation work, the major aim was to utilize X-ray crystallography to study the nucleosomal interactions of chromatin-interacting factors, to address the two opposing aspects of chromatin regulation. The Fox family of proteins with a conserved winged-helix DNA-binding domain like the globular domain of the linker histones, recognizes a signature DNA sequence with an off-dyad position on the nucleosome. Owing to its highly unstable nature, recombinant bacterial expression and purification of full-length human FOXA proteins has proven especially challenging. We pursued random truncations and have purified a library of FOXA1 truncated protein constructs that could serve as controls for biochemical characterization. We have addressed the soluble purification of full-length FOXA1 using a maltose binding protein (MBP)-tag. Alternatively, we have purified a shorter, functionally relevant construct, lacking the N-terminus. Both constructs demonstrated sequence dependent binding to cognate FOXA1-DNA. However, in contrast to previous work, stable binding to nucleosomes was not apparent, thereby warranting further optimization for in vitro binding studies. Members of the HMGN subfamily of proteins, capable of dynamic interactions with chromatin, display a cooperative two-to-one stoichiometry of binding to the acidic patch of the nucleosome. Despite the highly conserved nucleosome binding domain between variants, under physiological conditions, the two HMGN molecules associated with a given nucleosome are always of the same variant type. We have optimized and purified all but one of the human HMGN variants in large quantity using recombinant bacterial expression systems. We have screened conditions to crystallize the complex of HMGN1 or HMGN2 with nucleosomes and obtained crystals that diffracted X-rays up to 7 Å resolution. Optimization of crystallization and crystal stabilization conditions could further improve the diffraction quality. Different linker histone variants display distinct gene-modulating activities and localization on chromatin. Given our recent success in our lab solving the structure of an H1x-nucleosome assembly [Adhireksan et al., unpublished data], for this dissertation work we purified the human H1.5, H1t, H1T2 and HILS linker histone variants and crystallized the four nucleosome-linker histone assemblies. X-ray diffraction data sets for the different complexes range from 3.0 to 3.3 Å resolution. Although in some cases electron density maps indicate clearly the presence of bound linker histone, the degree of disorder in the linker histones is substantial. We propose that further experiments utilizing heavy atom labeling and phasing approaches should enable the solving of atomic models for these four different nucleosome-linker histone assemblies. This could reveal novel aspects of variant-specific linker histone structure and activity. Here we have focused largely on developing structural biology platforms that could aid in the characterization of nucleosome-protein interactions with atomic detail. The above projects provide useful insights into purification of highly disordered human proteins, nucleosome assembly crystallization strategies, techniques to improve diffraction and strategies to address the challenges involved in solving large protein-DNA complex structures.