Biophysical and structural studies of aggregation of nucleosome core particle with charge modified histone proteins.

• Main Author: Liu, Ying.
• Other Authors: Lars Nordenskiold
• Format: Theses and Dissertations
• Language: English
• Published: 2012
• Subjects: DRNTU::Science::Biological sciences
• Online Access: http://hdl.handle.net/10356/50715
• Institution: Nanyang Technological University

The core histone proteins H2A, H2B, H3 and H4 constitute the spool for the DNA hierarchical packaging in eukaryotes and form the basic chromatin structural unit of the nucleosome core particle (NCP). The positive charges on the histone H4 N-terminal tails extensively interact with DNA through electrostatic interactions, and with the H2A-H2B dimer of adjacent NCPs through structural specific interactions. Therefore, reduction of the H4 tail positive and the H2A core negative charges through acetylation or mutations would interrupt DNA-histone or inter-nucleosome associations. Such processes resemble the opening up of chromatin fiber structure that consequently leads to increased DNA accessibility and active gene transcription. In this work the biophysical properties of cation induced aggregation of NCPs containing various charge mutated histone proteins were studied. Five inducing agents including divalent cations Mg2+ and Ca2+, trivalent cation CoHex3+ and natural polyamines Spd3+ and Spm4+ were used. The positive charges displayed by K5, K8, K12 and K16 on the H4 tail were neutralized individually or together, by KQ mutations or acetylation. The aggregation potency of NCPs containing such modified histones was studied with precipitation assay and static/dynamic light scattering methods. In addition, the ‘acidic patch’ of the H2A globular domain consisting mainly D90, E91 and E92 were neutralized by DS and ET mutations. The structural feature of the NCP aggregates containing modified H4 or H2A was studied with small angle X-ray scattering. Preliminary investigations were also carried out with a 12-mer nucleosome array containing both modified H4 and H2A with analytical ultracentrifugation to understand the inter-nucleosome interactions and the effect of these charges on the chromatin condensation. The results suggest that the aggregation capacity of NCPs, with partial neutralizations on the H4 tails, significantly decreases as compared to the native NCP. The effect of acetylation is more recognizable than the KQ mutation. Among all the NCP variants, the tetra-acetylated NCP produces the most pronounced effect. Furthermore, structural studies confirm an overall columnar-hexagonal phase of the aggregated NCP phase. The long-range columnar NCP-NCP stacking and the inter-column association were distorted in the NCP aggregates containing charge modifications. The findings altogether imply that these charges are crucial; and removal of the charges significantly interrupts inter-nucleosome interactions and influences the supramolecular structures resembling higher order chromatin packaging. Moreover, the preliminary studies with the double modified 12-mer nucleosome array also suggest an additive effect of the charged residues on the H4 tail and H2A core.