Contribution of non-histone proteins to chromatin structure and stability
Browne, Martin J. G.
MetadataShow full item record
This item's downloads: 83 (view details)
Histones are responsible for packaging the genomes of almost all eukaryotes into fundamental repeating nucleosome units. The packaging must facilitate compaction into the cell nucleus but also enable dynamic access to the genome. A variety of mechanisms exist for targeting enzymes to undertake local opening of chromatin such as at active genes or for DNA repair. However, larger scale transitions in chromatin also occur where extended genome regions have altered chromatin organisation. This often involves abundant non-histone chromatin proteins that switch chromatin between states that are not well understood at the structural level. The contribution of highly basic non-histone chromatin proteins in vitro has been investigated using the HMGA2 protein implicated in human stem cell chromatin opening, and the Hematodinium DVNP protein which is suggested to replace histones as the dominant packaging protein in this dinoflagellate. These two proteins are compared to histone H1 which stabilises nucleosome structure. Considerable technical difficulties were overcome to establish a strategy for production of HMGA2 without nucleic acid contamination. HMGA2 binding to free DNA and nucleosome substrates is investigated in initial attempts to determine whether this is cooperative and how it affects nucleosome structure using electrophoretic mobility and microscale thermophoresis assays. Separately, genes for the four Hematodinium core histones were designed and used for recombinant expression. They behave aberrantly in histone complex formation, possibly due to extended H2A and H4 tails, but can be assembled into nucleosome-like structures. Nucleosomes were mixed in vitro with the Hematodinium DNA binding protein DVNP, which appears to subsume the nucleosomal DNA packaging role in vivo, and the cooperative binding effects were investigated. Together, this work suggests a mechanism by which highly basic non-histone chromosomal proteins can interact in a non-specific manner and by mass action lead to displacement or modification of nucleosome organisation to generate alternative genome packaging. In addition isotope labeled recombinant histone H2AX was purified for use in quantitative mass spectrometry and quantitative western blotting to determine the abundance in a selection of cell lines that are important in studying DNA damage response.
This item is available under the Attribution-NonCommercial-NoDerivs 3.0 Ireland. No item may be reproduced for commercial purposes. Please refer to the publisher's URL where this is made available, or to notes contained in the item itself. Other terms may apply.
The following license files are associated with this item: