Abstract
Chromatin was one of the first sub-cellular structures to be described in early microscopy studies (Heitz 1928). Chromatin is formed from DNA, RNA, histones and other associated proteins. Within the nucleus, chromatin is arranged as compacted heterochromatin and decompacted euchromatin. During cell division, interphase chromatin compacts to form the mitotic chromosomes. The arrangement of chromatin within cells, with respect to its compaction state, has a number of implications for processes like transcription, DNA replication, DNA repair and more recently, in vision of nocturnal animals.Most work on the study of chromatin compaction has been done with in vitro assembled nucleosome arrays. Chromatin has been shown to compact when the concentration of polyvalent cations increases, possibly due to negation of the repulsive forces of the negatively charged poly-phosphate DNA backbone. However, these in vitro studies do not necessarily reflect the in vivo chromatin environment. An example of this difference can be seen in the formation of the 30nm fibre in nucleosome arrays, which has not been found within mammalian nuclei, where it adopts a ‘beads on a string’ structure, even in more compact regions.
The work in this thesis studies chromatin compaction in mammalian cells using three different approaches. In the first part, I have performed experiments to study charge-based compaction of chromatin in intact nuclei. Studies on permeabilised cells indicate that compaction of chromatin increases with an increase in concentration of polyvalent cations. This increase in chromatin compaction is charge and concentration dependent. The results show that the concentration required for maximal compaction in cells is similar to that required for nucleosome arrays assembled in vitro. Increasing the free intracellular concentration of Ca2+ also led to compaction of chromatin in intact cells.
In the second part of the thesis, I use ATP depletion, in vivo, as a model system to induce chromatin compaction in HeLa cells. Either inhibition of transcription, or inhibition of kinases, does not prevent this increase of chromatin compaction caused by ATP depletion. The ATP dependent change in compaction is also seen in mitotic chromosomes. There is a difference in charge distribution within the cell on ATP depletion, with an increase in Ca2+ and changes in localisation of spermine4+, which could explain the increase in chromatin compaction.
In the third and final results section, I used a quantitative proteomics approach to systematically identify changes in the abundance of chromatin-associated proteins in HeLa cells, when chromatin is decompacted by Trichostatin A (TSA) treatment. TSA causes increased acetylation of lysine residues in histones, which leads to a loss of positive charge, and concominant decompaction of chromatin. Using MS based proteomics, I have identified a list of proteins that could potentially mediate TSA-dependent chromatin compaction.
| Date of Award | 2013 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Angus Lamond (Supervisor) |