Graduate Thesis Or Dissertation
 

Towards the understanding of the function of the histone "tails" with respect to the structure, stability, and function of chromatin

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  • By using immobilized trypsin, I have been able to prepare well-defined, stable trypsinized nucleosomes. The difficulties of lacking of control in the extent of trypsinization, which were encountered in previous studies with the use of free trypsin, have been eliminated. The nucleosomes and oligonucleosomes prepared by immobilized trypsin are suitable for biochemical and biophysical studies to analyze the function of the histone N-terminal regions ("tails"), which are removed by trypsin treatment, on chromatin structure and stability. Studies were first conducted using the trypsinized nucleosome core particles to examine the role of the histone tails in the stabilization of the nucleosome core particle. While it was found that these tails have little effect on either the nucleosome dissociation or the conformational transition in salt, they play a very important role in determining thermal stability of the nucleosome. The differential effects of selective removal of these tails also provided more insight about histone-DNA interactions in the nucleosome core particle. Experiments have also been carried out to investigate the change in structure and hydration of nucleosome core particles which may be associated with the salt-dependent conformational transition. Changes in the tertiary structures are suggested to be responsible for the salt-dependent transition. Roles of the histone tails in determination of nucleosome positions along specific DNA sequences were examined by analysis of nucleosome positioning on a specific eukaryotic gene sequence (Lytechinus Variegatus 5S rRNA gene) after in vitro nucleosome reconstitution with native and trypsinized histone octamers. Data obtained indicate that the histone tails are not required for nucleosome positioning. Results also seem to restrict the portions of histones which are responsible for determining nucleosome positions to the globular regions of (H3/H4)₂ tetramer, and possibly H2B. Studies with different DNA templates strongly suggest that the most important determinants of nucleosome positioning are the mechanical properties (such as bending and flexibility) of the DNA molecule. Taking together, it seems that the N-terminal tails of the histones may play roles in stabilizing both nucleosome structure and the higher-order structure of chromatin.
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