The reversible acetylation of histones is thought to play a role in chromatin processing, including transcription, replication and repair. Studies on the acetyltransferases, responsible for acetylating the nucleosomal core histones, have resulted in characterization of these enzymes. However, very little is known about the properties and distribution of histone deacetylase. The reversible inhibition of histone deacetylase by butyrate was employed to permit studies on the chromatin-bound histone deacetylase of HeLa cells using endogenous [3H]-acetyl labelled polynucleosomes containing the enzyme. These were prepared in the presence of 50mM butyrate and histone deacetylase was assayed upon removal of the inhibitor. It was found that active enzyme is present only in association with a high molecular weight complex. This deacetylase-containing complex is relatively resistant to digestion with micrococcal nuclease. No activity is found on mononucleosomes or oligonucleosomes. Up to 90% of labelled acetyl groups are removed from histone deacetylase complexes incubated in the absence of butyrate, indicating that denaturation of the histone deacetylase is kept to a minimum using the techniques developed in this study. Free histones are a poor substrate under these conditions, but histones in mononucleosomes are deacetylated when they are incubated with histone deacetylase complex. Histone deacetylase remains bound to this complex in 1-2 M NaCl and does not dissociate from it during its reaction with acetylated core hisones. Under typical nuclease digestion conditions, the histone deacetylase complex contains DNA with a size distribution of 5-11 kilobase pairs and a variety of nonhistone proteins. Comparison of the protein composition of histone deacetylase complexes with that of nuclear matrix preparations shows some similarities. Taken together, the results on the chromatographic behaviour, the DNA fragment sizes, and the protein composition of the deacetylase complex suggest that protein-protein interactions may be important in maintaining its structure and also in the binding of the deacetylase itself to the complex Later research efforts were concerned with characterization of the histone deacetylase complex. The effect of J3-mercaptoethanol and neocuproine on histone deacetylase was examined in view of the fact that these reagents are known to disrupt chromosome scaffolds. HeLa cell histone deacetylase complex partially dissociates in 10 mM B-mercaptoethanol, resulting in a loss of non-histone proteins. The presence of 10 mM J3-mercaptoethanol during the partial micrococcal nuclease digestion of HeLa cell nuclei, results in a very low yield of histone deacetylase complex, with a correspondingly large increase in the production of small oligonucleosomes and mononucleosomes. Histone deacetylase activity on endogenous labelled histone is strongly inhibited by either 1 or 10 mM J3-mercaptoethanol or 3 mM neocuproine. The loss of histone deacetylase activities is not due to an inactivation of the enzyme, but appears to be a consequence of the disruption of the structure of the histone deacetylase complex. Histone H4 in histone deacetylase complex prepared from HeLa cell nuclei by micrococcal nuclease digestion was more highly acetylated than H4 in bulk nucleosomes. Restriction enzyme analysis of the DNA associated with the histone deacetylase complex revealed neither an enrichment nor depletion of major satellite sequences in this material. In view of these findings, histone deacetylase appears to be associated with a high molecular weight chromatin complex which may be a site of rapid acetyl group turnover. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Diss. (sammanfattning) Umeå : Umeå universitet, 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Oct. 13, 2008). Includes bibliographical references.

Thesis (Ph.D.)--Uniformed Services University of the Health Sciences, 2006 / Typescript (photocopy)

Canavan’s Disease is a fatal Central Nervous System disorder caused by genetic defects in the enzyme – aspartoacylase and currently has no effective treatment options. We report additional phenotypes in a stringent preclinical aspartoacylase knockout mouse model. Using this model, we developed a gene therapy strategy with intravenous injections of the aspartoacylase gene packaged in recombinant adeno associated viruses (rAAVs). We first investigated the CNS gene transfer abilities of rAAV vectors that can cross the blood-brain-barrier in neonatal and adult mice and subsequently used different rAAV serotypes such as rAAV9, rAAVrh.8 and rAAVrh.10 for gene replacement therapy. A single intravenous injection rescued lethality, extended survival and corrected several disease phenotypes including motor dysfunctions. For the first time we demonstrated the existence of a therapeutic time window in the mouse model. In order to limit off-target effects of viral delivery we employed a synthetic strategy using microRNA mediated posttranscriptional detargeting to restrict rAAV expression in the CNS. We followed up with another approach to limit peripheral tissue distribution. Strikingly, we demonstrate that intracerebroventricular administration of a 50-fold lower vectors dose can rescue lethality and extend survival but not motor functions. We also study the contributions of several peripheral tissues in a primarily CNS disorder and examine several molecular attributes behind pathogenesis of Canavan’s disease using primary neural cell cultures. In summary, this thesis describes the potential of novel rAAV-mediated gene replacement therapy in Canavan’s disease and the use of rAAVs as a tool to tease out its pathological mechanism.