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Testis-specific protein (TSP) variability has been examined in the three-spined stickleback, Gasterosteus aculeatus and related species including Gasterosteus wheatlandi, Punqitius pungitius, and Aulorhynchus flavidus, in order to determine if such proteins can act as molecular markers for different species of Gasterosteiformes and for different populations of a single species. Cytochemistry of the sperm histones of Gasterosteus aculeatus revealed that these basic proteins can be classified as intermediate sperm histones according to Bloch's (1969 and 1976) categories. Electron microscopy indicates that the chromatin in the nucleus condenses in a granular pattern as the somatic histones of the spermatogonia are replaced by the sperm histones in the spermatid during spermiogenesis in this teleost. The testis specific proteins (TSP's) of G. aculeatus and related species were characterized by electrophoresis on polyacrylamide gels and by hydrolysis of the amidoblack stained bands from the gel and subsequent amino acid analysis. The compositional analysis revealed that all the TSP's of the fish in the order Gasterosteiformes were intermediate type containing histidine, lysine, and arginine amino acid residues. However, the TSP's of different species could be distinguished by their electrophoretic mobilities on polyacrylamide gels and by differences in the amino acid composition. Apparently TSP's can act as molecular markers to distinguish these particular teleosts. To establish the electrophoretic pattern for the TSP's of mature, breeding G. aculeatus, the developmental profile was investigated over the course of a season for a population of these fish in Jericho Pond, Vancouver, B.C. As the testis matures, somatic histones are replaced gradually by one or several rapidly moving TSP's. Some protein bands that are present in fish with immature testes are removed by the time the electrophoretic pattern typical for mature males occurs during the breeding season. This pattern shows only rapidly moving TSP's and very low levels of somatic histones. Experiments using alkaline phosphatase indicated that the electrophoretic profile of the multiplicity of TSP'.s of mature fish was not due to differences in charge because of phosphorylation of serine side chains in TSP bands. In addition to acting as molecular markers for different species, the TSP's showed an electrophoretic profile in anadromous G. aculeatus that differed from the profile in freshwater fish with respect to band morphology and also the number of bands seen on long gels. Here, too, the similarity of the freshwater and anadromous TSP profiles was reinforced by similar digestion patterns with cyanogen bromide, confirming the presence of methionine in these proteins. However, different populations of anadromous G. aculeatus either from British Columbia or from Quebec were indistinguishable by electrophoretic analysis. Moreover the electrophoretic and amino acid analysis could not distinguish between G. aculeatus from different freshwater populations. There was no discernable trend for the multiplicity of TSP's from such populations. However, in the fish from two freshwater lakes there was an indication that the benthic forms (bottom dwellers) might have reached sexual maturity later than the limnetic forms (top dwellers) as the limnetic animals lost their somatic histones before the benthics did. From these analyses, TSP's apparently can act as molecular markers between different species of Gasterosteiformes, and to a lesser extent between anadromous and freshwater forms of G. aculeatus. This agrees with the findings of Mann et. al. ( 1982) that the spermatid/sperm-specific proteins of the frog genus Xenopus can distinguish between different species of the genus and somewhat between different subspecies of Xenopus laevis. Both the Gasterosteiformes and anuran TSP's are of the intermediate type. In several instances, particular species of fish gave anomalous results. For example, an anadromous G. aculeatus showed the presence of a band typical for the TSP of G. wheatlandi on polyacrylamide gel electrophoresis. Perhaps this is due to hybridization between Gasterosteidae of different species. Finally, incubation of TSP preparations at 37 °C indicated the presence of endogenous protease at neutral pH. Such a protease was not active at acid pH and therefore did not interfere with the electrophoretic analysis. / Science, Faculty of / Zoology, Department of / Graduate

During spermatogenesis in rainbow trout, a synchronous development of stem cells → spermatocytes → spermatids → mature sperm occurs, with replacement of the histones in chromatin by protamines. PART A: Histone methylation. Along with histone acetylation and phosphorylation, methylation of specific lysyl residues of histones H3 and H4 can be observed. This histone methylation occurs predominantly in the large diploid stem cells and primary spermatocytes, which actively synthesize DNA and histones. In spermatids, histone methylation is minimal and so probably has no role in the replacement of histones by protamine. Other labelling experiments suggest that histone H4 methylation is a late event in the cell cycle, occurring after the synthesis, acetylation and deacetylation of histone H4. This methylation may be necessary for histone phosphorylation or chromatin condensation prior to cell division. PART B: Chromatin subunit structure. When a sample of trout testis nuclei is digested with micrococcal nuclease, the DNA is cleaved almost entirely to discrete fragments approximately 200 base pairs long and multiples thereof. The same DNA fragments can be obtained when isolated chromatin, as opposed to intact nuclei, is nuclease digested. These DNA fragments can also be found in discrete chromatin "subunits" isolated from nuclease-digested nuclei. Sedimentation through sucrose gradients, or velocity sedimentation in an analytical ultracentrifuge separates these chromatin subunits into 11S (monomer), 16S (dimer), 22S (trimer) etc. species. Subunits can also be fractionated on a Sepharose 2B column equilibrated and run in low salt. High salt (>40 mM NaCl) or divalent cations (≃5 mM) cause subunit precipitation. Chromatin subunits have a protein:DNA ratio of approximately 1.2 and contain all the histones, including the trout-specific histone H6. There are however no detectable nonhistone chromosomal proteins. Mg⁺⁺ precipitates of the 11S chromatin monomers, when pelleted, are thin and clear, while oligomer Mg⁺⁺ pellets are thick and white. This could reflect a more symmetrical or ordered packing of 11S monomers, which are deficient in histone Hi. This histone may crosslink the larger oligomers, resulting in a disordered Mg⁺⁺ complex. These results are consistent with the subunit model of chromatin structure, based on 200 base pair long regions of DNA associated with histones. These subunits would be separated by nuclease-sensitive DNA spacer regions, and crosslinked by histone Hi. Testis consisting predominantly of early spermatids (meiotic tissue, containing mainly nucleohistone) gives similar yields of DNA fragments and 11S subunits. Later stage testis (protamine has replaced the histones) however, gives no DNA fragments or 11S subunits. This presumably reflects large differences in structure between nucleoprotamine and nucleohistone. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

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

Bibliography: pages 110-126. / This thesis describes methodology for the reconstitution of the chicken erythrocyte octamer from acid-denatured histones or the natural H3-H4 tetramer and H2A-H2B dimers. Oligomeric properties of reconstituted octamers were elucidated during column chromatographic and chemical cross-linking studies. The conformational identity of the natural and reconstituted octamers was demonstrated by the ability of all preparations to crystallise as helical octamer tubes. The application of the reconstitution methodology in addressing fundamental problems of chromatin research, was demonstrated during subsequent studies, namely (i) The reconstitution of hybrid histone octamers containing a structural variant of a specific histone. These studies were undertaken to study the effect on histone-histone interactions in hybrid octamers of which erythrocyte H2B was substituted for by sea urchin sperm H2B(l) or erythrocyte H3 and H4 were substituted for by dethiolated H3 and sea urchin sperm H4 respectively. (ii) The reconstitution of an octamer suitable for the sitespecific derivatisation of a specific histone, or covalently labelled with aurothiomalate in a specific histone complex. These studies were concluded to represent general labelling strategies which may be of use in crystallographic or physico-chemical studies of nucleosome structure.

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Histone proteins were examined during development in the meiotic elongate and a genetically close line N6HT. Histones were also extracted from an F1 (el x N6Ht). Comparisonc beteen the histone samples from each line of N6HT and elongate, and the F1 for leaf, root, and stem were inconclusive. A tassel sample form elongate exhibited a markedly slower migrating band that was not present in N6HT. The histone profiles of elongate and N6HT also differed. Each line N6HT and elongate exhibited three protein bands in the H1 region. Maize histone samples have been shown to exhibit four major H1 bands. The possibility exists that an H1 protein altered in its molecular weight and possibly in its interaction with the chromosome is present in elongate.

L'organisation des génomes eucaryotes sous forme de chromatine constitue un élément de régulation essentiel de tous les processus cellulaires dépendants de l'ADN. Les facteurs intervenant sur cette organisation jouent donc un rôle crucial dans le bon fonctionnement et le maintien de l'identité des cellules et l'intégrité du matériel génomique. Le complexe NuA4/TIP60 est capable d'agir sur l'organisation de la chromatine de deux façons distinctes : premièrement en acétylant les histones H2A et H4 via sa sous-unité KAT5/Tip60, conduisant à une structure chromatinienne plus relâchée et accessible; deuxièmement en incorporant le variant d'histone H2A.Z dans la chromatine, conférant des propriétés particulières aux régions du génome concernées. NuA4/TIP60 joue ainsi un rôle central dans la régulation de nombreux processus cellulaires, en particulier l'expression des gènes et la réparation des dommages à l'ADN. Le complexe est composé d'au moins 17 sous-unités chez l'humain; les propriétés et fonctions de certaines de ces sous-unités restent à préciser dans le but de mieux comprendre comment NuA4/TIP60 régule l'organisation de la chromatine. Dans la première partie de mes travaux de doctorat présentés ici, nous avons cherché à clarifier la fonction du chromodomaine de KAT5/Tip60, la sous-unité catalytique du complexe. En effet des observations contradictoires avaient été rapportées dans la littérature, en particulier en ce qui concerne la capacité du chromodomaine à reconnaître des marques d'histones spécifiques. Nos résultats suggèrent que ce domaine régule plutôt l'activité acétyltransférase du complexe indépendamment des marques d'histones. Nous avons également caractérisé des mutations de KAT5/Tip60, dont l'une dans le chromodomaine, liées à un syndrome neurodéveloppemental chez plusieurs patients. Dans une deuxième partie, nous nous sommes intéressés à l'incorporation du variant d'histone H2A.Z au sein de la chromatine par NuA4/TIP60 et par un autre complexe, SRCAP. Nos résultats suggèrent que NuA4/TIP60 favorise l'un des paralogues de H2A.Z, H2A.Z.2, par rapport à H2A.Z.1, contrairement à SRCAP. Nous avons également identifié des partenaires spécifiques pour chaque paralogue de H2A.Z qui permettent d'expliquer une partie des rôles différents joués par ces paralogues dans la régulation de la transcription. Dans leur ensemble ces travaux contribuent à améliorer notre compréhension de la façon dont le complexe NuA4/TIP60 affecte l'organisation chromatinienne, et comment des perturbations de cette fonction peuvent entraîner des conséquences pathologiques sérieuses. / Eucaryotic genomes take the shape of chromatin, the organization of which affects all DNA-based cellular processes. Hence, factors involved in this organization are critical for maintaining proper cell function, identity, and genome integrity. The NuA4/TIP60 complex affects chromatin organization through two different mechanisms: first by acetylating histones H2A and H4 in chromatin, increasing its relaxation and accessibility; second by incorporating the histone variant H2A.Z into chromatin, assigning distinct properties to given genomic regions. NuA4/TIP60 therefore acts as a central regulator of many cellular processes, in particular gene expression and DNA damage repair. NuA4/TIP60 comprises at least 17 subunits, the functions and properties of many of which still need elucidating in order to better understand how the complex regulates chromatin structure. In the first part of my PhD project presented hereby, we aimed to clarify the function of KAT5/Tip60, the catalytical subunit of NuA4/Tip60. Contradictory results had been previously reported regarding the chromodomain ability to bind specific histone marks. Our results suggest that this domain instead regulates the acetyltransferase activity of NuA4/Tip60 independently of histone marks. We have also characterized mutations in KAT5/Tip60, one of them inside the chromodomain, linked to a rare neurodevelopmental syndrome. In the second part, we were interested in the incorporation of the histone variant H2A.Z in chromatin by NuA4/TIP60 as well as another complex, SRCAP. Our results suggest that NuA4/TIP60 favors one of the two H2A.Z paralogs, H2A.Z.2, over H2A.Z.1, as opposed to SRCAP which binds both equally. We also identified specific interactors for each paralog, which could explain in part how H2A.Z.1 and H2A.Z.2 regulate gene expression differently. Overall this work contributes to a better understanding of how NuA4/TIP60 regulates chromatin organization, and how disruption of these functions can lead to serious pathological outcomes.

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