As a centromere-specific protein complex in direct contact with the DNA, CenH3-containing nucleosomes are generally thought to act as the distinguishing epigenetic mark of active centromere location. Confusingly, seemingly disparate models have been proposed for the structure of CenH3 nucleosomes. The most widely supported model is an octameric structure that, like histone H3 nucleosomes, contains two subunits of each histone. Another more contentious, yet persistent model is the hemisome model proposed for fly and human CenH3 nucleosomes. In this case it is suggested that CenH3 nucleosomes contain only single subunit of each histone. One reason for this lack of consensus is that seemingly contradicting models are often proposed, even with material from the same organism, with little overlap in experimental approaches. For example, the proposed hemisome model for fly and human CenH3 nucleosomes is predominantly based on atomic force microscopy (AFM) imaging where the height of nucleosomes on a surface is measured. These AFM measurements are the main data used by protagonists for the hemisome model. However, data supporting an octameric model for human, and other, CenH3 nucleosomes is largely based on biochemical analysis of nucleosomes prepared in vitro, with little cross-over in the methodology used to generate data to support either model. In order to reach a consensus the same analyses needs to be applied to CenH3 nucleosomes assembled in vitro or extracted from cells. Here, recombinant Schizosaccharomyces pombe CENP-ACnp1 and H3 histones expressed and purified from E. coli have been assembled into nucleosomes. To our knowledge this is the first time that recombinant S. pombe nucleosomes have been produced, allowing the stoichiometry and composition of these nucleosomes to be examined in detail by a variety of biochemical and biophysical assays. The application of AFM has enabled the height of these recombinant nucleosomes to be measured and tests the ability of AFM to infer stoichiometry using defined material. The intriguing conclusion is that octameric CenH3 nucleosomes uniquely behave as tetrameric “hemisomes” as defined by AFM. In recent years the contribution of DNA sequence to directing H3 nucleosome location has received a great deal of interest. Since CENP-ACnp1 nucleosomes wrap DNA differently to H3 nucleosomes their preference for sequences that produce a stable nucleosome is expected to be altered. The development of protocols to assemble recombinant CENP-ACnp1 nucleosomes in vitro has also been used here to assess the contribution of primary DNA sequence to CENP-ACnp1 nucleosome positioning. CENP-ACnp1 and H3 nucleosomes were reconstituted on genomic DNA at low density and the resulting nucleosomal DNA from CENP-ACnp1 and H3 particles compared by Illumina sequencing. The stability of CENP-ACnp1 and H3 nucleosomes on specific ‘H3’ and ‘CENP-ACnp1’ sequences was cross-checked. Comparing these data with in vivo CENP-ACnp1 nucleosome positions has allowed the contribution of primary DNA sequence to CENP-ACnp1 nucleosome positioning to be explored.

Der SET-Bereich befindet sich unter den verschiedenen Proteinsequenzbereichen, die mit epigentischer Regulation hauptsächlich durch die Präsenz von Trihtorax (trxG) und Polycomb (PcG) Gruppen von Chromatinmodifikatoren in Zusammenhang gebracht werden. Nach der Entdeckung des ersten SET-Bereichs vor einigen Jahren, welcher die Histon-Lysin-Methyltransferase (Su(var)39) enthält, wurde den Proteinen mit SET-Bereich sehr viel Aufmerksamkeit geschenkt. Obwohl die Histon-Lysin-Methylierung schon länger als 30 Jahre bekannt ist, war ihre Funktion vor diesem überragenden Ergebnis größten Teils unbekannt. In meiner Arbeit beschreibe ich die kombinatorische und funktionale Charakterisierung von 3 Hefe Proteinkomplexen durch die Anwendung von proteomischer SEAM (Sequential Epitope Tagging and Mass Spectrometry). Zwei dieser Komplex enthalten einen SET-Bereich und die Dritte ist der Rad6 Komplex aus S. pombe (Sp_Rad6C). Der Set1 Komplex (Set1C) beinhaltet 8 Bausteine, methylisiert Lysin 4 in Histon H3 und ist die erste, entdeckte Histone H3 Lysin 4 Methyltransferase. Es beinhaltet ein Ash2 Homolog (Bre2), einen bekannten Baustein von trxG. Kürzlich wurden Rad6 beinhaltende Komplexe gezeigt, die in engem Bezug zu der H3-K4 und H3-K79 Methylation durch ubiquitinierung von Histon H2B und die Etablierung von trans-histonen Signalwegen stehen. Unsere Analysen von Sp-Rad6C führten zu mehreren interessanten Ansichten. Der Set3 Komplex (Set3C) hat keine feststellbare Aktivität einer Methyltransferase, enthält jedoch zwei Histon deacetylasen (HDACs) ? eine klassische HDAC (Hos2) und eine NAD-abhängige HDAC (Hst1). Unsere funktionelle Analyse von Set3C zeigt, dass Set3C bei der Regulierung des meiotischen Genexpressionsprogramms in knospenden Hefen (S. cerevisiae) beteiligt ist. Evolutionbiologisch betrachtet, ist die Spalt-Hefe (S. pombe) sehr weit von S. cerevisiae entfernt und wird meist als ein besserer Modellorganismus fur höhere Eukaryoten angesehen. In einem Versuch, unser Wissen uber andere Organismen zu vergrößern, haben wir ähnliche Untersuchungen in S. pombe unternommen und haben herausgefunden, dass Set1C in beiden Hefen sehr stark konserviert ist. Darüberhinaus waren die Set1-Ash2 Verbindungen konserviert und wir nehmen an, dass auch in höheren Eukaryoten Set1-ahnliche Methyltransferasen Ash2-ahnliche Proteinen angehören. Dies wurde später durch mehrere Studien von anderen Gruppen bestätigt, die an Säugetieren arbeiten. Was Set3C anbelangt, wurden unsere weiteren Analysen nur durch vergleichende Proteomik beschränkt. Wir zeigen, dass der proteomische Kern von Set3C in Spalt-Hefe konserviert wird. Im Gegensatz zu Set3C in S. cerevisiae, beinhaltet diese in S. pombe nur eine HDAC, die zur Hos2 Familie gehört,. Die präsentierte Arbeit hat auch viele Auswirkungen auf die übergreifende Organisation von Proteomen. Wir beschreiben verschiedene Beispiele von gemeinsamen Komponenten zwischen unterschiedlichen Komplexen und prägen den Begriff "proteomischer Hyperlink". Wir waren in der Lage zu zeigen, dass proteomische Kerne sogar für unwesentliche Proteinkomplexe hoch konserviert sind. Die generelle proteomische Schaltung über proteomische Hyperlinks scheint jedoch verworrener und unvorhersehbar zu sein. Wir schlussfolgern, dass die Erschaffung von zuverlässigen, detailierten, proteomischen Abbildungen, welche auf dem Wissen von niederen Organismen fundieren, zur Zeit nicht möglich ist.

In the last two decades the study of changes in the genome function that are not induced by changes in DNA has consolidated a strong research field called ”epigenetics”. Chromatin state changes play an essential role in the regulation of transcription of many genes, thus controlling cell differentiation. A large part of these changes is due to histone modifications that alter the accessibility of the DNA. Current state of the art visualization methods for the analysis of epigenetic data sets are not suited to represent the relationship between the combinatorial pattern of histone modifications and their regulatory effects.

The N-terminal tails of histone proteins protrude from the nucleosome core and are extensively post-translationally modified. These modifications are proposed to affect many DNA-based processes such as transcription, DNA replication and repair. Post-translational modifications on histone tails do not act independently but are subject to crosstalk. One example of crosstalk is on histone H3 between lysine 14 (H3K14) and trimethylated lysine 4 (H3K4me3), a modification found at the 5’ end of most active or poised genes. In this work, Western blots and chromatin immunoprecipitation (ChIP) experiments show that different amino acid substitutions at histone H3 position 14 cause varying degrees of H3K4me3 loss, indicating that H3K14 is not essential for H3K4me3 but acts as a modulator of H3K4me3 levels. A neighbouring residue, H3P16 is also important for H3K4me3 and may operate in concert with H3K14 to control H3K4me3. These crosstalk pathways have gene-specific effects and the levels of H3K4me3 are influenced to different extents on genes that fall into functionally distinct classes. A model is proposed to explain how H3K14/H3P16 may exert these varying effects on H3K4me3 at individual genes. In addition to its ability to regulate H3K4me3, H3K14 also influences the levels of two modifications on H3K18, acetylation and monomethylation. A ChIP-sequencing experiment has shown that H3K18me1, a previously uncharacterised modification in S. cerevisiae, is widely distributed throughout the genome and correlates strongly with histone H3 levels. The potential for a functional acetyl/methyl switch at H3K18 is explored. Together, these data indicate that, with gene-specific effects, crosstalk between histone modifications may be even more complex than originally thought.