HMG-Proteine sind Architekturelemente des Chromatins und regulieren durch ihre Bindung an das Chromatin auf verschiedene Weise DNA-abhängige Prozesse wie Replikation, Transkription und DNA-Reparatur. Um zu verstehen, wie HMG-Proteine ihre vielfältigen Funktionen erfüllen können, wurde mit Hilfe von EGFP- und DsRed2-Fusionsproteinen ihre Funktion in vivo untersucht. Im Wesentlichen wurde dabei mit Hilfe von Bleichtechniken ihr dynamisches Verhalten charakterisiert. Daneben wurde für die HMGN-Proteine ihr bislang unbekanntes Expressionsverhalten in Tumorzellen bestimmt. So konnte für die HMGN-Proteine gezeigt werden, dass bestimmte Tumorzelllinien (HT-29, FTC-133, MCF-7, RPMI 8226, 697, Ishikawa, LNCap) eine relativ erhöhte Expression von HMGN2 aufweisen, die mit der Tumordifferenzierung korreliert. Eine relativ verringerte Expression von HMGN1 steht dagegen in Mammakarzinomen und Non-Hodgkin-Lymphomen in direktem Zusammenhang mit der Aggressivität der Tumore. Somit kann die HMGN-Expression bei diesen Tumoren als diagnostischer Marker verwendet werden. FRAP-Analysen mit EGFP-Fusionsproteinen führten zu der Erkenntnis, dass HMGN1, HMGN2, HMGA1a, HMGA1b und HMGB1 sich sehr schnell durch den Zellkern bewegen und nur transient an das Chromatin gebunden sind. Es konnte gezeigt werden, dass die spezifischen DNA/Chromatin-Bindungsmotive im Wesentlichen entscheiden, wo die Bindung der HMG-Proteine in vivo erfolgt, ihre Verweildauer im Euchromatin, Heterochromatin und zellzyklusabhängig dann aber durch Modifikationen (Phosphorylierungen, Acetylierungen) reguliert wird. Dies wurde beispielhaft durch punktmutierte und deletierte Fusionsproteine, sowie durch Inkubation der Zellen mit spezifischen Drogen für die HMGA1a-Proteine gezeigt. FRAP-Analysen haben außerdem gezeigt, dass die Spleißvarianten hHMGA1a und hHMGA1b unterschiedliche kinetische Parameter besitzen. Dies zeigt, dass beiden Varianten unterschiedliche Funktionen zugesprochen werden können. Die gefundenen spezifischen, transienten Verweildauern der einzelnen HMG-Proteine führen zu einem Modell eines dynamischen Chromatin-Netzwerkes, wobei alle HMG-Proteine in Wechselwirkungen innerhalb eines dynamischen Chromatinprotein-Cocktails DNA-abhängige Prozesse regulieren können. Die jeweiligen, wie hier gezeigt, durch Modifikationen regulierten Verweildauern der HMG-Proteine bestimmen darüber, welche anderen Chromatinproteine wie lange am Chromatin verbleiben und bestimmte Funktionen, wie beispielsweise die Modifikation der Core-Histone, übernehmen können. Die dynamischen Parameter einzelner HMG-Proteine erklären so, wie diese Proteine ihre vielfältigen Funktionen als Architekturelemente und bei der Regulation DNA-abhängiger Prozesse erfüllen können. Einige Vertreter, wie die HMGB1-Proteine, bewegen sich so schnell durch den Zellkern, dass ihre kinetischen Parameter durch das beschränkte zeitliche Auflösungsvermögen konfokaler Mikroskope der älteren Generation nicht erfassbar sind. Die Bestimmung von Dosis-Wirkungs-Beziehungen von Drogen, welche die kinetischen Parameter von HMGB1-Proteinen beeinflussen können, ist inzwischen mit Mikroskopen der neuen Generation möglich. Im Verlaufe der Arbeit zeigte sich, dass andere verwendete Fluorophore wie DsRed2 die kinetischen Eigenschaften von HMG-Fusionsproteinen beeinflussen können. Durch eine erhöhte Verweildauer können auch sehr transiente Interaktionen sichtbar gemacht werden. Wie gezeigt wurde, kann eine erhöhte Verweildauer aber auch zur Verdrängung anderer Proteine führen, die die gleichen Bindungsstellen benutzen und so eine Modulation des Chromatins bewirken. Die Nutzung von DsRed-Fluorophoren ermöglicht interessante neue Erkenntnisse. Diese müssen aber stets vor dem Hintergrund eines veränderten dynamischen Verhaltens der Fusionsproteine interpretiert werden. Zusammengenommen liefern die hier vorgestellten Ergebnisse zur Dynamik der HMG-Proteine grundlegende Informationen, die zur Klärung ihrer Funktion bei Chromatinmodulationen, etwa bei Differenzierungsprozessen oder der Entstehung von Tumorzellen entscheidend beitragen. Die Erkenntnis, dass diese Proteine lediglich transiente Interaktionen mit ihren Bindungspartnern eingehen können, sind im Hinblick auf die Behandlung von Tumoren, bei denen HMG-Proteine im Vergleich zu Normalgewebe häufig überexprimiert sind, von großer Bedeutung. / HMG proteins are architectural chromatin proteins that regulate different DNA dependent processes such as replication, transcription and DNA repair. To understand how HMG proteins manage to fulfill their multiple functions they were investigated in vivo with the help of EGFP and DsRed2 fusion proteins. Using photobleaching techniques their dynamic properties were characterized in detail. Furthermore, the expression pattern of HMGN proteins in tumor cell lines was investigated for the first time. As presented in this thesis, it was found that HMGN2 proteins exhibited an elevated expression level in some tumor cells (HT-29, FTC-133, MCF-7, RPMI 8226, 697, Ishikawa, LNCap) correlating with the tumor differentiation status. In contrast a reduced expression of HMGN1 found in Mammacarcinoma and Non-Hodgkin-Lymphoma correlated with tumor aggressiveness. Therefore the analyses of HMGN expression may be a suitable diagnostic marker at least in the tumors investigated. FRAP analyses with cells expressing EGFP fusion proteins revealed that HMGN1, HMGN2, HMGA1a, HMGA1b and HMGB1 move very rapidly through the cell nucleus and only bind transiently to chromatin. It was demonstrated that the decision where HMG proteins bind in vivo is essentially mediated by their specific DNA binding motifs. However, the individual residence times in eu- or heterochromatin and chromosomes are regulated by protein modifications (phosphorylation, acetylation). This has been demonstrated using point mutated and truncated HMGA fusion proteins and by the application of specific drugs as well. FRAP analyses also indicated that the splice variants HMGA1a and HMGA1b exhibit different kinetic properties. This supports the view that both variants have different functions. The kinetic parameters characteristic for each HMG protein lead to a model of a dynamic chromatin network in which all HMG proteins are able to regulate DNA dependent processes via multiple interactions with other proteins as components of a cocktail of dynamic chromatin proteins. In this model, individual residence times of all HMG proteins which are regulated by secondary modifications would determine how long other chromatin modulating proteins could reside on chromatin. Therefore the dynamic parameters of the HMG proteins directly affect the capability of other proteins to modulate chromatin structure, e.g. by modifications of core histones. This explains the multiple functions of HMG proteins in chromatin packaging and function. The kinetic parameters of some rapidly moving members of the HMG protein family, such as HMGB1, are beyond the time resolution capacities of most confocal microscopes. However, novel setups of modern confocal microscopes are now capable to determine the dynamic parameters of HMGB proteins and allow investigations of drug induced effects on HMGB dynamics. Control experiments revealed that other fluorophors such as DsRed2 modulate the dynamic parameters of HMG fusion proteins. Due to an increased residence time of HMG DsRed2 fusion proteins it is possible to monitor even very transient interactions. Moreover, it could be observed that this increased residence time may interfere with binding of other proteins (i.e. proteins which occupy the same binding sites) leading to a reorganization of chromatin. Thus, fusion proteins with DsRed fluorophores may be used as helpful tools to investigate protein functions. However, results should always be considered against the background of DsRed modulated kinetics and thus they should be interpreted very carefully. Taken together the results presented in this thesis provide novel information about the dynamic behaviour of HMG proteins which is crucial to understand how chromatin is modulated during differentiation processes or development of neoplasia. Their transient interactions with DNA or other proteins and the fact that overexpression correlates with tumor progression might be relevant for the development of novel strategies for tumor treatment.

Thesis advisor: Anthony T. Annunziato / The project I am presenting aimed to: 1. Elucidate the pattern of post- translational modification on the different variants of newly synthesized histones H3 in mammalian cells; 2. Reveal whether the acetylation of residue K56 on newly synthesized H3 histones plays a role in the incorporation of the histone into chromatin in mammalian cells; and 3. Determine whether the acetylation of residue K56 on newly synthesized H3 histones plays a role in the incorporation of the histone specifically in replicating chromatin in mammalian cells. The experiments to answer these questions were performed using HEK293 cells with inducible expression of FLAG-histones, enabling us to control the synthesis of new histones of interest and to detect and analyze their presence and relative levels in the cells. The results suggest that the acetylation of lysine 56 on histone H3 may play a positive role in the incorporation of the histone into new chromatin, and lack of acetylation may be reducing the efficiency of incorporation compared to acetylated histones. / Thesis (MS) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

CpG islands (CGIs) are short GC rich sequences with a high frequency of CpGs that are associated with the active chromatin mark H3K4me3. Most occur at gene promoters and are often free of cytosine methylation. Recent work has begun to clarify the functional significance of CGIs with respect to chromatin structure and transcription. In particular, proteins associated with histone-modifying activities, such as Cfp1 and Kdm2a, bind specifically to non-methylated CGIs via their CxxC domains. For example, artificial promoterless CpG-rich sequences integrated at the 3’ UTR of genes recruit Cfp1 and generate novel peaks of H3K4me3 in mouse ES cells without apparent RNA polymerase recruitment. There is also evidence that G+C-rich DNA recruits H3K27me3, a gene silencing mark. In this thesis I am exploring the constraints on DNA sequence and genomic location that are required to impose both H3K4me3 and H3K27me3 at CGI sequences. Showing that the generation of novel peaks of H3K4me3 and H3K27me3 over a promoter-less CpG rich sequence in a gene desert region is independent of it’s location in the genome extends earlier findings. These findings suggest that shared features of the primary DNA sequence at CGIs directly influence chromatin modification. Thus CGIs are not passive footprints of other cellular mechanisms, but play an active role in setting up local chromatin structure. However, the relative contribution of CpG frequency versus G+C content remains unclear. Therefore a sequence was generated that contains low levels of CpGs, comparable to the bulk genome, but has a G+C content similar to that of CGIs (Low CpG / High G+C). When this sequence was inserted into a gene desert neither marks of H3K4me3 or H3K27me3 were formed, indicating the importance of CpGs. Surprisingly, the reverse sequence with a high CpG frequency similar to that of CGIs and a low G+C content similar to that of the bulk genome (High CpG / Low G+C) did not establish H3K4me3 or H3K27me3 either. However, it was found that this sequence becomes heavily methylated in contrast to CGI-like sequences that remained unmethylated when introduced into a gene desert. This finding suggests that a high G+C content is important for keeping CGI-like sequences methylation free. Upon insertion of this High CpG / Low G+C sequence into mouse ES cells that were devoid of the de-novo DNA methyltransferases 3a and 3b (Dnmt3a/3b -/-) both H3K4me3 and H3K27me3 marks were established at the inserted sequence. This discovery confirms the importance of CpGs for setting up local chromatin structure.

Enhancers are non-coding DNA sequences which are able to activate the expression of a gene in a specific tissue manner and at a precise stage during embryonic development. First identified almost 40 years ago, our growing understanding of enhancers has transformed the concept of gene regulation to recognise the key role of these sequences in the expression of many genes. Moreover, the identification of human diseases caused by genetic variation in non-coding enhancer elements highlights the importance of characterising enhancers in order to understand human disease. However, enhancers are often located far from the promoter they influence and the mechanisms through which enhancers govern gene expression remain unclear. The most widely accepted model for the action of distal enhancers involves the formation of a chromatin loop, in which the enhancer and promoter physically interact at the loop base. The kinetics or molecular basis for the formation of enhancer/promoter loops is unknown and it remains unclear whether this mechanism of enhancer communication is universal, or indeed whether it is the most pervasive. The aim of my PhD is to investigate further the mechanism of action of distal enhancers in the regulation of developmental genes. Using chromatin profiling during the differentiation of embryonic stem cells to neural progenitor cells in order to see which Shh enhancer is active in neural progenitor cells (NPCs), I report the identification of a novel long-range enhancer for Shh - Shh-Brain- Enhancer-6 (SBE6) – that is located 100kb upstream of Shh and that is required for the proper induction of Shh expression during a neural differentiation programme. SBE6 enhances Shh expression during the differentiation of neural progenitor cells (NPCs) and is active in the brain of developing zebrafish and mouse embryos. Next, using a super-resolution 3D-FISH based approach to study the enhancer-driven activation of the Sonic hedgehog gene (Shh) I have identified a novel mechanism of longrange enhancer regulation that is incompatible with the looping model. Instead, gene activation is associated with an increase in nuclear distance between Shh and Shh-Brain- Enhancers. Using a synthetic biology approach I have determined that the chromatin unfolding is regulated specifically by the Shh-Brain-Enhancer and is mediated by the recruitment of transcription factor SIX3 and Poly (ADP-Ribose) Polymerase 1. Chromatin decondensation upon gene activation has been observed previously in Drosophila polytene chromosomes. I suggest an analogous decompaction is driven by Shh-Brain-Enhancer to promote the activation of Shh in mouse neural progenitor cells. This ‘chromatin unfolding’ model represents a new mechanism of long-range enhancer-promoter communication in addition to the looping and tracking models.

Orientador: Marion Burkhardt de Koivisto / Banca: Maricy Apparício Ferreira / Banca: Janaína Torres Carreira / Resumo: A avaliação de espermatozoides em tecnologias de reprodução assistida raramente analisa a integridade do DNA, crucial para o desenvolvimento embrionário. A técnica do azul de toluidina permite identificar alterações da cromatina com avaliação concomitante da morfometria espermática. O método foi descrito em diversas espécies, mas ao conhecimento dos autores, ainda não foi relatado em gatos. O objetivo deste estudo foi verificar a aplicabilidade da técnica de coloração de azul de toluidina em avaliar as anormalidades de DNA de espermatozoides epididimários (cabeça, corpo e cauda) de gatos. Investigar ainda se houve correlação entre as variáveis: condensação do DNA, morfologia e morfometria da cabeça espermática. Para este propósito, os índices de alteração de DNA obtidos pela técnica de azul de toluidina e laranja de acridina foram comparados, observando correlação de 65,38% (p<0,001). A estabilidade da cromatina aumentou significativamente da região da cabeça (92,06%) do epidídimo para a cauda (97,94%, p=0,0023), mas não houve diferença entre as regiões do corpo e da cauda, demonstrando que os espermatozoides provenientes destas regiões já possuem maturidade reprodutiva. Não houve correlação entre a anormalidade do DNA e a morfologia espermática como nas demais espécies, mas sim com a morfometria. Observou-se diminuição significativa do tamanho da cabeça do espermatozoide durante a passagem pelas três regiões epididimárias (p < 0,0001). A porcentagem de espermatozoides com cr... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Sperm selection in assisted reproductive technologies rarely evaluates the DNA integrity, which is crucial to the embryo's development. The toluidine blue technique allows identification of chromatin alterations, simultaneously with evaluation of sperm morphometry. The method has been described in many species, but to the authors' knowledge, it has yet to be described in cats. The objective of this study was to verify the applicability of the toluidine blue technique in analyzing DNA abnormalities of epididymal sperm (caput, corpus and cauda) in cats and further investigating if there was correlation between the variables: DNA condensation, morphology and morphometry of the sperm head. For this purpose, the DNA alteration indexes obtained by both toluidine blue and acridine orange techniques were compared and a 65.38% (p < 0.001) correlation was observed. The chromatin stability increased significantly in the head region of the epididymis (92.06%) in relation to the cauda (97.94%, p = 0.0023), however there was no difference between the caput and cauda regions, which demonstrates that sperm coming from these region are already mature. There was no correlation between the DNA abnormality and the sperm morphology as observed in other species, however there was correlation to morphometry. A significant decrease of the sperm head size was observed during the passage of the three epididymal regions (p < 0.0001). The percentage of sperm with deficient chromatin condensation decre... (Complete abstract click electronic access below) / Mestre

The centromere is a chromosomal locus required for accurate segregation of sister chromatids during cell division. They are maintained epigenetically in most eukaryotes, by incorporating the H3 variant CENP-A, and can, in rare instances, change location on the chromosome throughout generations. Centromeres are transcribed, and an active transcription chromatin signature is required for centromere maintenance. For this reason, insight into the nature of this so-called “centrochromatin” is essential for understanding a centromere’s place in the chromosome. The body of work contained in this thesis shows my efforts to understand the centromere in the context of chromatin, revealing interactions and new evidence for repressive chromatin domains with centromere activity, in two different vertebrate models: chicken DT40 cells and human HeLa cells. Centromeres are generally embedded within large domains of heterochromatic repetitive sequences in most eukaryotes, and mapping “centrochromatin” to high-resolution has proven difficult. However, chromosomes 5, 27 and Z of Gallus gallus are not located within repeat arrays, and are fully sequenced. CENP-A distribution on these centromeres has been mapped by ChIP-seq, and I have performed ChIP against selected histone modifications as part of a collaboration. While levels of heterochromatin are naturally quite low in these centromeres, I have shown that repressive polycomb chromatin instead is enriched in these non-repetitive centromeres, suggesting a replacement of one silenced chromatin state with another. Additional mapping of these centromeres showed a pattern of active chromatin marks distinct from that reported for human cells, which exhibited dynamic distribution throughout the cell cycle. Furthermore, conditionally generated neocentromeres in DT40 cells revealed that centrochromatin formation lowers, but does not eliminate, active transcription. To directly study the interaction of polycomb and heterochromatin with centrochromatin, I used a synthetic Human Artificial Chromosome (HAC), which allows for specific conditional targeting of chromatin modification enzymes, allowing manipulation of the underlying chromatin. Enrichment of the polycomb chromatin state on the HAC centromere, by EZH2 tethering, reduced its active transcriptional chromatin signature, but did not impair its actual transcription or mitotic activity. However, direct tethering of polycomb secondary silencing effector PRC1 caused centromere loss, and this effect was mimicked with homologous heterochromatin factors, indicating that centromeres can subsist within repressive chromatin domains, but are lost when direct repression is applied. To understand the contribution of the local repressive heterochromatin to centromere stability, I erased heterochromatin marks from the HAC centromere by tethering JMJD2D (an H3K9me3 demethylase): long-term (but not short-term) heterochromatin loss impaired CENP-A assembly, perturbed mitotic behaviour, and resulted in significant HAC mis-segregation. These results strongly suggest that local heterochromatin is essential to maintain normal CENP-A dynamics and centromere function. Together with previous observations, these data suggest that a repressive chromatin environment contributes to centromere stability, and that centromeres likely have natural mechanisms to maintain their transcriptional activity within such domains.

Histone post-translational modifications (PTMs) are key determinants of the local chromatin landscape and critical for regulation of eukaryotic gene expression. These histone marks are deposited by a vast number of chromatin modifying enzymes and preferentially recognized by specific associated histone reader domains. Recognition of histone PTMs by histone reader domains is important for either targeting these complexes to chromatin or regulating their enzymatic activity once there. The Polycomb repressive complex 1 and 2 (PRC1 and PRC2) are two such chromatin modifying complexes that are critical for developmental gene repression. The enzymatic activity of PRC2 is tightly regulated by many histone reader domains whereas the PRC1 complex is targeted to chromatin through these domains. In this thesis, I explore how PRC1 and PRC2 functions are regulated by histone reader domains. I identify a previously unrecognized histone reader domain within the PRC2 complex, the EZH2 SANT1 domain, which has important implications for regulating PRC2 enzymatic activity. In addition, I explore the mechanism through which the CBX8 chromodomain targets the PRC1 complex to chromatin. Together, these studies provide significant insight into the regulation of chromatin modifying complexes by histone reader domains and how this occurs via multiple mechanisms.

The Distal-less (Dlx) homeobox genes of mammals are expressed in many tissues of the developing organism including the limbs, craniofacial skeleton and the forebrain. In the forebrain, Dlx1, Dlx2, Dlx5 and Dlx6 play a critical role in driving tangential migration of GABAergic progenitors from the ventral telencephalon to their final destinations, notably the neocortex and the striatum. These Dlx genes are organised into convergently transcribed clusters with short intergenic regions that contain notable cis regulators elements (CREs) that drive Dlx expression in unique subdomains of the developing ventral telencephalon. Previous studies have characterised Dlx regulation including but not limited to the direct activation of these CREs by effector proteins. However, to date very little work has been done to examine how the forebrain Dlx genes may be regulated at the level of the chromatin. To explore this, I used in silico and in vivo methods to examine some key histone modifications of the Dlx1/2 and Dlx5/6 bigene clusters in the developing forebrain; namely H3K27Ac, H3K4me3, H3K4me1 and H3K27me3. I found that within the Dlx expressing ganglionic eminences (GE), at midgestation, the Dlx loci are marked by bivalent chromatin which is enriched in both permissive H3K4me3 and repressive H3K27me3 marks. By performing ChIP-qPCR on the GE tissue of embryonic mice with targeted deletions of enhancer CREs, I found that these CREs play unique roles in shaping the chromatin. Removal of one of these CREs has widespread effects on the chromatin at both loci. Since these changes in chromatin signatures do not accompany significant changes in expression of histone modifying genes, we believe these CREs play yet-to-be determined roles in recruiting the modifying proteins to the loci, thereby establishing bivalent chromatin to fine-tune Dlx expression.

Post-translational histone modifications (PTMs) such as methylation of lysine residues influence chromatin structure and function. PTMs are involved in different cellular processes such as DNA replication, transcription and cell differentiation. Deregulations of PTM patterns are responsible for a variety of human diseases including acute leukemia. DOT1 enzymes are highly conserved histone methyltransferases that are responsible for methylation of lysine 79 on histone H3 (H3K79). Most eukaryotes contain one single DOT1 enzyme, whereas African trypanosomes have two homologues, DOT1A and DOT1B, which methylate H3K76 (H3K76 is homologous to H3K79 in other organisms). DOT1A is essential and mediates mono- and di-methylations, whereas DOT1B additionally catalyzes tri-methylation of H3K76. However, a mechanistic understanding how these different enzymatic activities are achieved is lacking. This thesis exploits the fact that trypanosomes possess two DOT1 enzymes with different catalytic properties to understand the molecular basis for the differential product-specificity of DOT1 enzymes. A trypanosomal nucleosome reconstitution system was established to analyze methyltransferase activity under defined in vitro conditions. Homology modeling allowed the identification of critical residues within and outside the catalytic center that modulate product-specificity. Exchange of these residues transferred the product-specificity from one enzyme to the other and revealed regulatory domains adjacent to the catalytic center. This work provides the first evidence that few specific residues in DOT1 enzymes are crucial to catalyze methyl-state-specific reactions. These results have also consequences for the functional understanding of homologous enzymes in other eukaryotes. / Posttranslationale Histonmodifizierungen (PTMs), wie beispielsweise die Methylierung von Lysinseitenketten, beeinflussen maßgeblich die Struktur und Funktion von Chromatin. PTMs spielen eine wichtige Rolle in verschiedensten zellulären Prozessen, darunter DNA Replikation, Transkription oder Zelldifferenzierung. Darüber hinaus liegt ein verändertes PTM-Muster einer Vielzahl humaner Erkrankungen zugrunde, wie z.B. der akuten myeloischen Leukämie. DOT1-Enzyme sind hochkonservierte Histonmethyltransferasen, die für die Methylierung von Lysin 79 in Histon H3 (H3K79) verantwortlich sind. Im Gegensatz zu den meisten Eukaryoten, die lediglich ein einziges DOT1-Enzym besitzen, finden sich zwei homologe Proteine in afrikanischen Trypanosomen (DOT1A und DOT1B), die Lysin 76 in Histon H3 (H3K76) methylieren (H3K76 ist homolog zu H3K79 in anderen Organismen). DOT1A ist essentiell und katalysiert Mono- und Di-Methylierungen, wohin gegen DOT1B darüber hinaus eine Trimethylierung an H3K76 setzen kann. Derzeit fehlt jegliches mechanistische Verständnis darüber, wie beide Enzyme diese unterschiedliche Produktspezifität erreichen. Die vorliegende Dissertation macht sich den Umstand zunutze, dass Trypanosomen zwei DOT1-Methyltransferasen mit unterschiedlichen katalytischen Eigenschaften besitzen, um Einblicke in die molekulare Grundlage der unterschiedlichen Produktspezifität zu erlangen. Zunächst wurde ein Rekonstitutionssystem für Nukleosomen aus Trypanosomen etabliert, das es ermöglichte die Methyltransferase-Aktivitäten unter definierten in vitro Bedingungen zu analysieren. Homologiemodelle erlaubten die Identifikation von wichtigen Aminosäurepositionen innerhalb und außerhalb des katalytischen Zentrums der Enzyme, die einen Einfluss auf die Produktspezifität haben. Ein Austausch der Aminosäuren an diesen Positionen führte zu einer Umwandlung der Produktspezifität und offenbarte gleichzeitig DOT1A- und DOT1B-spezifische regulatorische Domänen, die an das katalytische Zentrum angrenzen. Diese Arbeit liefert erste Hinweise, dass wenige maßgebliche Aminosäuren in DOT1-Enzymen für den H3K76-Methylierungsgrad während der Katalyse entscheidend sind. Darüber hinaus haben die hier dargestellten Ergebnisse ebenfalls Konsequenzen für das funktionale Verständnis der homologen Enzyme in anderen Eukaryoten.

Regulation of gene expression is vital for proper growth and prevention of disease states. In eukaryotes this regulation occurs in the context of chromatin which creates an inherent barrier for the binding of trans-acting factors, such as transcription factors and RNA polymerase. This dissertation focuses on the role of transcriptional activators and chromatin remodeling coactivators in the regulation of the repressible acid phosphatase gene PHO5. Our studies show that histone methylation at lysine 4 of histone H3 is required for the full repression of PHO5and GAL1-10. We show that bromodomains, a domain conserved in chromatin remodeling coactivators, may function to stabilize binding. Finally, we present a strategy using DNA methyltransferases as in vivo probes to detect DNA-protein interactions and examine chromatin structure. We extend this strategy to zinc-finger proteins which can be engineered to bind to any desired DNA sequence as a means of targeting methylation with potential use in epigenetic silencing.