Universiẗat, Diss., 2004--München.

Techn. University, Diss., 2005--Dresden.

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.

The family of trypanosomatid parasites, including the human pathogens Trypanosoma brucei and Leishmania, has evolved sophisticated strategies to survive in harmful host environments. While Leishmania generate a safe niche inside the host’s macrophages, Trypanosoma brucei lives extracellularly in the mammalian bloodstream, where it is constantly exposed to the attack of the immune system. Trypanosoma brucei ensures its survival by periodically changing its protective surface coat in a process known as antigenic variation. The surface coat is composed of one species of ‘variant surface glycoprotein’ (VSG). Even though the genome possesses a large repertoire of different VSG isoforms, only one is ever expressed at a time from one out of the 15 specialized subtelomeric ‘expression sites’ (ES). Switching the coat can be accomplished either by a recombination-based exchange of the actively-expressed VSG with a silent VSG, or by a transcriptional switch to a previously silent ES. The conserved histone methyltransferase DOT1B methylates histone H3 on lysine 76 and is involved in ES regulation in T. brucei. DOT1B ensures accurate transcriptional silencing of the inactive ES VSGs and influences the kinetics of a transcriptional switch. The molecular machinery that enables DOT1B to execute these regulatory functions at the ES is still elusive, however. To learn more about DOT1B-mediated regulatory processes, I wanted to identify DOT1B-associated proteins. Using two complementary approaches, specifically affinity purification and proximity-dependent biotin identification (BioID), I identified several novel DOT1B-interacting candidates. To validate these data, I carried out reciprocal co-immunoprecipitations with the most promising candidates. An interaction of DOT1B with the Ribonuclease H2 protein complex, which has never been described before in any other organism, was confirmed. Trypanosomal Ribonuclease H2 maintains genome integrity by resolving RNA-DNA hybrids, structures that if not properly processed might initiate antigenic variation. I then investigated DOT1B’s contribution to this novel route to antigenic variation. Remarkably, DOT1B depletion caused an increased RNA-DNA hybrid abundance, accumulation of DNA damage, and increased VSG switching. Deregulation of VSGs from throughout the silent repertoire was observed, indicating that recombination-based switching events occurred. Encouragingly, the pattern of deregulated VSGs was similar to that seen in Ribonuclease H2-depleted cells. Together these data support the hypothesis that both proteins act together in modulating RNA-DNA hybrids to contribute to the tightly-regulated process of antigenic variation. The transmission of trypanosomatid parasites to mammalian hosts is facilitated by insect vectors. Parasites need to adapt to the extremely different environments encountered during transmission. To ensure their survival, they differentiate into various specialized forms adapted to each tissue microenvironment. Besides antigenic variation, DOT1B additionally affects the developmental differentiation from the mammalian-infective to the insect stage of Trypanosoma brucei. However, substantially less is known about the influence of chromatin-associated proteins such as DOT1B on survival and adaptation strategies of related Leishmania parasites. To elucidate whether DOT1B’s functions are conserved in Leishmania, phenotypes after gene deletion were analyzed. As in Trypanosoma brucei, generation of a gene deletion mutant demonstrated that DOT1B is not essential for the cell viability in vitro. DOT1B deletion was accompanied with a loss of histone H3 lysine 73 trimethylation (the lysine homologous to trypanosomal H3K76), indicating that Leishmania DOT1B is also solely responsible for catalyzing this post-translational modification. As in T. brucei, dimethylation could only be observed during mitosis/cytokinesis, while trimethylation was detectable throughout the cell cycle in wild-type cells. In contrast to the trypanosome DOT1B, LmxDOT1B was not essential for differentiation in vitro. However, preliminary data indicate that the enzyme is required for effective macrophage infection. In conclusion, this study demonstrated that the identification of protein networks and the characterization of protein functions of orthologous proteins from related parasites are effective tools to improve our understanding of the parasite survival strategies. Such insights are a necessary step on the road to developing better treatments for the devastating diseases they cause. / Vertreter der Familie der Trypanosomatidae einschließlich der humanpathogenen Trypanosoma brucei und Leishmania Arten entwickelten eine Reihe von ausgeklügelten Strategien, um in ihren Wirten zu überleben. Während sich Leishmanien eine sichere Nische in den Makrophagen ihrer Wirte aufbauen, lebt Trypanosoma brucei ausschließlich extrazellulär im Blutkreislauf der Säugetiere. Dort ist der Parasit ständig dem Angriff des Immunsystems ausgesetzt. Um sein Überleben zu sichern, wechselt er regelmäßig seine variablen Oberflächenproteine (VSG), eine Strategie, die auch als antigene Variation bekannt ist. Obwohl das Genom des Parasiten über ein enormes Repertoire an VSG Genen verfügt, wird immer nur eine einzige Art von einer von 15 spezialisierten telomerproximalen Expressionsstellen (ES) transkribiert. Um die VSG-Zelloberfläche zu wechseln, können Trypanosomen das VSG Gen der aktiven ES gegen ein inaktives VSG aus dem gigantischen Repertoire mittels Rekombination eintauschen. Eine weitere Möglichkeit ist der Transkriptionswechsel zu einer zuvor stillen ES. Die konservierte Histonmethyltransferase DOT1B katalysiert die Methylierung von Histon H3 am Lysin 76 und ist an der ES-Regulation beteiligt. DOT1B gewährleistet den transkriptionell inaktiven Status der ES und beeinflusst die Kinetik eines transkriptionellen ES Wechsels. Die molekularen Komponenten, die DOT1B diese regulatorischen Funktionen an der ES ermöglichen, sind jedoch noch unbekannt. Um mehr über die von DOT1B vermittelten Mechanismen zu erfahren, ist es notwendig, DOT1B-assoziierte Proteine zu identifizieren. Durch die Anwendung von komplementären biochemischen Proteinaufreinigungsmethoden gelang es mir, mehrere potentielle Proteininteraktionen zu DOT1B zu entdecken. Um die Daten zu validieren, führte ich weitere Proteinaufreinigungen mit den vielversprechendsten Kandidaten durch. Eine Interaktion zwischen DOT1B und der Ribonuklease H2 konnte bestätigt werden - eine Interaktion, die noch nie zuvor in anderen Organismen beschrieben wurde. In Trypanosomen gewährleistet Ribonuklease H2 die Genomintegrität, indem das Enzym RNA-DNA-Hybride auflöst. Diese Strukturen können zudem, wenn sie nicht richtig prozessiert werden, antigene Variation initiieren. In dieser Studie wurde daher außerdem DOT1B’s Beitrag zu diesem Weg der Initiation der antigenen Variation analysiert. In der Tat konnte gezeigt werden, dass DOT1B RNA-DNA-Hybride moduliert und die Genomintegrität sowie VSG-Wechselrate beeinflusst. Die Tatsache, dass in DOT1B-Mutanten VSG Isoformen von den unterschiedlichsten Genomregionen exprimiert wurden, deutet darauf hin, dass rekombinations-basierte Ereignisse dem VSG-Wechsel zu Grunde lagen. Da in den DOT1B-Mutanten ähnliche VSG exprimiert wurden wie in Ribonuklease H2-Mutanten, kann vermutet werden, dass beide Proteine bei der Modulation der RNA-DNA-Hybride zusammenwirken, um antigene Variation zu regulieren. Trypanosomen und Leishmanien werden mittels Insektenvektoren auf den nächsten Säugerwirt übertragen. Sie müssen daher nicht nur im Säugerwirt überleben, sondern sich auch an die extrem unterschiedliche Umgebung im Vektor anpassen. Dafür differenzieren sich die Parasiten in speziell angepasste Zellstadien. Zusätzlich zu der antigenen Variation beeinflusst DOT1B die Entwicklungsdifferenzierung in Trypanosoma brucei. In Leishmanien hingegen ist über den Einfluss von chromatin-assoziierten Proteinen wie DOT1B auf die Überlebens- und Anpassungsstrategien wesentlich weniger bekannt. Um herauszufinden, ob die Funktionen von DOT1B in Leishmanien konserviert sind, wurden Phänotypen nach Gendeletion analysiert. Wie auch in Trypanosoma brucei konnte gezeigt werden, dass DOT1B für das Überleben der Parasiten nicht essentiell ist. Die Deletion von DOT1B ging mit einem Verlust der Trimethylierung von Histon H3 am Lysin 73 (dem zum trypanosomalen H3K76 homologen Lysin) einher, was darauf hinweist, dass DOT1B auch in Leishmanien allein für die Katalyse dieser posttranslationalen Modifikation verantwortlich ist. Wie in Trypanosoma brucei konnte eine Dimethylierung nur in der Mitose/Zytokinese beobachtet werden, wobei die Trimethylierung während des gesamten Zellzyklus in Wildtyp-Zellen nachweisbar war. Im Gegensatz zum trypanosomalen DOT1B war LmxDOT1B für die Differenzierung in vitro entbehrlich. Vorläufige Daten zeigen jedoch, dass das Enzym für eine wirksame Makrophageninfektion wesentlich ist. Zusammenfassend zeigte diese Studie, dass die Identifizierung von Proteinnetzwerken und die Charakterisierung von Funktionen orthologer Proteine aus verwandten Parasiten wirksame Werkzeuge sind, um unser Verständnis der Überlebensstrategien der Parasiten zu verbessern. Solche Erkenntnisse sind ein notwendiger Schritt auf dem Weg zu effektiveren Behandlungsmethoden für die verheerenden Krankheiten, die diese Parasiten verursachen.

Mammals have two orthologs, Mll and Trx2, for the Drososphila protein Trithorax (TRX), which is the founding member of the trithorax group (TrxG) of epigenetic regulators. TrxG proteins are characterized by an evolutionary conserved SET domain. A major function of all SET domain- containing proteins is to modulate gene activity, but the underlying mechanisms are poorly understood. Apparently TRX, Mll and Trx2 are histone H3 lysine 4 specific methyltransferases. So far all evidence points to roles in expression of specific target genes. However, target genes and function of the epigenetic regulator Trx2 were still unknown. Homozygous trx2 mutant embryos arrest in development because of severe and widespread defects {Glaser, 2005 #296}. Thus mouse embryonic stem (ES) cells carrying a null mutation of trx2 were used as an alternative model system to address the implication of Trx2 in differentiation. This study showed that Trx2 is redundant for ES cell self-renewal. Homozygous trx2 knockout ES cells did not exhibit cell cycle defects. However, loss of Trx2 resulted in reduced proliferation and increased apoptosis rates in trx2-/- ES cells. Due to the fact that differentiation requires an appropriate rate of population growth, trx2-/- cells were affected adversely upon in vitro differentiation. Neurogeneic differentiation of trx2 mutant cells generated fewer mature neurons than wild type cells. Moreover a temporal delay in the developmental progression to differentiation became apparent. Cardiac differentiation of trx2-/- cells confirmed the developmental defect and temporal delay. Notably differentiation of trx2-/- cells was merely delayed or impaired but it was not absent, implying that Trx2 is not required for gene expression programs specific for neurons or cardiac myocytes. We propose that differentiation of trx2-/- ES cells is impaired because apoptosis is disturbing differentiation. Apart from analyzing the phenotype of trx2 mutant cells, this work was focused on the identification of Trx2 target genes. Oligonucleotide expression arrays were used to identify genes whose expression levels were affected by the absence of Trx2. In general, loss of Trx2 function resulted in more genes with decreased than increased expression levels. This is consistent with the hypothesis that Trx2 functions as a transcriptional activator. Comparison of gene expression profiles for constitutive and conditional trx2 mutant cells enabled a distinction between direct and indirect target genes for Trx2. As a result Magoh2 was identified as the key candidate target gene for Trx2. Interaction between Trx2 and Magoh2 suggested a potential regulatory role for Trx2 in alternative splicing. Furthermore this work provided evidence that Trx2 could be involved in the maintenance of CpG island promoter gene expression, thus providing a potent regulatory mechanism for ubiquitously expressed genes.

Mammals have two orthologs, Mll and Trx2, for the Drososphila protein Trithorax (TRX), which is the founding member of the trithorax group (TrxG) of epigenetic regulators. TrxG proteins are characterized by an evolutionary conserved SET domain. A major function of all SET domain- containing proteins is to modulate gene activity, but the underlying mechanisms are poorly understood. Apparently TRX, Mll and Trx2 are histone H3 lysine 4 specific methyltransferases. So far all evidence points to roles in expression of specific target genes. However, target genes and function of the epigenetic regulator Trx2 were still unknown. Homozygous trx2 mutant embryos arrest in development because of severe and widespread defects {Glaser, 2005 #296}. Thus mouse embryonic stem (ES) cells carrying a null mutation of trx2 were used as an alternative model system to address the implication of Trx2 in differentiation. This study showed that Trx2 is redundant for ES cell self-renewal. Homozygous trx2 knockout ES cells did not exhibit cell cycle defects. However, loss of Trx2 resulted in reduced proliferation and increased apoptosis rates in trx2-/- ES cells. Due to the fact that differentiation requires an appropriate rate of population growth, trx2-/- cells were affected adversely upon in vitro differentiation. Neurogeneic differentiation of trx2 mutant cells generated fewer mature neurons than wild type cells. Moreover a temporal delay in the developmental progression to differentiation became apparent. Cardiac differentiation of trx2-/- cells confirmed the developmental defect and temporal delay. Notably differentiation of trx2-/- cells was merely delayed or impaired but it was not absent, implying that Trx2 is not required for gene expression programs specific for neurons or cardiac myocytes. We propose that differentiation of trx2-/- ES cells is impaired because apoptosis is disturbing differentiation. Apart from analyzing the phenotype of trx2 mutant cells, this work was focused on the identification of Trx2 target genes. Oligonucleotide expression arrays were used to identify genes whose expression levels were affected by the absence of Trx2. In general, loss of Trx2 function resulted in more genes with decreased than increased expression levels. This is consistent with the hypothesis that Trx2 functions as a transcriptional activator. Comparison of gene expression profiles for constitutive and conditional trx2 mutant cells enabled a distinction between direct and indirect target genes for Trx2. As a result Magoh2 was identified as the key candidate target gene for Trx2. Interaction between Trx2 and Magoh2 suggested a potential regulatory role for Trx2 in alternative splicing. Furthermore this work provided evidence that Trx2 could be involved in the maintenance of CpG island promoter gene expression, thus providing a potent regulatory mechanism for ubiquitously expressed genes.