Methylation of lysine residues in histone tails is an intensively studied epigenetic signal that regulates transcription throughout development. Methylation of histone 3 lysine 4 (H3K4) is usually associated with promoters of actively transcribed genes whereas H3K27 or H3K9 methylation silences genes. Yeast possess only one H3K4 methyltransferase, Set1. In contrast, there are six enzymes capable of catalyzing this modification in mammals implying a certain specialization or division of labor. The present study examined the functions of the mouse H3K4 methyltransferase paralogs, Mixed Lineage Leukemia 1 (Mll1) and Mll2, during neural differentiation and reprogramming of neural stem (NS) cells to induced pluripotency. We could show that Mll2 is required for differentiation of embryonic stem (ES) cells to neural progenitors and identified Nuclear transport factor 2-like export factor 2 (Nxt2) as essential target gene. Mll2 trimethylated the Nxt2 promoter in ES cells in order to allow for transcriptional upregulation during subsequent neural differentiation. Additionally, Mll2 prevented apoptosis of differentiating cells by regulating B cell leukemia/lymphoma 2 (Bcl2) levels. Mll1 could replace Mll2 after the first steps of cell commitment towards epiblast stem (EpiS) cells. While Mll2 activity was only required briefly when ES cells started to differentiate, the influence of Mll1 seemed to increase with developmental progression. It stabilized the NS cell state by regulating expression of the neural transcription factor Orthodenticle homolog 2 (Otx2). Thereby, Mll1 impeded early steps of reprogramming to induced pluripotency and its inactivation increased the efficiency. Besides their specificity for certain target genes, both enzymes also differed in their activity. The major function of Mll1 was to prevent silencing by H3K27 methylation and possibly recruitment of transcription factors. In contrast, Mll2 conducted H3K4 trimethylation of its target genes. Importantly, once established in NS cells, the expression of Nxt2 became independent of promoter H3K4 methylation. Thus, Mll2 and its target gene Nxt2 represent an example for H3K4 methylation functioning as priming mechanism rather than for fine-tuning or maintenance of transcription levels.

Methylation of lysine residues in histone tails is an intensively studied epigenetic signal that regulates transcription throughout development. Methylation of histone 3 lysine 4 (H3K4) is usually associated with promoters of actively transcribed genes whereas H3K27 or H3K9 methylation silences genes. Yeast possess only one H3K4 methyltransferase, Set1. In contrast, there are six enzymes capable of catalyzing this modification in mammals implying a certain specialization or division of labor. The present study examined the functions of the mouse H3K4 methyltransferase paralogs, Mixed Lineage Leukemia 1 (Mll1) and Mll2, during neural differentiation and reprogramming of neural stem (NS) cells to induced pluripotency. We could show that Mll2 is required for differentiation of embryonic stem (ES) cells to neural progenitors and identified Nuclear transport factor 2-like export factor 2 (Nxt2) as essential target gene. Mll2 trimethylated the Nxt2 promoter in ES cells in order to allow for transcriptional upregulation during subsequent neural differentiation. Additionally, Mll2 prevented apoptosis of differentiating cells by regulating B cell leukemia/lymphoma 2 (Bcl2) levels. Mll1 could replace Mll2 after the first steps of cell commitment towards epiblast stem (EpiS) cells. While Mll2 activity was only required briefly when ES cells started to differentiate, the influence of Mll1 seemed to increase with developmental progression. It stabilized the NS cell state by regulating expression of the neural transcription factor Orthodenticle homolog 2 (Otx2). Thereby, Mll1 impeded early steps of reprogramming to induced pluripotency and its inactivation increased the efficiency. Besides their specificity for certain target genes, both enzymes also differed in their activity. The major function of Mll1 was to prevent silencing by H3K27 methylation and possibly recruitment of transcription factors. In contrast, Mll2 conducted H3K4 trimethylation of its target genes. Importantly, once established in NS cells, the expression of Nxt2 became independent of promoter H3K4 methylation. Thus, Mll2 and its target gene Nxt2 represent an example for H3K4 methylation functioning as priming mechanism rather than for fine-tuning or maintenance of transcription levels.

Epigenetic mechanisms are of fundamental importance for resolving and maintaining cellular identity. The mechanisms regulating muscle stem and progenitor cell identity have ramifications for understanding all aspects of myogenesis. The epigenetic mechanisms regulating muscle stem cells are therefore important aspects for understanding the regulation of muscle regeneration and maintenance. Important roles for the trithorax H3K4 histone methyltransferase (HMT) MLL1 have been established for early embryogenesis, and for hematopoietic and neural identity. Here, using a conditional Mll1 knockout (KO), we find that in vivo, MLL1 is necessary for efficient muscle regeneration, and for maintenance and proliferation of muscle stem and progenitor cells. Loss of Mll1 in cultured myoblasts reveals an essential role for expression of the myogenic specification gene Pax7. Mll1 KO results in a minor decrease in Pax7 mRNA and a strong decrease of Pax7 protein. While MLL1 was found to bind the Pax7 promoter, Mll1 KO results in a minor decrease of H3K4me3 at Pax7, supporting a recognized non-HMT role for Mll1 at Pax7. Microarray analysis of mRNA expression in Mll1 KO myoblasts finds that Myf5 is the most strongly downregulated of all genes, unexpectedly, mRNA expression of previously identified MLL1 targets are unaffected by loss of MLL1 in myoblasts. Pax7 activates Myf5 expression through recruitment of a H3K4 HMT, and in Mll1 KO myoblasts expression of, and H3K4me3 at Myf5 is lost. Exogenous Pax7 rescues Myf5 expression and H3K4me3 at Myf5 in the absence of MLL1, indicating that Myf5 expression is conditional on Pax7, but not MLL1. We also show that Myf5 DNA is methylated in non-myogenic cells, and in satellite stem cells that have never expressed Myf5, but is not methylated in satellite cells that are committed to the myogenic lineage, indicating that demethylation of Myf5 may be a fundamental step in myogenic commitment. Intriguingly, Myf5 promoter DNA becomes remethylated in Mll1 KO myoblasts. This work finds that Pax7 expression and myogenic identity is partly dependent on MLL1 expression. Further, evidence is uncovered that myogenic commitment is initiated by demethylation of Myf5. These findings add to the understanding of the epigenetic mechanisms that regulate and define muscle stem cells.

Genetisch bedingte Veränderungen im Wnt-Signalweg sind in der Tumorigenese des Darms von zentraler Bedeutung. Mutationen des Wnt-Effektormoleküls β-Catenin in den adulten Stammzellen des Darmepithels führen zu unkontrollierter Proliferation und Expansion der Darmstammzellen und initiieren die Tumorentstehung. Auch in fortgeschrittenen Darmtumoren unterstützt die Wnt-Signalgebung maßgeblich das Tumorwachstum und den Erhalt von Tumorstammzellen. Nach erfolgreicher chemotherapeutischer Behandlung treten oftmals Tumorrezidive auf, für deren Entstehung therapieresistente Tumorstammzellen verantwortlich gemacht werden. Trotz intensiver Forschung fehlen in der Darmkrebstherapie nach wie vor Behandlungsansätze zur gezielten Therapie der Tumorstammzellen. Ziel dieser Dissertation ist es, unser Verständnis der molekularen Regulationsmechanismen in Kolonkarzinomen zu erweitern und die Entwicklung rationaler Behandlungsstrategien zu fördern. Ich konnte die Histonmethyltransferase Mll1 als entscheidenden Faktor in der epigenetischen Regulation humaner und muriner Darmkrebsstammzellen und -tumore identifizieren. Humane Kolonkarzinome weisen eine erhöhte Mll1-Expression auf, die mit dem Level an nukleärem β-Catenin korreliert. Im adulten Darmepithel ist Mll1 insbesondere in den Lgr5+ Stammzellen exprimiert und maßgeblich an der Wnt/β-Catenin-induzierten Stammzellexpansion sowie der Tumorentstehung beteiligt. Der konditionelle Verlust von Mll1 im murinen Darmkrebsmodell verhindert die β-Catenin-induzierte Tumorigenese. Mll1 unterstützt die Selbsterneuerungsfähigkeit und Proliferation der Tumorstammzellen, indem es die Expression von essentiellen Stammzellgenen wie dem Wnt-abhängigen Stammzellmarker Lgr5 aufrechterhält. Eine Inhibition der Mll1-Funktion in der Darmkrebstherapie kann eine gezielte Eliminierung der Tumorstammzellen ermöglichen, wodurch das fortschreitende Tumorwachstum unterbunden und die Bildung von Rezidiven verhindert werden kann. / Genetic mutations inducing aberrant activity of Wnt signalling are causative for intestinal tumorigenesis. Mutations of the Wnt effector molecule β-catenin in adult stem cells of the intestinal epithelium drive uncontrolled proliferation, expand the stem cell pool and initiate tumor formation. In advanced tumors, aberrant Wnt signalling promotes tumor growth and maintains cancer stem cells. The cancer stem cells are highly resistant to conventional chemotherapy and frequently initiate tumor relapse after completion of treatment. Despite extensive research, we are still lacking efficient therapies for colon cancer that specifically eliminate the cancer stem cells. This dissertation aims to expand our knowledge on molecular gene regulatory mechanisms in colon cancer cells to promote the identification and future development of rational therapies for colon cancer patients. I identified the histone methyltransferase Mll1 as an epigenetic regulator in human and mouse intestinal cancer stem cells and tumors. Human colon carcinomas with nuclear β-catenin exhibit high levels of Mll1. In the adult intestinal epithelium of mice, Mll1 is highly expressed in the Lgr5+ stem cells and is a prerequisite for the oncogenic Wnt/β-catenin-mediated stem cell expansion and tumorigenesis. Conditional knockout of Mll1 in an intestinal mouse tumor model prevents the β-catenin-driven intestinal tumorigenesis. Knockdown of Mll1 impairs the self-renewal and proliferation of colon cancer sphere cultures and halts tumor growth in xenografts. Mechanistically, Mll1 sustains the expression of intestinal stem cell genes including the Wnt/β-catenin target gene Lgr5 by antagonizing gene silencing through polycomb repressive complex 2-mediated H3K27 tri-methylation. Interfering with Mll1 function can efficiently eliminate colon cancer stem cells, and has potential as a rational therapy for colon cancer.

The mixed lineage leukemia (MLL) proteins regulate an array of developmental and differentiation processes. Similar to other members of the SET1 family, association of MLL1-4 with Ash2L, RbBP5 and WDR5, collectively termed the MLL core complex, is required for MLL mediated histone H3 Lys-4 di/tri-methylation. Each member of the core complex has a unique role in modulating the activity of MLL1. WDR5 is key in nucleating the formation of the core complex by acting as a structural scaffold, whereas Ash2L and RbBP5 are responsible for stimulating MLL methyltransferase activity. Currently, the structural and biochemical mechanisms utilized by the core complex to regulate MLL1 activity are unknown. Through structural and biochemical dissection of the core complex we have assigned specific functions to core complex subunits and have identified the minimal structural requirements for methyltransferase activity. Furthermore, through structure based drug design, we have identified a peptidomimetic inhibitor of MLL1 methyltransferase activity.

The mixed lineage leukemia (MLL) proteins regulate an array of developmental and differentiation processes. Similar to other members of the SET1 family, association of MLL1-4 with Ash2L, RbBP5 and WDR5, collectively termed the MLL core complex, is required for MLL mediated histone H3 Lys-4 di/tri-methylation. Each member of the core complex has a unique role in modulating the activity of MLL1. WDR5 is key in nucleating the formation of the core complex by acting as a structural scaffold, whereas Ash2L and RbBP5 are responsible for stimulating MLL methyltransferase activity. Currently, the structural and biochemical mechanisms utilized by the core complex to regulate MLL1 activity are unknown. Through structural and biochemical dissection of the core complex we have assigned specific functions to core complex subunits and have identified the minimal structural requirements for methyltransferase activity. Furthermore, through structure based drug design, we have identified a peptidomimetic inhibitor of MLL1 methyltransferase activity.

The mixed lineage leukemia (MLL) proteins regulate an array of developmental and differentiation processes. Similar to other members of the SET1 family, association of MLL1-4 with Ash2L, RbBP5 and WDR5, collectively termed the MLL core complex, is required for MLL mediated histone H3 Lys-4 di/tri-methylation. Each member of the core complex has a unique role in modulating the activity of MLL1. WDR5 is key in nucleating the formation of the core complex by acting as a structural scaffold, whereas Ash2L and RbBP5 are responsible for stimulating MLL methyltransferase activity. Currently, the structural and biochemical mechanisms utilized by the core complex to regulate MLL1 activity are unknown. Through structural and biochemical dissection of the core complex we have assigned specific functions to core complex subunits and have identified the minimal structural requirements for methyltransferase activity. Furthermore, through structure based drug design, we have identified a peptidomimetic inhibitor of MLL1 methyltransferase activity.

The mixed lineage leukemia (MLL) proteins regulate an array of developmental and differentiation processes. Similar to other members of the SET1 family, association of MLL1-4 with Ash2L, RbBP5 and WDR5, collectively termed the MLL core complex, is required for MLL mediated histone H3 Lys-4 di/tri-methylation. Each member of the core complex has a unique role in modulating the activity of MLL1. WDR5 is key in nucleating the formation of the core complex by acting as a structural scaffold, whereas Ash2L and RbBP5 are responsible for stimulating MLL methyltransferase activity. Currently, the structural and biochemical mechanisms utilized by the core complex to regulate MLL1 activity are unknown. Through structural and biochemical dissection of the core complex we have assigned specific functions to core complex subunits and have identified the minimal structural requirements for methyltransferase activity. Furthermore, through structure based drug design, we have identified a peptidomimetic inhibitor of MLL1 methyltransferase activity.

No description available.

Récemment, des analyses dans divers organismes eucaryotes ont révélé que l'ensemble du génome est transcrit et produit en plus des ARNs messagers, une grande variété d’ARNs non codants de différentes longueurs. Les ARNs non codants de plus de 200 nucleotides, classés comme longs ARNs non codants (LARNnc), représentent la classe la plus abondante de transcripts non codants. Les études des fonctions des LARNnc suggèrent que beaucoup d'entre eux seraient impliqués dans la régulation de la transcription. L'objectif de ma thèse de doctorat était d'élucider les mécanismes de la régulation transcriptionnelle médiée par des LARNnc dans différents systèmes eucaryotes. Dans mon premier projet, j'ai étudié le rôle d'un long ARN non codant antisens dans la régulation transcriptionnelle du gène PHO84, codant un transporteur de phosphate à haute affinité, chez S. cerevisiae. Des études antérieures ont montré que la suppression d’une proteine de l’exosome Rrp6 entraîne une augmentation de l'expression antisens et la répression de PHO84. Il a été suggéré que la perte de Rrp6 entraîne une stabilisation antisens au locus PHO84, entraînant le recrutement de l'histone de-acétylase Hda1 et la répression de PHO84. Cependant, le mécanisme par lequel Rrp6p régule la transcription de PHO84 n’était pas connu. En combinant des méthodes à l’échelle de cellule unique, des approches biochimiques et génétiques, nous avons montré que les niveaux d'ARN antisens sont régulés principalement lors de l'élongation par le complexe Nrd1-Nab3-Sen1, qui nécessite Rrp6 pour un recrutement efficace à l`extrémité 3`de PHO84. De plus, nous révélons l'expression anticorrelé du sens et de l'antisens, En résumé, nos données suggèrent que la transcription antisens régule le seuil d'activation du promoteur PHO84. Dans mon second projet, j'ai étudié les rôles des ARNs dérivés des amplificateurs (ARNa) dans la regulation de la transcription. En utilisant les cellules de cancer du sein MCF7 comme système modèle, nous avons cherché à déterminer comment les ARNa induits par l'oestrogène (E2) participent à la régulation de la transcription médiée par le recepteur d’oestrogène (ERα) au niveau de l'allèle unique. À l'aide de l’hybridation fluorescente à l’échelle de molécule unique (smFISH), nous avons révélé qu`après induction d'E2, les ARNa sont induits avec une cinétique similaire à celle des ARNm cibles, sont localisés exclusivement dans le noyau, principalement associés à la chromatine, et sont moins abondants que les ARNm. De manière surprenante, nous avons constaté que les ARNa sont rarement co-transcrits avec leurs loci cibles, indiquant que la transcription active des gènes ne nécessite pas la synthèse continue ou l'accumulation d'ARNa sur l'amplificateur. En outre, en utilisant des mesures de la distance à sous-diffraction, nous avons démontré que la cotranscription des ARNa et des ARNm se produit rarement dans une boucle amplificateurpromoteur. De plus, nous avons révélé que la transcription basale d'ARNa n'exige pas ERα ou l'histone méthyltransférase MLL1 qui active l'amplificateur par la mono-méthylation H3K4. Dans l'ensemble, nos résultats ont montré que les ARNa peuvent jouer un rôle lors de l'activation du promoteur, mais ne sont pas nécessaires pour maintenir la transcription de l'ARNm ou pour stabiliser les interactions amplificateur-promoteur. / Transcription is the initial step in gene expression and is subject to extensive regulation. Recently, analyses in diverse eukaryotes have revealed that in addition to protein coding genes, transcription occurs throughout the noncoding genome, producing non-coding RNAs of various lengths. Non-coding RNAs longer than 200 nucleotides, classified as long non-coding RNAs (lncRNAs), represent the most abundant class of non-coding transcripts, whose functions however are poorly understood. Recent studies suggest that many lncRNAs might have roles in transcription regulation. The goal of my PhD thesis was to elucidate the mechanisms of lncRNA mediated transcription regulation in different eukaryotic systems. For my first project, I investigated the role of an antisense long noncoding RNA in transcription regulation of the high-affinity phosphate transporter gene PHO84 in the unicellular eukaryote S. cerevisiae. Previous studies showed that deletion of the nuclear exosome component Rrp6 results in increased antisense expression and repression of PHO84. It was suggested that the loss of Rrp6 results in antisense stabilization at the PHO84 locus, leading to recruitment of the histone de-acetylase Hda1 and repression of PHO84. However, most of the mechanistic details of how Rrp6p functions in regulating PHO84 transcription were not understood. Combining single cell methods with biochemical and genetic approaches, we showed that antisense RNA levels are regulated primarily during transcriptional elongation by the Nrd1-Nab3-Sen1 complex, which requires Rrp6 for efficient recruitment to the 3’end of PHO84. Furthermore, we reveal anti-correlated expression of sense and antisense, which have distinct modes of transcription. In summary, our data suggest a model whereby antisense transcriptional read-through into the PHO84 promoter regulates the activation threshold of the gene. For my second project, I investigated the roles of enhancer derived RNAs (eRNAs). eRNAs are lncRNAs transcribed from enhancers that have been suggested to regulate transcription through different mechanisms, including enhancer-promoter looping, RNA polymerase elongation, and chromatin remodeling. However, no coherent model of eRNA function has yet emerged. Using MCF7 breast cancer cells as a model system, we sought to determine how estrogen (E2) induced eRNAs participate in estrogen receptor alpha (ERα) mediated transcription regulation at the single allele level. Using single molecule fluorescent in situ hybridization (smFISH), we revealed that upon E2 induction eRNAs are induced with similar kinetics as target mRNAs, but are localized exclusively in the nucleus, mostly chromatin associated, and are less abundant than mRNAs. Surprisingly, we found that eRNAs are rarely co-transcribed with their target loci, indicating that active gene transcription does not require the continuous synthesis or accumulation of eRNAs at the enhancer. Furthermore, using sub-diffraction-limit distance measurements, we demonstrated that co-transcription of eRNAs and mRNAs rarely occurs within a closed enhancer-promoter loop. Moreover, we revealed that basal eRNA transcription does not require ERα or the histone methyltransferase MLL1, which activates the enhancer through H3K4 mono-methylation. Altogether, our findings showed that eRNAs may play a role during promoter activation, but are not required to sustain mRNA transcription or stabilize enhancer-promoter looping interactions.