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.

Zusammenfassung Die Asteraceae Brachycome dichromosomatica ist eine besonders geeignete Spezies, um B-Chromosomen zu analysieren. Die auf den B-Chromosomen-lokalisierte 45S rDNA wurde auf Ursprung und Funktion untersucht. Die Mikrodissektion von B-Chromosomen und PCR-Amplifikation ermöglichte es, B-Chromosomen-spezifische ITS2-Sequenzen der 45S rDNA zu erhalten. Auffallend bei dieser Analyse waren zwei beständige Differenzen zwischen den Sequenzen von A- und B-Chromosomen. Phylogenetische Untersuchungen identifizierten keine Spezies, die eine ITS2-Sequenz hatte, die ähnlicher zu der B-Chromosomen-ITS2-Sequenz war als die A-Chromosomen-ITS2-Sequenz von B. dichromosomatica. Es wurde ein Ursprung der B-Chromosomen in der Zeit vor der Ausbildung der vier Cytodeme von B. dichromosomatica postuliert. Die Analyse der Assoziationen von Mikro-B-Chromosomen mit dem Nukleolus ergab, dass 70% der Mikro-B-Chromosomen nicht mit dem Nukleolus assoziierten. Die hohe Frequenz von nichtassoziierten Mikro-B-Chromosomen weist auf eine Inaktivität der Mikro-B-Chromosomen-lokalisierten 45S rDNA hin. Die Immunfluoreszenzmarkierung zeigte, dass sich das Chromatin der A- und B-Chromosomen deutlich in der euchromatischen Histon-H3-Methylierung unterscheidet. Während die A-Chromosomen deutliche Immunfluoreszenzsignale aufwiesen, zeigten die Mikro-B- und Standard-B-Chromosomen nur eine schwache Markierung mit Antikörpern gegen Histon H3K4me1,2,3, H3K9me3 und H3K27me2,3. Die heteropygnotischen, mit Tandem-Repeats angereicherten Mikro-B-Chromosomen waren dabei noch weniger mit diesen euchromatischen Markierungen gekennzeichnet als die Standard-B-Chromosomen. Keine Unterschiede zwischen den A- und B-Chromosomen wurden für die heterochromatischen Markierungen Histon H3K9me1,2 und H3K27me1 gefunden, was darauf hinweist, dass die B-Chromosomen nicht spezifisch durch zusätzliche heterochromatische Histonmarkierungen gekennzeichnet sind. / Summary The Asteraceae Brachycome dichromosomatica is a suitable species for the analysis of B chromosomes (Bs). The origin and activity of micro B-located 45S rDNA of was analysed. Microisolation of Bs and PCR with internal transcribed spacer 2 (ITS2)-specific primers succeeded in the isolation of B-specific ITS2-sequences. ITS2 was sequenced for micro B, large B and A chromosomes, and conserved differences were identified between sequences originating from A and both types of Bs. Phylogenetic analysis did not identify a species that contained an ITS2 sequence that was more similar to either of the B’s sequences than that of the B. dichromosomatica A chromosomes (As). Thus, an origin of the Bs from As at a time prior to the divergence of the four cytodemes of B. dichromosomatica is suggested. Because 70% of micro Bs did not co-localize with the nucleolus I conclude that micro B-located 45S rDNA is not constitutively transcribed. Immunofluorescence demonstrates that the chromatin in A and both types of Bs differs markedly in euchromatic histone H3 methylation marks. While A chromosomes are labelled brightly, the micro B and large Bs are faintly labelled with antibodies against H3K4me2/3, H3K9me3 and H3K27me2/3. The heteropycnotic, tandem-repeat enriched micro Bs were even less labelled with euchromatic histone H3 methylation marks than large Bs. No differences between A and Bs were found as to the heterochromatic marks H3K9me1/2 and H3K27me1, indicating that Bs are not additionally labelled by heterochromatin typical histone H3 modifications. 1

Abstract 2-oxoglutarate-dependent dioxygenases (2-OGDDs) are an enzyme family that contains many enzymes that modify chromatin in extensive ways. These enzymes include several histone lysine demethylases (KDMs) and TET enzymes that convert methylated cytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) ultimately leading to DNA demethylation. Disturbed DNA and histone methylation are found in many cancers. However, the role of KDMs and TETs behind these oncogenic changes has so far not been fully investigated. This study focused on the role of these chromatin-modifying enzymes in cancers with special emphasis on enzyme kinetic studies. Cancers with inactivating fumarate hydratase (FH), succinate dehydrogenase (SDH) and isocitrate dehydrogenase (IDH) mutations accumulate fumarate, succinate and R-2-hydroxyglutarate, respectively. In this study we showed how these cancer-associated 2-oxoglutarate (2-OG) analogues can inhibit the TET enzymes and many of the KDMs leading to lower 5-hmC levels and increased H3K27 and H3K9 methylation on chromatin, respectively. We also characterized kinetic properties of acute myeloid leukaemia (AML)-associated TET2 mutants and found that their ability to bind 2-OG or iron was impaired leading to diminished catalytic activity. Tumours are often hypoxic due to inadequate vasculature and blood supply. The TET enzymes and KDMs require oxygen for the reactions they catalyse. We determined the oxygen affinity of TETs and many KDMs and found that a H3K27 demethylase KDM6A has a remarkably low affinity for oxygen indicating that it is inactivated in hypoxic tumours and tissues. H3K27 methylation was found to be increased in hypoxic cells and this blocked cell differentiation. Altogether, these studies shed light on the mechanisms behind the altered DNA and histone methylation found in several cancers with hypoxic conditions or FH, SDH and IDH mutations. Altered DNA and histone methylation has previously been associated with progression of cancer, such as epithelial-to-mesenchymal transition (EMT). We now linked catalytic inhibition of 2-OGDDs to disturbed DNA and histone methylation that can account for altered cell differentiation, EMT and increased aggressiveness and invasiveness of cancers. / Tiivistelmä 2-oksoglutaraatista riippuvaiset dioksygenaasit ovat entsyymiperhe, johon kuuluu useita entsyymejä, jotka muokkaavat kromatiinin epigeneettisiä merkkejä monin tavoin. Näitä entsyymejä ovat mm. DNA:n demetylaatioon vaikuttavat TET-entsyymit sekä useat histonidemetylaasit. Vaikka muutoksia DNA:n ja histonien metylaatiotasoissa on havaittu useissa syövissä, ei näiden entsyymien roolia muutosten taustalla ole vielä tutkittu. Tämä tutkimus kohdistui näiden epigenetiikkaan vaikuttavien entsyymien roolin ymmärtämiseen syövissä keskittyen erityisesti kyseisten entsyymien kinetiikkaan. Useissa syövissä on havaittu fumaraattihydrataasin, sukkinaattidehydrogenaasin ja isositraattidehydrogenaasien aktiivisuuteen vaikuttavia mutaatioita, jotka johtavat fumaraatin, sukkinaatin ja R-2-hydroksiglutaraatin kertymiseen syöpäsoluihin. Tässä tutkimuksessa osoitimme, kuinka nämä karsinogeeniset 2-oksoglutaraattianalogit voivat inhiboida TET-entsyymejä ja histonidemetylaaseja, mikä alentaa 5-hydroksimetyylisytosiinitasoja ja lisää histonien metylaatiota. Näytämme myös, kuinka tietyillä akuutissa myelooisessa leukemiassa esiintyvillä TET2-mutanteilla on heikentynyt kyky sitoa 2-oksoglutaraattia tai rautaa, mikä johtaa entsyymien aktiivisuuden laskuun. Kasvainkudoksissa happipitoisuudet ovat usein matalia nopean kasvun ja puutteellisen verisuonituksen vuoksi. TET-entsyymit ja histonidemetylaasit vaativat happea katalysoimissaan reaktioissa. Määritimme TET-entsyymien ja monien histonidemetylaasien riippuvuutta hapesta ja osoitimme, että H3K27-histonidemetylaasi KDM6A on erittäin riippuvainen hapesta, mikä osoittaa, ettei se pysty toimimaan kasvaimissa ja kudoksissa, joissa happipitoisuudet ovat matalia. Huomasimme, että vähähappisissa olosuhteissa solujen H3K27 metylaatio on lisääntynyt, mikä johti erilaistumisen estymiseen soluissa. Tämä tutkimus paljasti uusia mekanismeja useista syövistä löytyneiden muuntuneiden DNA:n ja histonien metylaatiotasojen taustalla. Häiriintynyt DNA:n ja histonien metylaatio on aiemmin yhdistetty syöpien etenemiseen, erityisesti solujen erilaistumisen häiriintymisen kannalta. Tässä tutkimuksessa yhdistimme 2-oksoglutaraatista riippuvaisten entsyymien inhibition häiriintyneeseen DNA:n ja histonien metylaatioon, joka voi johtaa muuntuneeseen solujen erilaistumiseen ja lopulta lisääntyneeseen syöpien aggressiivisuuteen ja invasiivisuuteen.

Indiana University-Purdue University Indianapolis (IUPUI) / This dissertation describes structure-function studies of CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, in order to determine the functional significance of Cfp1 protein domains and properties. Cfp1 is an important regulator of chromatin structure and is essential for mammalian development. Murine embryonic stem (ES) cells lacking Cfp1 (CXXC1-/-) are viable but demonstrate a variety of defects, including hypersensitivity to DNA damaging agents, reduced plating efficiency and growth, decreased global and gene-specific cytosine methylation, failure to achieve in vitro differentiation, aberrant histone methylation, and subnuclear mis-localization of Setd1A, the catalytic component of a histone H3K4 methyltransferase complex, and tri-methylated histone H3K4 (H3K4me3) with regions of heterochromatin. Expression of wild-type Cfp1 in CXXC1-/- ES cells rescues the observed defects, thereby providing a convenient method to assess structure-function relationships of Cfp1. Cfp1 cDNA expression constructs were stably transfected into CXXC1-/- ES cells to evaluate the ability of various Cfp1 fragments and mutations to rescue the CXXC1-/- ES cell phenotype. These experiments revealed that expression of either the amino half of Cfp1 (amino acids 1-367) or the carboxyl half of Cfp1 (amino acids 361-656) is sufficient to rescue the hypersensitivity to DNA damaging agents, plating efficiency, cytosine and histone methylation, and differentiation defects. These results reveal that Cfp1 contains redundant functional domains for appropriate regulation of cytosine methylation, histone methylation, and in vitro differentiation. Additional studies revealed that a point mutation (C169A) that abolishes DNA-binding activity of Cfp1 ablates the rescue activity of the 1-367 fragment, and a point mutation (C375A) that abolishes the interaction of Cfp1 with the Setd1A and Setd1B histone H3K4 methyltransferase complexes ablates the rescue activity of the 361-656 Cfp1 fragment. In addition, introduction of both point mutations (C169A and C375A) ablates the rescue activity of the full-length Cfp1 protein. These results indicate that retention of either DNA-binding or Setd1 association of Cfp1 is required to rescue hypersensitivity to DNA damaging agents, plating efficiency, cytosine and histone methylation, and in vitro differentiation. In contrast, confocal immunofluorescence analysis revealed that full-length Cfp1 is required to restrict Setd1A and histone H3K4me3 to euchromatic regions.

No description available.

Mi trabajo ha estado enfocado en la implicación de los diferentes mecanismos epigenéticos de PML-RARa en la inducción de la leucemia promielocítica aguda (APL).En particular yo estudié el rol de MBD1, un miembro de la conservada familia de proteinas capaces de unirse al DNA metilado, demostrando que desempeña un papel importante en la progresión de la leucemia. De hecho, mostré que MBD1 es recruida por PML-RARa a sus promotores diana a través de los mecanismos mediados por HDAC3, participando por tanto en la represión transcripcional. Además, investigué hasta donde la metilación de la H3K27 mediada por Polycomb contribuye a la tumorgénesis mediada por PML-RARa. Demostré que PML-RARa dirige al PRC2 hacia el locus del tumor supresor causando la metilación de la H3K27. Fue interesante ser capaz de mostrar que tanto la metilación del DNA como la de las histonas era requerida para mantener el aberrante silencio génico. Esto apuntaba hacia una intercomunicación entre estos diferentes marcadores epigenéticos contribuyendo a la patología molecular de la leucemia. Resumiendo, estos resultados nos proporcionan elementos nuevos para comprender los mecanismos moleculares esenciales en la tumorgénesis y progresión de la APL. / My work was focused on the involvement of different epigenetic mechanisms in PML-RARa-induced acute promyelocytic leukemia (APL). In particular, I studied the role of MBD1, a member of a conserved family of proteins able to bind methylated DNA, demonstrating that has an important function in leukemia progression. Indeed, I showed that MBD1 is recruited by PML-RARa to its target promoters through an HDAC3-mediated mechanism, thus participating in transcriptional repression.. Furthermore, I investigated how far Polycomb-mediated H3K27 methylation contributes to PML-RARa mediated tumorigenesis. I demonstrated that PML-RARa targets the PRC2 to tumor suppressor loci causing H3K27 methylation. Interestingly, I was able to show that both DNA and histone methylation are required to maintain PML-RARa aberrant gene silencing, pointing towards a crosstalk among these different epigenetic layers that contributes to the molecular pathology of leukemia. In summary these results provide new insights into the molecular mechanisms underlying APL tumorigenesis and progression.