Les facteurs de transcription ETS sont divisés en sous-familles en fonction de leurs similitudes en matière de séquence protéique, de séquences de liaison à l'ADN et d’interactions avec différents cofacteurs. Ils sont régulés par des signaux extracellulaires et contribuent à divers processus cellulaires, dont la prolifération cellulaire et la transformation tumorale. Les gènes de la famille ETS sont fréquemment ciblés par des processus oncogéniques que ce soit des translocations chromosomiques ou des gains du nombre de leurs copies. Le gène PU.1/SPI1 est également ciblé par des mutations ponctuelles inactivatrices dans les hémopathies myéloïdes humaines. Nous avons étudié une mutation somatique récurrente du gène PU.1/SPI1 (c.676C>G, p.Q226E), identifiée chez environ 6% des patients atteints d’une macroglobulinémie de Waldenström (MW), un syndrome lymphoprolifératif B chronique rare. La mutation modifie les caractéristiques de liaison à l'ADN de la protéine mutante, passant des séquences classiques reconnues par SPI1 à des séquences reconnues par d’autres protéines ETS comme ETS1, et d’une liaison à des régions enhancer à une liaison à des régions promotrices. La liaison accrue du mutant de SPI1 aux régions promotrices active des programmes transcriptionnels impliquant des voies de signalisation intracellulaire généralement favorisées par d'autres membres de la famille ETS. Les conséquences fonctionnelles de cette mutation sont une augmentation de la prolifération cellulaire et une diminution de la différenciation lymphoïde B terminale dans une lignée cellulaire modèle et des échantillons primaires de MW. Nous décrivons ici un mécanisme de subversion oncogénique de la fonction d’un facteur de transcription suite à la modification subtile de la spécificité de liaison à l'ADN de la protéine mutante, menant à un arrêt de différenciation. La démonstration qu'une mutation somatique ponctuelle peut modifier l'équilibre de liaison d’un facteur de transcription à l’échelle du génome fournit un paradigme mécanistique sur la façon dont les mutations faux sens dans les gènes codant pour des facteurs de transcription pourraient être oncogéniques dans les tumeurs humaines. / The ETS-domain transcription factors are divided into subfamilies based on protein similarities, DNA binding sequences and interaction with cofactors. They are regulated by extracellular clues and contribute to a variety of cellular processes, including proliferation and transformation. ETS genes are targeted by oncogenic processes through chromosomal translocations and copy number gains. The PU.1/SPI1 gene is also targeted by inactivating point mutations in human myeloid malignancies. We investigated a recurrent somatic missense mutation (Q226E) of the PU.1/SPI1 gene in Waldenström macroglobulinemia, a human B-cell lymphoproliferative disorder. The mutation changes DNA binding of the mutant protein from classical SPI1 to ETS1-like sequences, shifting the balance from binding to promoter regions from enhancers. Increased binding by mutant SPI1 at promoters activates gene expression of intracellular signaling pathways typically promoted by other ETS factor family members. The functional consequences are decreased terminal B-cell differentiation in a model cell line and primary samples. In summary, we describe oncogenic subversion of transcription factor function through subtle alteration DNA binding specificity leading to differentiation arrest. The demonstration that a somatic point mutation subtly changes the balance of genome binding provides a mechanistic paradigm for how missense mutations in transcription factor genes may be oncogenic in human tumors.

B cells are capable of producing antibodies of diverse antigen specificities and effector functions to counter infection by a wide range of pathogens. The diversification of immunoglobulin (Ig) is achieved through a series of programmed DNA recombination and mutagenic events during B cell maturation. A key factor involved in the Ig diversification process is Activation Induced Cytidine Deaminase (AID). AID is a B cell specific enzyme that is critical for three distinct pathways of Ig diversification: class switch recombination, somatic hypermutation and Ig gene conversion. AID functions by deaminating cytosine to uracil in target DNA at the Ig loci. Although essential for effective immunity, the mutagenic activity of AID needs to be confined to the Ig loci in order to protect genomic integrity, but the underlying mechanism is not fully understood. In this study, I show that two lymphoid specific transcription factors, PU.1 and IRF4, play important roles in regulating AID function in chicken B cells. PU.1 and IRF4 have been implicated in many aspects of B cell development and function. The two factors could form a heterodimer and regulate target gene expression cooperatively. However, we found that PU.1 and IRF4 appear to have different impacts on AID function. We show that PU.1 is important for the expression of AID gene in chicken B cells, and the regulation appears to involve direct interaction of PU.1 with the AID gene. By comparison, IRF4 plays a minor role in AID expression. On the other hand, both PU.1 and IRF4 are required for efficient gene conversion that is mediated by AID at the Igλ locus. Moreover, the gene dosage of PU.1 is critical for AID function, since a severe gene conversion defect is observed in PU.1+/- cells. The function of PU.1 and IRF4 in AID-mediated gene conversion involves binding sites for the PU.1/IRF4 complex within a regulatory element at the Igλ locus. Future studies will be directed at understanding how PU.1 and IRF4 regulate AID-mediated gene conversion. / text

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PU.1 downregulation within haematopoietic stem and progenitor cells (HSPCs) is the primary mechanism for the development of acute myeloid leukaemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. p53 is a well known tumor suppressor that is often mutated in human haematologic malignancies including AML and adds to their aggressiveness; however its genetic deletion does not cause AML in mouse. Deletion of p53 in the PU.1ure/ure mice (PU.1ure/ure p53-/- ) results in more aggressive AML with shortened overall survival. PU.1ure/ure p53-/- progenitors express significantly lower PU.1 levels. In addition to URE deletion we searched for other mechanisms that in absence of p53 contribute to decreased PU.1 levels in PU.1ure/ure p53-/- mice. We found involvement of Myb and miR-155 in downregulation of PU.1 in aggressive murine AML. Upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukaemic cell growth similarly to PU.1 rescue. The MYB/miR-155/PU.1 axis is a target of p53 and is activated early after p53 loss as indicated by transient p53 knockdown. Furthermore, deregulation of both MYB and miR-155 coupled with PU.1 downregulation was observed in human AML, suggesting that MYB/miR-155/PU.1 mechanism may be involved...

9 Abstract Enhancers are distal cis - regulatory DNA sequences that regulate (enhance) transcription of the respective gene driven by its promoter. Enhancers are found in non-coding DNA upstream or downstream of the gene coding sequence, or in introns or coding regions that are located up to hundreds kb away from the gene. Superenhancers are newly discovered clusters of multiple enhancers that play a vital role in activating tissue-specific genes, determining cell identity and regulating differentiation. PU.1 is the transcription factor (TF) that is necessary for normal haematopoiesis, specifically for the development of myeloid and lymphoid blood lineages. Distinct levels of PU.1 induce differentiation of hematopoietic cells into different cell lineages whereby disruption of PU.1 levels leads to leukemogenesis. High PU.1 levels stimulate macrophage development, while intermediate levels stimulate the development of granulocytes. This diploma thesis seeks to contribute to addressing the interesting biological question of what are the regulatory mechanisms to ensure that granulocytic genes are activated only at the intermediate concentration of PU.1, whereas macrophage genes are activated only at its high levels. The aim of this diploma thesis was to create a series of reporter vectors carrying regulatory...

Hematopoiesis is coordinated by a complex regulatory network of transcription factors among them PU.1 (Spi1, Sfpi1) and GATA-1 represent key molecules. GATA-1 and PU.1 bind each other on DNA to block each others transcriptional programs to prevent development of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells, transformed erythroid precursors that are blocked from completing the late stages of erythroid differentiation, co-express GATA-1 and PU.1 and as my and others data document, are able to respond to molecular removal (down-regulation) of PU.1 or addition (up-regulation) of GATA-1 by inducing terminal erythroid differentiation. We provide novel evidence that downregulation of GATA-1 or upregulation of PU.1 induces incompletely differentiation into cell cycle arrested monocytic-like cells. Furthermore, PU.1- dependent transcriptome is negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) that encode additional key hematopoietic transcription factors. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb that are co-occupied by PU.1 and GATA-1 in the leukemic blasts. Furthermore, transcriptional regulation of these loci by...

Hematopoiesis is coordinated by a complex regulatory network of transcription factors among them PU.1 (Spi1, Sfpi1) and GATA-1 represent key molecules. GATA-1 and PU.1 bind each other on DNA to block each others transcriptional programs to prevent development of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells, transformed erythroid precursors that are blocked from completing the late stages of erythroid differentiation, co-express GATA-1 and PU.1 and as my and others data document, are able to respond to molecular removal (down-regulation) of PU.1 or addition (up-regulation) of GATA-1 by inducing terminal erythroid differentiation. We provide novel evidence that downregulation of GATA-1 or upregulation of PU.1 induces incompletely differentiation into cell cycle arrested monocytic-like cells. Furthermore, PU.1- dependent transcriptome is negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) that encode additional key hematopoietic transcription factors. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb that are co-occupied by PU.1 and GATA-1 in the leukemic blasts. Furthermore, transcriptional regulation of these loci by...

No description available.

Veränderungen in der lokalen Chromatinstruktur beeinflussen die dynamische Regulation von Genen, welche für die Differenzierung notwendig sind. PU.1 ist ein Master-Transkriptionsfaktor in der Hämatopoese und wird streng reguliert, um ein zelllinienspezifisches Expressionsmuster zu erzielen. Hohe Konzentrationen von PU.1 sind für myeloische Differenzierung erforderlich. In B-Zellen wird PU.1 mittelstark exprimiert und muss aktiv runterreguliert werden, um eine Ausdifferenzierung der multipotenten Vorläuferzellen zu T-Zellen zu ermöglichen. Derzeit ist wenig über die Regulierung von PU.1 in T-Zellen bekannt. Darüber hinaus wurde eine abnormale Expression von PU.1 in verschiedenen Leukämieerkrankungen beobachtet. Mittels eines genome-wide Chromatin-Interaktions-Screens konnten wir einen cis-Repressor mit insulierender Kapazität identifizieren, welcher mittels eines Chromatinloops die Promotoraktivität von PU.1 in T-Zellen, jedoch nicht in myeloischen oder B-Zellen blockiert. Sowie Looping als auch Insulation erfordern die Bindung des Chromatin-Regulatorprotein CTCF. Im Gegensatz zu normalen myeloischen Zellen finden wir, dass Krebszellen aus myeloischen Leukämie Patienten diese T-Zell-spezifische repressive Chromatinstruktur aufweisen, was einen räumlichen Kontakt des Insulator mit dem PU.1 Promotor ermöglicht. Die Ergebnisse dieser Arbeit beschrieben das CTCF gesteuerte „long distance looping“ als ein neuer molekularer epigenetischer Mechanismus, um Transkriptionsfaktor PU.1 in T-Zellen runterzuregulieren, und zeigen zum ersten mal, dass Krebszellen die Chromatinstruktur anderer Zelllinien imitieren können, um die Expression von Differenzierungsgenen zu blockieren. / Alterations in the local chromatin structure orchestrate the dynamic regulation of differentiation promoting genes. PU.1 is a master transcription factor in hematopoiesis. PU.1 gene must be tightly regulated to achieve lineage specific expression pattern. High levels of PU.1 are required for myeloid commitment: it is expressed at intermediate level in B-cells and must be actively silenced to permit T cell development from early multipotent progenitors. However, little is known of how PU.1 is regulated in T-cells. Moreover, aberrant PU.1 expressions have been observed in multiple leukemias. Using a genome-wide chromatin interaction screen we identified a cis-repressor with insulating capacity that undergoes long-distant chromatin looping to block PU.1 promoter activity in T cells but not myeloid or B cells. Looping and repression requires binding of the chromatin regulator protein CTCF. In contrast to normal myeloid cells, we found that cancer cells from myeloid leukemia patients adopt the T cell specific repressive chromatin structure bringing the insulator into spatial contact with the PU.1 promoter. These results identify CTCF controlled long-distant insulator looping as a novel mechanism to silence lineage-opposing transcription factor expression, and reveal that cancer cells can mimic the chromatin confirmation of another lineage to block expression of differentiation driving genes.

Als Schlüsselfaktor des hämatopoietischen Systems spielt PU.1 eine ent-scheidende Rolle in der Entwicklung der meisten hämatopoietischen Li-nien. Das PU.1 Expressionslevel bestimmt das Differenzierungspotential hämatopoietischer Stammzellen und Vorläufer. In den unterschiedlichen Zelltypen werden verschiedene Expressionsstärken etabliert. Wie diese zelltypischen Expressionslevel vonPU.1 generiert werden, ist bisher weit-gehend unbekannt. In der vorliegenden Doktorarbeit wurde mit Hilfe eines transgenen Maus-modells die cis-regulatorische Einheit von PU.1 definiert, um mit nachfol-genden molekularbiologischen und genomweiten Ansätzen Mechanismen der zellspezifischen Regulation von PU.1 zu aufzuzeigen. Die Definition der cis-regulatorischen Einheit von PU.1 erfolgte mit Hilfe eines transgenen Mausmodells, welches ein humanes PU.1 BAC Kons-trukt trägt. Es konnte gezeigt werden, dass humanes und murines PU.1 substituierbar sind und den gleichen Regulationsmechanismen unterlie-gen. Mit Hilfe genomweite DNaseI hypersensitivity Analysen, Methylie-rungs- und Bindungsstudien konnte ein neuer Regulationsmechanismus beschrieben werden, der eine spezifische kombinatorische Interaktion verschiedener cis-regulatorischer Elemente erfordert. Durch Reportergenassays in verschiedenen Zelltypen war es möglich, einen myeloischen Enhancer zu identifizieren. Es konnte gezeigt werden, dass PU.1 mit zelltyp-spezifischen Transkriptionsfaktoren interagiert, um unterschiedliche Bindungsmuster an seinen regulatorischen Elementen zu etablieren. Dadurch kommt es zu den spezifischen Expressionstärken von PU.1 / The transcription factor PU.1 occupies a central role in controlling myeloid and early B cell development and its correct lineage-specific expression is critical for the differentiation choice of hematopoietic progenitors. However, little is known of how this tissue-specific pattern is established. We previously identified an upstream regulatory cis-element (URE) whose targeted deletion in mice decreases PU.1 expression and causes leukemia. We show here that the URE alone is insufficient to confer physiological PU.1 expression, but requires the cooperation with other, previously unidentified elements. Using a combination of transgenic studies, global chromatin assays and detailed molecular analyses we present evidence that PU.1 is regulated by a novel mechanism involving cross-talk between different cis-elements together with lineage-restricted autoregulation. In this model, PU.1 regulates its expression in B cells and macrophages by differentially associating with cell-type specific transcription factors at one of its cis-regulatory elements to establish differential activity patterns at other elements.