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.

Tissue-resident macrophages (MΦ) such as microglia, Kupffer and Langerhans cells derive from Myb-independent yolk sac (YS) progenitors generated before the emergence of hematopoietic stem cells (HSCs). Myb-independent YS-derived resident MΦ self-renew locally, independently of circulating adult monocytes and HSCs. In contrast, adult blood monocytes as well as infiltrating, gut and dermal MΦ derive from Myb-dependent HSCs and are less proliferative. These findings are derived from the mouse, using gene knock-outs and lineage tracing, but their applicability to human development has not been formally demonstrated. Here I use a human pluripotent stem cell (hPSC) differentiation model of hematopoiesis, capable of monocyte/MΦ production over prolonged periods of time, as a tool to investigate human mononuclear phagocyte ontogeny. Using a transcriptomic approach I showed that hiPSC-derived monocytes/MΦ (iPS-Mo/MΦ) produced early in differentiation (first weeks) are more proliferative and less immunologically mature than iPS-Mo/MΦ produced at a later time point. I therefore hypothesised either that iPS-Mo/MΦ only become fully mature after several weeks of differentiation or that there are two developmentally distinct waves of MΦ produced over time. By comparing the transcription profile of iPS-Mo/MΦs to that of primary adult blood monocytes and fetal microglia I then showed that early and late iPS-Mo/MΦs were transcriptionally closer to fetal microglia than to adult blood monocytes. To further investigate if iPS-Mo/MΦs are indeed of the same developmental origin as MYB-independent MΦ such as microglia, I used a CRISPR-Cas9 knock-out strategy to show for the first time, that human iPS-Mo/MΦs develop in a MYB-independent, RUNX1 and SPI1 (PU.1)-dependent fashion. This result makes human iPS-Mo/MΦs developmentally related to, and a good model for, MYB-independent tissue-resident \Macros such as alveolar and kidney MΦs, microglia, Kupffer and Langerhans cells. Interestingly, while MYB was not required for the generation of iPS-Mo/MΦs, its knock-out resulted in an increase in iPS-Mo/MΦ production. To investigate this increase I developed two methods for quantifying the differentiation bottleneck occurring during hiPSC differentiation to iPS-Mo/MΦs. Those techniques highlighted a potential increase in progenitor cell generation in MYB KO cells and thus lay foundation to improve our technical understanding of EB differentiation and will enable enhanced manipulation of the EB model.

CCCTC-binding factor (CTCF) can both activate as well as inhibit transcription by forming chromatin loops between regulatory regions and promoters. In this regard, Ctcf binding on the non-methylated DNA and its interaction with the Cohesin complex results in differential regulation of the H19/Igf2 locus. Similarly, a role for CTCF has been established in normal hematopoietic development; however its involvement, despite mutations in CTCF and Cohesin complex were identified in leukemia, remains elusive. CTCF regulates transcription dependently on DNA methylation status and can if bound block interactions of enhancers and promoters. Here, we show that in hematopietic cells CTCF binds to the imprinting control region of H19/Igf2 and found that chromatin remodeller Smarca5, which also associates with the Cohesin complex, facilitates Ctcf binding and regulatory effects. Furthermore, Smarca5 supports CTCF functionally and is needed for enhancer-blocking effect at imprinting control region. We identified new CTCF-recognized locus near hematopoietic regulator SPI1 (PU.1) in normally differentiating myeloid cells together with members of the Cohesin complex. Due to DNA methylation, CTCF binding to the SPI1 gene is reduced in AML blasts and this effect was reversible by DNA methylation inhibitor 5-azacitidine.

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