Rôle du facteur de transcription Ikaros dans la répression de la voie Notch lors de la différenciation des cellules T murines

La voie Notch joue un rôle majeur dans le contrôle des aspects fondamentaux de la vie multicellulaire,comme la prolifération ou la différenciation. Il est clairement établi que cette voie est requise pour la maturation des cellules T, et que la dérégulation de cette voie est oncogénique. Le gène Notch1 est la cible de mutations activatrices dans plus de 50% des leucémies aiguës lymphoblastiques de type T (LAL-T). Cependant, les mécanismes régulant la voie Notch lors du développement des cellules T sont encore incompris. Mon équipe a montré que le facteur de transcription Ikaros, qui est également un suppresseur de tumeur, réprime la transcription du gène Hes1, gène cible de Notch le mieux caractérisé, par fixation compétitive sur des séquences de liaison à l ADN communes. De plus, les souris IkL/L, générées au laboratoire et déficientes pour Ikaros, développent des tumeurs thymiques associées à une hyperactivation de la voie Notch. Il existe donc une corrélation entre l absence d Ikaros et l hyperactivation de la voie Notch. Mon objectif a été d étudier le rôle d Ikaros dans l extinction de la voie Notch au cours de la différenciation des cellules T. Je me suis attachée à déterminer si l hypothèse de compétition entre Ikaros et la voie Notch pouvait être étendue à d autres gènes. Pour le démontrer, j ai identifié d une part les gènes régulés par Ikaros dans des cellules T leucémiques issues des souris IkL/L (où la voie Notch est hyperactivée), et d autre part les gènes régulés par Ikaros dans ces mêmes lignées où j ai surexprimé Ikaros. De façon intéressante, les gènes les plus activés par Notch sont ceux qui sont le plus réprimés par Ikaros. Mes résultats de ChIP-Sequencing montrent qu Ikaros et RBPJ se fixent sur l ADN au même endroit pour la moitié des gènes corégulés par Notch et Ikaros. Par ailleurs, j ai identifié quelques gènes cibles pour lesquels Ikaros entre en compétition directe avec RBPJ pour la fixation de séquences consensus communes. De plus, j ai montré que la plupart des gènes cibles communs sont aussi dérégulés in vivo, au cours de la maturation des cellules T chez les souris IkL/L par rapport aux souris sauvages. Ces recherches permettent non seulement d établir un nouveau mode de régulation de la voie Notch au cours de la différenciation des cellules T, mais aussi de caractériser les gènes importants pour leur transformation, ainsi que de déterminer le stade de transformation.The Notch signalling pathway plays a major role in controlling fundamental aspects of multicellular life including proliferation, stem cell maintenance, differentiation and death. It has a central role in the haematopoietic system and the requirement for Notch activation in thymocyte differentiation is well established. Moreover, when deregulated, the Notch pathway is oncogenic as more than 50% of the T Lymphoblastic Acute Leukemia (T-ALL) display activating mutations in the Notch gene. However, the mechanisms that could regulate the Notch pathway during T cell differentiation are still poorly understood. Ikaros is a zinc finger transcription factor, predominantly expressed in haematopoietic cells; it functions as a transcriptional repressor and a tumor suppressor. Our data show that Ikaros-deficient mice develop T cell lymphomas, which are associated with an early activation of the Notch pathway. Using the Notch target gene Hes1 as a model, we recently reported that Ikaros and RBPJ, the transcriptional mediator of Notch signalling, compete for binding to two elements in the Hes1 promoter in immature thymocytes. These results indicate that Ikaros functions as a transcriptional checkpoint to repress the Notch target gene Hes1 expression in T cells. The molecular basis of this repression could be due to the similarity between the sequences recognized by Ikaros and RBPJ. To determine if Ikaros repression plays a central role in silencing Notch target genes during T ymphopoiesis, I have studied its capacity to regulate the transcription of Notch target genes on a global scale in gain- and loss-of-function systems. My results indicate that Ikaros represses most of Notch induced genes. Furthermore, both Ikaros and RBPJ bind to the promoters of half the common target genes, exactly at the same place. I have also demonstrated on several genes that Ikaros directly competes with RBPJ in order to bind to same consensus sequence in the promoter of these common target genes. Finally, loss of Ikaros causes the deregulation of a significant number of Notch target genes during T cell differentiation, before the cells get transformed. These data suggest a new molecular pathway by which the Notch pathway is regulated during T cell development. Ikaros competes with RBPJ to repress a set of common target genes, and this mechanism plays a central role in silencing Notch target genes during T lymphopoiesis. Furthermore, the loss of Ikaros would cause a deregulation of the Notch target genes and this could lead to leukemogenesis.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Ikaros Represses the Transcriptional Response to Notch Signaling in T-Cell Development ▿ †

Notch activity is essential for early T-cell differentiation, but aberrant activity induces T-cell transformation. Thus, Notch target genes must be efficiently silenced in cells where Notch activity is no longer required. How these genes are repressed remains poorly understood. We report here that the Ikaros transcription factor plays a crucial role in repressing the transcriptional response to Notch signaling in T-cell development. Using the Notch target gene Hes-1 as a model, we show that Ikaros and RBP-Jκ, the transcriptional mediator of Notch signaling, compete for binding to two elements in the Hes-1 promoter in immature thymocytes. This antagonistic interaction likely occurs at the CD4− CD8− CD3− double-negative 4 (DN4) stage, where Ikaros levels and binding to the Hes-1 promoter increase sharply and wild-type thymocytes lose their capacity to transcribe Hes-1 upon Notch stimulation. Nonresponsiveness to Notch signaling requires Ikaros, as Ikaros-deficient DN4 and CD4+ CD8+ double-positive (DP) cells remain competent to express Hes-1 after Notch activation. Further, Hes-1 promoter sequences from Ikaros-deficient DP cells show reduced trimethylated H3K27, a modification associated with silent chromatin. These results indicate that Ikaros functions as a transcriptional checkpoint to repress Notch target gene expression in T cells

Oncogenic activation of the Notch1 gene by deletion of its promoter in Ikaros-deficient T-ALL

The Notch pathway is frequently activated in T-cell acute lymphoblastic leukemias (T-ALLs). Of the Notch receptors, Notch1 is a recurrent target of gain-of-function mutations and Notch3 is expressed in all T-ALLs, but it is currently unclear how these receptors contribute to T-cell transformation in vivo. We investigated the role of Notch1 and Notch3 in T-ALL progression by a genetic approach, in mice bearing a knockdown mutation in the Ikaros gene that spontaneously develop Notch-dependent T-ALL. While deletion of Notch3 has little effect, T cell–specific deletion of floxed Notch1 promoter/exon 1 sequences significantly accelerates leukemogenesis. Notch1-deleted tumors lack surface Notch1 but express γ-secretase–cleaved intracellular Notch1 proteins. In addition, these tumors accumulate high levels of truncated Notch1 transcripts that are caused by aberrant transcription from cryptic initiation sites in the 3′ part of the gene. Deletion of the floxed sequences directly reprograms the Notch1 locus to begin transcription from these 3′ promoters and is accompanied by an epigenetic reorganization of the Notch1 locus that is consistent with transcriptional activation. Further, spontaneous deletion of 5′ Notch1 sequences occurs in approximately 75% of Ikaros-deficient T-ALLs. These results reveal a novel mechanism for the oncogenic activation of the Notch1 gene after deletion of its main promoter