Implication du facteur IKAROS dans la régulation des gènes cibles de la voie NOTCH dans les cellules érythroïdes

IKAROS is a critical transcription factor in hematopoiesis. It facilitates the chromatin binding of many important co-factors required for chromatin organization during cell renewal and lineage commitment. IKAROS is particularly important in lymphoid cells whereby it is involved in the repression of target genes of the NOTCH signaling pathway. IKAROS is also important in the development of other hematopoietic lineages, including the erythroid cells, in which it facilitates the passage of a fetal to adult globin in the embryo through the recruitment of the chromatin remodeling complexes NuRD and BAF. Under normal conditions, the NOTCH signaling pathway represses development of erythroid cells. It is therefore important to precisely understand how the NOTCH target genes are regulated during passage from hematopoietic progenitor to the enucleated circulating erythrocyte. IKAROS has been demonstrated to be an important regulator of Hes1 gene expression in hematopoietic cells of different lineages. Hes1 is the major effector target of the NOTCH pathway and IKAROS actively participates in its repression. In erythroid cells, the regulation of Hes1 imposed by IKAROS is required for terminal differentiation. We therefore investigated the importance of IKAROS in the regulation of NOTCH-targeted genes in erythroid cells. The combined effect of the mutation of IKAROS with NOTCH signaling was particularly investigated in these cells. To define how IKAROS influences the regulation of NOTCH target genes, we performed the bioinformatics analysis of a RNA-sequencing performed in murine erythroid cells activated or not for NOTCH signaling and whereby IKAROS is absent. We identified genes influenced by IKAROS expression and by NOTCH, and defined the effect of the combination of the absence of IKAROS expression and NOTCH pathway activation. Two particular expression patterns were identified and characterized the combined effect of the absence of IKAROS and NOTCH pathway activation in erythroid cells.  Indeed, the absence of IKAROS either favors the overexpression of NOTCH target genes or prevents their response to NOTCH pathway activation. To understand how IKAROS could have an opposite effect on different NOTCH target genes we analysed the effect of IKAROS on their regulation. Among the genes repressed by IKAROS are the target genes of NOTCH Cdkn1a (encoding the P21WAF1/CIP1 protein) and Trp53 (encoding the TP53 protein), whose expression increases strongly when IKAROS is mutated and the NOTCH pathway is activated. Prdm16 and Nrarp are, instead, requiring IKAROS expression for their activation by NOTCH. The characterization of these NOTCH target genes suggests that IKAROS can work in partnership with the NuRD complex to influence the expression. The chromatin characterization of these genes led us to posit that the IKAROS-NuRD could act as a ‘platform’ to impose a bivalent chromatin organization associated with poised transcription. Then, the regulation imposed by IKAROS-NuRD would be required for the physiological activation of NOTCH targets upon external signaling. Finally, since in embryonic stem cells the Ten-Eleven Translocation (TET) enzymes are reported to be frequently associated to bivalent chromatin in order to prevent DNA hypermethylation, we assessed whether IKAROS could interact and ‘use’ TET enzymes to regulate NOTCH target genes. We determined that IKAROS can co-immunoprecipitate with the TET1 enzymes. We show that IKAROS influences both recruitment and activity of TET1 to different NOTCH target genes and favors the accumulation of hydroxymethylcytosine (5-hmC) to these genes. 5-hmC can be considered as a mark of transcriptional pausing/bivalence. Thus, these studies bring new information on the mechanism used by IKAROS to influence gene regulation in hematopoietic cells. Our results suggest that IKAROS primary function is to organize a bivalent chromatin and to promote transcriptional pausing to multiple NOTCH target genes. IKAROS is required to set the epigenetic and promoter organization for rapid activation upon NOTCH signaling.

Keyphrases: NuRD, IKAROS, TET1, NOTCH, Transcriptional poising