GENOME-WIDE ANALYSIS OF TRANSCRIPTION FACTOR NETWORKS IN MYELOID DIFFERENTIATION AND ACUTE MYELOID LEUKAEMIA

One of the most frequent genetic abnormalities underlying the pathogenesis of acute myeloid leukaemia (AML) is a reciprocal translocation between chromosomes 8 and 21 which results in the generation of a chimeric gene that encodes for the AML1/ETO fusion protein. AML1/ETO has the capacity to block myeloid differentiation, thus leading to the accumulation of immature precursor cells. The fusion protein functions as an aberrant DNA binding transcription factor, therefore altering the normal gene expression profile of the cell. Moreover, it interacts with a number of other transcription factors involved in myeloid differentiation. In this thesis, a murine haematopoietic stem/precursor cell line was exploited to study AML1/ETO functions. The binding pattern of AML1/ETO was investigated by ChIP-sequencing and correlated to the binding profiles of AML1, PU.1 and C/EBPα transcription factors. The distribution of three histone marks (H3K4me1, H3K4me3 and H3K27ac) upon expression of AML1/ETO was also analysed. The results showed that AML1/ETO expression is associated with modifications in the binding profile of the three transcription factors and in the distribution of chromatin marks, that changes in gene expression are associated with such modifications, and more rarely, with AML1/ETO binding. High-throughput profiling of miRNAs expression revealed the presence of two miRNAs up-regulated by AML1/ETO, miR-322* and miR-351, which are likely candidates for having a role in the block of differentiation, since their inhibition restores the ability of AML1/ETO-expressing cells to differentiate. Analysis of the AML1/ETO-associated gene expression profile predicted a modulation of the cell motility and cell localization. To further explore these functions, in vitro migration assays and in vivo homing experiments were performed. These assays confirmed that AML1/ETO endows the cell with an enhanced motility and an impaired homing of cells to haematopoietic organs. Bioinformatics analysis of AML1/ETO target genes and their regulatory regions revealed that a modification in the intensity of AML1, PU.1 and C/EBPα binding correlates with changes in gene expression. Sequence analysis showed that STAT6 and other STAT factors could synergize with AML1/ETO in the regulation of gene expression. Taken together, these results describe how AML1/ETO expression influences its target genes and how the three transcription factors included in the study, chromatin modifications, microRNAs, and putative co-regulators are involved in such regulation. In addition, alterations in cell motility and localization, functions not yet described for AML1/ETO, were identified.