Regulation of the LIFR and gp130 genes by the RUNX1 transcription factor

The RUNX1 transcription factor is an important regulator of haematopoiesis and has been found to influence gene activity at both the transcriptional and chromatin levels. In leukaemia, particularly Acute Myeloid Leukaemia, RUNX1 activity is frequently altered by point mutations and chromosomal rearrangements, the most common being the t(8;21) translocation that produces a RUNX1-ETO fusion protein. While RUNX1 is generally associated with gene activation, RUNX1-ETO mainly acts as a transcriptional repressor and its presence is therefore associated with altered expression of RUNX1 target genes. Previous microarray analysis performed in our laboratory identified both the LIFR and gp130 genes as novel putative RUNX1 targets. LIFR together with gp130 forms a heterodimeric receptor complex that mediates LIF signalling, and in doing so controls various cellular processes including cellular development, differentiation and inflammatory responses. However, despite their important biological roles little is known about regulation of the LIFR and gp130 genes. Bioinformatic analysis identified potential RUNX1 binding sites in the promoters of both the LIFR and gp130 genes, and this study therefore examined the hypothesis that LIFR and gp130 are RUNX1 target genes and their altered expression in leukemic cells in which RUNX1 is disrupted may therefore contribute to leukaemia. The LIFR gene is regulated by alternate promoters, with a so called 'general' and 'placental' promoter previously described. Analysis of LIFR mRNA across a number of cell types demonstrated that activity of the placental promoter is limited to cell lines of placental origin, while the general promoter is active in a range of cell types, including myeloid cells. This was confirmed by analysis of the chromatin status of the two promoters, which found that the general, but not placental promoter is assembled into highly acetylated histones in myeloid cells. The expression of the gp130 mRNA was detected across all cells lines examined. Reporter analysis demonstrated that both the placental and general promoters can be activated by RUNX1 in placental and myeloid cell lines respectively. In addition, the gp130 promoter was activated by RUNX1 in myeloid cell lines. In contrast, RUNX1-ETO repressed both the LIFR and gp130 promoters in myeloid cells. Further, binding of RUNX1 to the endogenous LIFR and gp130 promoters was confirmed by chromatin immunoprecipitation, suggesting that the endogenous promoters are targeted by RUNX1. In support of this RUNX1 knockdown reduced LIFR and gp130 expression in myeloid cell lines, and decreased expression of the placental LIFR transcript in placental cells. Put together, the data presented in this thesis demonstrates that RUNX1 regulates expression of the LIFR and gp130 promoters, suggesting that activity of the LIFR/gp130 receptor complex is likely to be altered in leukaemic cells in which RUNX1 is altered.