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Abstract
Aberrant stem cell-like gene regulatory networks are a feature of leukaemogenesis. The ETS-related gene (ERG), an important regulator of normal haematopoiesis, is also highly expressed in T-cell acute lymphoblastic leukaemia (T- ALL) and acute myeloid leukaemia (AML). However, the transcriptional regulation of ERG in leukaemic cells remain poorly understood. In order to discover transcriptional regulators of ERG, I employed a quantitative mass spectrometry (MS)-based method to identify factors binding the ERG +85 stem cell enhancer region in MOLT-4 and KG-1 cells. Using this approach, I identified a number of known transcription factors (TF) bound to the ERG +85 enhancer in leukaemic cells along with previously unknown binders, ETV6 and IKZF1. I confirmed that ETV6 and IKZF1 were also bound in vivo at the ERG +85 enhancer in both leukaemic cells and in healthy human CD34+ haematopoietic stem and progenitor cells (HSPCs). Knockdown experiments confirmed that ETV6 and IKZF1 are transcriptional regulators of ERG and a number of genes regulated by a densely interconnected network of the heptad TF. Furthermore, I showed that ETV6 and IKZF1 expression levels are positively correlated with expression of a number of heptad genes in AML and high expression of all nine genes confers poorer overall prognosis.
Protein phosphorylation is known to regulate the transcriptional activity of genes. Aberrant protein phosphorylation can lead to numerous haematological malignancies, but few studies have focused on phosphorylations of DNA bound TF at regulatory DNA. By adapting the reverseChIP method I develop an approach to identify and quantify of site-specific phosphorylation if TF on DNA in vitro using MOLT-4 and KG-1 cells. I have identified phosphorylated DNA bound TFs and 28 novel phosphorylation sites that are uniquely enriched at the ERG+85 enhancer under a leukaemia context. Oncoprotein ETS1 was identified as exhibiting changes in phosphorylation when bound to DNA compared to the nuclear input, this finding was also validated by western blot.
This work significantly expands existing knowledge of the regulation of ERG over-expression driven by the ERG+85 enhancer in leukaemic and HSPCs cells. In addition, both reverseChIP methods can be utilised to study other regulatory regions of interest.