Characterisation of Oncogenic LMO Transcriptional complexes

Abstract

The LIM only (LMO) family of proteins are transcriptional regulators that are critical for the regulation of many developmental processes. In early red blood cell development one family member, LMO2, regulates blood cell development (erythropoiesis) through involvement in a transcriptional complex that also contains Tal1, E2a proteins, Gata1 and LDB1. Two components of this complex, Tal1 and LMO2, along with their homologues Lyl1, LMO1 and LMO3, are oncogenes in T cell acute lymphoblastic leukaemia (T ALL). Complexes similar to the erythropoietic LMO2 transcriptional complex are thought to drive the onset of T ALL. This thesis investigates the protein protein interactions of potential constituents of both normal and oncogenic complexes. A full set of tethered LMO-LDB1 sub-complexes was designed, generated, purified and characterised using SEC/MALLS, CD spectropolarimetry and 1D 1H-NMR. The proteins were folded, predominantly monomeric and may be more stable than previously generated versions. Attempts to crystallise the previously uncharacterised LMO1-LDB1 and LMO3-LDB1 constructs are described. GST-pulldown and EMSA experiments were used to show that LMO1–3-LDB1 but not LMO4 LDB1 can bind to Tal1 E2a and Lyl1 E2a heterodimers in the absence of DNA and to characterise heterodimer DNA binding of the higher order complex. These data indicate that the bHLH proteins likely undergo dimer exchange, such that in the presence of DNA, Tal1 E2a proteins form substantial amounts of homodimers as well as heterodimers, but heterodimer formation is favoured by interaction with LMO-LDB1 proteins. No evidence of Lyl1 E2a heterodimer formation on DNA was observed by EMSA, but this heterodimer was stabilised through higher order complex formation with LMO2-LDB1. Binding affinities of LMO1–4-LDB1 to Tal1 E2a heterodimers indicate no binding by LMO4 and similar levels of binding for LMO1–3-LDB1. A protein engineering approach was thus used to generate more stable extended Tal1 E2a constructs, which would be useful for further characterisation of these interactions. Finally, it was shown that Gata2 and Gata3 bind with similar, but slightly lower affinity to LMO2. Also, only the C-terminal finger of Gata1 shows appreciable independent binding to DNA containing GATC/G motifs. The implications of this data for the mechanisms of transcriptional complex formation, and how variant complexes can up- and down-regulate different sets of genes throughout normal development and T-cell leukaemia are discussed.