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Title: The role of EVI-1 in cellular transformation and its biological activity in primary bone marrow cells
Author: Palmer, Susan A.
ISNI:       0000 0001 3466 0873
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2000
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The EVI-1 proto-oncogene, which is frequently activated in murine retrovirally induced leukaemias and human acute myeloid leukaemias by non-random chromosomal translocations involving 3q26-where the EVI-1 gene is located-encodes a transcriptional repressor. Repression is mediated by a 200 base pair proline-rich region designated Rp and also by a weaker second domain designated IR, which are both located between the two DNA binding domains (ZF1 and ZF2). Deletion mutagenesis studies, the results of which are reproduced in this thesis, reveal that the Rp, ZF1 and ZF2 domains are all required for optimal transformation of Rati fibroblasts. Moreover, it was demonstrated that several of these non-transforming evi-1 mutants, lacking either ZF1, Rp or both ZF1 and ZF2 (DNA binding defective mutants), could revert the transformed phenotype of evi-1-transformed Rati fibroblasts. Recent work has shown that evi-1 can bind the murine c-terminal binding protein 2 (mCtBP2) co-repressor and that evi-1 binds mCtBP2 through two conserved amino acid motifs residing in the Rp domain of the protein. The work described in this thesis has also shown that the integrity of these binding sites is crucial for evi-1- mediated repression and transformation in mammalian cells. Together these results suggested that DeltaZF1 and DeltaRp mutants with partially and fully intact DNA binding domains, respectively could inhibit transformation through a DNA binding competition for evi-1 binding sites. On the other hand, the DNA binding defective DeltaZF12 mutants may sequester CtBP proteins or other factors necessary for FLevi-1 regulation and in turn inhibit the biological activity of the wild type protein. In addition, until now no studies have addressed the significance of evi-1 functional domains for the biological activity of evi-1 in haematopoietic cells. Using retroviral infection of primary bone marrow cells it was shown that FLevi-1, the alternative evi-1 splice form A324, and DeltaRp and DeltaZF1 deletion mutants all blocked the production of erythroid and myeloid progenitors in an identical manner in methyl cellulose colony assays. The DeltaZF2 and DeltaZF12 mutants produced an intermediate phenotype. These results suggested that either distinct regions of the evi-1 protein are required for the transformation of fibroblasts and inhibition of haematopoietic cell colony formation or that the evi-1 proteins act in a dominant negative fashion over endogenous proteins required for haematopoiesis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Leukaemias; Cancer; Proto-oncogene