Abstract
MECOM (E), previously designated as EVI1 (Ecotropic Viral Integration site1) is a transcriptional repressor protein whose aberrant expression contributes to acute leukemia and transforms Rati fibroblast cells. Previously microarray studies identified Mecom (E) mediated changes in gene expression in Rati fibroblast cells and interestingly carbonic anhydrase III (calll) expression was greatly repressed in Mecom (E) expressed Rati fibrobalst cells. Initially Q-PCR and western blot analysis verified a 95-91% repression of calll by Mecom (E).MECOM (E) is known as a survival gene which is resistant to many stress inducers. Surprisingly, Rati fibroblasts cells expressing Mecom (E) (5.6 cells) show increased sensitivity to hydrogen peroxide (H2O2) induced apoptosis, demonstrated by elevated caspase 3 catalytic activity, which is due to decreased calll production. Targeted knockdown of calll alone in Rati fibroblast cells with dicer-substrate short inhibitory RNAs (siRNAs) also increases the sensitivity to H2O2, which occurs in the absence of any other changes mediated by Mecom (E) expression. Overexpression of calll in 5.61 cells reverses the phenotype, restoring H2O2 resistance.
The biological activity of calll is unknown but some studies suggest it is an anti-oxidant. Consistent to the above study, results from my work also show that calll acts as an antioxidant and is activity dependent upon two cysteine residues 181 and 186 which are known to be modified by glutathionylation in cells under oxidative stress.
To investigate the mechanism of Mecom (E) mediated calll repression, the rat calll gene promoter was characterised by deletion and site directed mutagenesis in Rati fibroblast cells. calll promoter reporter assay on different deletion constructs showed a) pA550 have a significant impact on promoter activity resulting a major loss of calll activity revealing the presence of negative regulator on this region; b) calll promoter activity was partially restored in p480 construct and c) pA137 results in a significant loss in promoter activity. Genomatrix analysis showed the presence of spl and ccaat transcription factor sites between A280 - A137. Reporter assay with site directed mutants on spl and ccaat binding sites revealed spl plays an important role in regulating calll expression in Rati fibroblast cells. Characterisation of -137 promoter region by Genomatrix analysis revealed the presence of ets, oct and srf as a potential transcription factors. Reporter assay on site directed mutants on ets, oct and srf transcription factors showed that pA137 calll promoter activity may be due to combinatorial activities of all the three transcription factors (ets, srf and oct).
Understanding the calll promoter activity in Rati fibroblast cells leeds to further investigation of Mecom (E) mediated transcriptional regulation of calll gene in Rati and derivative (Neo, 5.61 and 5.62) cells. As expected calll gene expression was repressed in 5.61 cells demonstrating that Mecom (E) either directly or indirectly modulates transcription of this gene in Rati fibroblasts cells. Genomatrix analysis on rat calll gene promoter showed the presence of multiple potential Mecom binding sites. Reporter assay with different deletion on calll gene promoter in Rati cell populations revealed that a) Mecom (E) still
represses calll promoter activity when all the known potential binding sites are eliminated, b) loss of calll repression occurs between -137 and -88 calll promoter region. Potential transcription factors such as sre, oct and ets mutant promoter activities were comparable to the calll wt promoter in the presence of Mecom (E) in each case suggesting that all 3 transcription factors co-operate to activate -137 promoter and that Mecom mediates its repression of calll promoter is through one or more of these factors. However, mutant promoters are further repressed in 5.61 cells which might suggest another mechanism; for example through epigenetic changes.
From these studies, we propose that Mecom (E) represses transcription of calll gene expression, leading to increased sensitivity to H202-induced apoptosis in Rati cells. Upon oxidative stress, calll undergoes S-glutathionylation on surface exposed cysteine residues due to increased cellular reactive oxygen species and thus protects cells from further oxidative damage. These findings might suggest the basis for the development of a novel therapeutic strategy for the treatment of leukemia’s and solid tumors where MECOM (E) is overexpressed.
| Date of Award | 2012 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Christopher Bartholomew (Supervisor) & Linda Scobie (Supervisor) |