Regulation of the EVI1 gene through 5´-end variability

The EVI1 gene plays important roles both in normal mammalian development and in malignant diseases of the hematopoietic system. Recently, the mRNA derived from it has been found to exhibit great variability at its 5`-end, with at least 6 different mRNA variants described. This suggests previously unrecognized, complex modes of regulation of the expression and the biological properties of the EVI1 protein product. This assumption is strongly supported by a small number of studies, one of which found that, of two EVI1 mRNA variants investigated, the expression of one, but not the other, was associated with a poor prognosis in myeloid leukemia. However, many basic aspects of the 5`-end variability of EVI1 remain still unexplored. In the first part of this project, we therefore plan to address some fundamental questions about the different EVI1 mRNA variants. Their complete sequences shall be obtained, their transcriptional start sites determined, and the possibility that some of them may code for protein products with alternative N-termini shall be explored. Their expression in different human tissues, and in response to physiological regulators of EVI1 will be measured. Finally, the stability of the different 5`-end variants and the efficiency with which they are translated into protein shall be assessed. The results of these studies will complement those of a parallel project (for which funding has been obtained from other sources), in which the expression of all six EVI1 mRNA variants will be measured in samples from patients with myeloid leukemia, and correlated with disease outcome. Whichever EVI1 5`-end variant/s will be found to have the strongest prognostic value, for further studies of the underlying mechanisms it will be essential to know, e.g., which protein variant is produced from ithem and with what efficiency. In the second part of the proposed studies, preliminary observations regarding the regulation of EVI1 by all-trans retinoic acid (ATRA), an important inducer of various developmental processes, shall be further extended. ATRA selectively upregulates some of the EVI1 transcript variants, but not others, and does so by increasing not their production, but their stability. This is an unusual and so far poorly characterized mechanism of gene regulation by ATRA that we plan to investigate using a convenient assay system that we have established for this purpose. Finally, experiments will be carried out to further our understanding of a previously described role of EVI1 in ATRA-induced neuronal differentiation.

The EVI1 gene plays important roles both in normal mammalian development and in malignant diseases of the hematopoietic system. Recently, the mRNA derived from it has been found to exhibit great variability at its 5`-end, with at least 6 different mRNA variants described. This suggests previously unrecognized, complex modes of regulation of the expression and the biological properties of the EVI1 protein product. This assumption is strongly supported by a small number of studies, one of which found that, of two EVI1 mRNA variants investigated, the expression of one, but not the other, was associated with a poor prognosis in myeloid leukemia. However, many basic aspects of the 5`-end variability of EVI1 remain still unexplored. In the first part of this project, we therefore plan to address some fundamental questions about the different EVI1 mRNA variants. Their complete sequences shall be obtained, their transcriptional start sites determined, and the possibility that some of them may code for protein products with alternative N-termini shall be explored. Their expression in different human tissues, and in response to physiological regulators of EVI1 will be measured. Finally, the stability of the different 5`-end variants and the efficiency with which they are translated into protein shall be assessed. The results of these studies will complement those of a parallel project (for which funding has been obtained from other sources), in which the expression of all six EVI1 mRNA variants will be measured in samples from patients with myeloid leukemia, and correlated with disease outcome. Whichever EVI1 5`-end variant/s will be found to have the strongest prognostic value, for further studies of the underlying mechanisms it will be essential to know, e.g., which protein variant is produced from ithem and with what efficiency. In the second part of the proposed studies, preliminary observations regarding the regulation of EVI1 by all-trans retinoic acid (ATRA), an important inducer of various developmental processes, shall be further extended. ATRA selectively upregulates some of the EVI1 transcript variants, but not others, and does so by increasing not their production, but their stability. This is an unusual and so far poorly characterized mechanism of gene regulation by ATRA that we plan to investigate using a convenient assay system that we have established for this purpose. Finally, experiments will be carried out to further our understanding of a previously described role of EVI1 in ATRA-induced neuronal differentiation.