Abstract 2205: The EVI1 human protein regulates its own transcription. Role of the different isoforms
Authors: Maicas Miren , Vazquez Iria , Cortes-Lavaud Xabier , Garcia-Sanchez Maria A. , Marcotegui Nerea , Urquiza Leyre , Cristobal Ion , Calasanz Maria J. , Odero Maria D. 
Published: August 2012

Abstract
The EVI1 transcription factor has an essential role in development and leukemogenesis. High expression of EVI1 is a negative prognostic indicator of survival in acute myeloid leukemia (AML) patients, irrespective of the presence of 3q26 rearrangements; however, the only known mechanisms that lead to EVI1 overexpression are 3q aberrations, and MLL oncoproteins, which activate the transcription of EVI1 in hematopoietic stem cells. Our aim was to study mechanisms of EVI1 expression at transcriptional level, and to determine the role of the different isoforms of this protein in leukemogenesis. Bioinformatic analysis of the promoter of the EVI1 gene identified 8 EVI1-potential binding sites. ChIP assay confirmed that EVI1 directly binds to two of these sites, suggesting that EVI1 could act as a regulator of its own expression. Luciferase assays showed that the EVI1-full length (145KDa) protein binds to the EVI1 promoter as a transcriptional activator. The EVI1 human locus codes for at least 4 different proteins: EVI1-145KDa; MDS1EVI1, with an additional PR domain; delta324, that lacks the zinc fingers 6 and 7; and RP-9 that lacks 9 amino-acids from the repressor domain. The best studied is the EVI1-145KDa protein; however, the functional significance of the other isoforms is unclear. This prompted us to determine whether the other isoforms had the same ability than EVI1-145KDa. We observed that the RP-9, delta324 and MDS1EVI1 isoforms repressed the transcription of EVI1 by binding to a more distal promoter region. To confirm these results we transfected HEK293T with different expression vectors. EVI1-145KDa overexpression increased the EVI1-1A, -1B and -1D transcripts. However, RP-9 decreased M/E, EVI1-1A, -1B and -1D, and delta324 decreased EVI1-1D. These results support the data obtained with the luciferase experiments; however, we found no differences at protein level. In addition, we analyzed at mRNA and protein level 16 cell lines and 18 samples of patients with AML. We found a positive correlation between the expression of EVI1-145KDa and MDS1EVI1 at mRNA and protein level. However, some of the patients with the same protein levels expressed different delta324 transcript levels, suggesting either a regulation of the protein expression at post-transcriptional level or protein stability. Cycloheximide experiments demonstrated that the delta324 isoform is more stable than the MDS1EVI1 and EVI1-145KDa proteins. Preliminary results show that knockdown of EVI1 caused a decrease in proliferation with an increase in apoptosis. Further experiments are in progress to determine the role of each isoform. In conclusion, our results suggest that the EVI1 isoforms oppositely regulate the expression of the EVI1 human gene by specific binding to the promoter motifs. This study opens new directions to further understand the mechanisms of EVI1 overexpressing leukemias.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2205. doi:1538-7445.AM2012-2205