Abstract
EVI1 (Ecotropic Viral Integration site I), which was originally identified as a myeloid transforming gene by means of retroviral insertional mutagenesis in mouse leukemia, encodes a nuclear DNA binding zinc finger protein. The presence of zinc fingers that are able to bind to specific sequences of DNA suggests that EVI1 is a transcriptional regulator; however, except a few, target genes of EVI1 are poorly functionally identified thus far. In this study we provide evidence that EVI1 directly induces the expression of Bcl-xL through the first set of zinc finger and thereby inhibits apoptosis. ChIP analysis showed that EVI1 binds to the Bcl-xL promoter in HT-29 cells, a colon carcinoma cell line, which expresses EVI1. The observation is also supported by the fact that EVI1 siRNA treated HT-29 cells, shows a down regulation of Bcl-xL expression and that over expression of EVI1 results in the induction of the Bcl-xL reporter construct. A set of EVI1 positive chronic myeloid leukemia (CML) samples also showed higher Bcl-xL expression with respect to EVI1 negative samples. Interestingly, co-expression of EVI1 with wild type, but not with dominant-negative form of PCAF, abolishes the effect of EVI1 on Bcl-xL, indicating that acetylation of EVI1 abrogates its ability not only to bind Bcl-xL promoter but also alleviate Bcl-xL activity. Finally we have shown that EVI1 expression regulates apoptosis in HT-29 cells, which is abrogated when HT-29 cells are transfected with EVI1 siRNA or PCAF. The result for the first time shows a direct pathway by which EVI1 can protect cells from apoptosis and also demonstrates that the pathway can be reversed when EVI1 is acetylated.
Highlights
One of the genes associated with both murine and human myeloid leukemia is EVI1 [1,2]
We found a significant increase in the activity with EVI1-DD where as no such change was observed with EVI1-DA (Figure 2E, bar 3 and 4) corroborating the chromatin immunoprecipitation (ChIP) data that the first set of zinc finger binds to Bcl-xL promoter and up regulates its activity
We found that the proto-oncogene EVI1 binds Bcl-xL promoter and up regulates its transcriptional activity and protein expression
Summary
One of the genes associated with both murine and human myeloid leukemia is EVI1 [1,2]. Over expression and aberrant expression of EVI1 was shown to be associated with most forms of human leukemia, as a consequence of chromosomal rearrangements involving 3q26.2, where the gene is mapped [3] and without cytogenetically detectable rearrangements of the EVI1 locus as a result of unknown mechanism [2,4]. The protein is highly conserved through evolution and encodes a repressor and an activator domain with two sets of zinc finger motifs [1,3,9,10] Both zinc finger domains of EVI1 recognize and bind to specific DNA consensus sequence in vitro and in vivo, of which some are characterized and some are not functionally characterized [3,11,12]. The exact mechanism by which EVI1 promotes cell proliferation and blocks apoptosis still remains unclear
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