Acute myeloid leukemia is a lethal illness whose molecular etiology is poorly understood, but which likely involves alterations in the ability of myeloid cells to control growth and execute normal maturation. The applicant's long-term goal is to understand the molecular mechanisms involved in this disease through the in-depth study of the zinc finger gene, Evi1. Evi1 has been implicated in acute myeloid leukemia both in mouse and human, but the mechanism by which it causes disease is not known. One possibility is that it causes abnormal responsiveness to differentiation-promoting cytokines, such as G-CSF. It is known to bind with high specificity to two sequences (GACAAGATA and GAAGATGAG) via two separate domains of zinc fingers, although the target genes on which it acts have not been described. The applicant hypothesizes that the mechanism by which the EVI1 protein causes leukemia is by altering the transcriptional regulation of certain target genes. The applicant asserts that through the identification and characterization of these genes he can begin to understand the exact mechanism by which EVI1 causes leukemia. This line of inquiry will also result in the establishment of viable bioassays with which he can perform structure-function studies. He shows in his Preliminary data that he has established two different approaches to identify EVI1 target genes, and has used these techniques to isolate several candidates. In this application, he proposes to further characterize these genes, and to study the role of EVI1 in their transcriptional regulation. He intends to improve upon his selection strategies, to identify greater numbers of targets that may be of relevance to EVI1 s role in hematopoiesis and leukemia. He also proposes to investigate the effect of EVI1 on transcriptional regulation, using the target genes in hand as substrates. In the second Specific Aim, the applicant wishes to address the hypothesis that EVI1 causes transcriptional repression, at least in part, through protein-protein interactions. It is known that the binding sites of EVI1 and GATA-1 overlap, and that EVI1 can repress the transcriptional activation properties of GATA1, leading to the hypothesis that EVI1 may contribute to leukemia by blocking GATA-1 function. However, EVI1 requires additional bases beyond the GATA motif for DNA binding, and thus can not bind to GATA sites described to date. Nonetheless, he has observed that EVI1 can repress GATA-1 activation of reporters that contain GATA motifs to which EVI1 fails to bind in vivo. These findings suggest that EVI1-mediated repression of transcription may be mediated by protein-protein interactions, rather than DNA-protein interaction. To further investigate the proteins to which EVI1 binds within the cell, he proposes to perform a two-hybrid screen in yeast. This will lead to the identification of proteins that bind to EVI1, and will likely yield significant insight into the mechanism by which EVI1 effects transcriptional repression, and by which it causes disease.
|Zhang, Yi; Stehling-Sun, Sandra; Lezon-Geyda, Kimberly et al. (2011) PR-domain-containing Mds1-Evi1 is critical for long-term hematopoietic stem cell function. Blood 118:3853-61|