IDENTIFICATION OF CANDIDATE TARGET GENES FOR EVI-1, A ZINC-FINGER ONCOPROTEIN, USING A NOVEL SELECTION STRATEGY

We have sought to identify and isolate target genes for the zinc finge r protein, EVI-1, which has been implicated in the genesis of myelogen ous leukemia both in mouse and human. We have approached this with a t wo-step selection: we first selected for genomic fragments of mouse DN A that bind to the protein with high affinity; second, we employed cDN A hybrid selection to identify gene sequences contained within these f ragments. We show that we have constructed a sublibrary of genomic fra gments that contains a significant fraction of the EVI-1-binding sites in the mouse genome, Our data has allowed us to estimate that there a re approximately 4300 binding sites per haploid genome in the mouse. W e further demonstrate that by using cDNA hybrid selection, it is relat ively straightforward to isolate cDNAs that correspond to genes embedd ed in the EVI-1-binding sublibrary. Several of these are novel, but ar e represented in databases of anonymous human or mouse cDNAs (expresse d sequence tags), One selected gene is Itpr2, encoding the inositol tr isphosphate type two receptor, which is transcriptionally regulated du ring myelopoiesis. Finally, using a chimeric EVI-1-VP16-fusion protein under the control of a tetracycline-regulated system, we have shown t hat this chimeric activator can directly regulate Itpr2.