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 finger protein, EVI-1, which has been implicated in the genesis of myelogenous leukemia both in mouse and human. We have approached this with a two-step selection: we first selected for genomic fragments of mouse DNA that bind to the protein with high affinity; second, we employed cDNA hybrid selection to identify gene sequences contained within these fragments. We show that we have constructed a sublibrary of genomic fragments that contains a significant fraction of the EVI-1-binding sites in the mouse genome. Our data has allowed us to estimate that there are approximately 4300 binding sites per haploid genome in the mouse. We further demonstrate that by using cDNA hybrid selection, it is relatively straightforward to isolate cDNAs that correspond to genes embedded in the EVI-1-binding sublibrary. Several of these are novel, but are represented in databases of anonymous human or mouse cDNAs (expressed sequence tags). One selected gene is Itpr2, encoding the inositol trisphosphate type two receptor, which is transcriptionally regulated during myelopoiesis. Finally, using a chimeric EVI-1-VP16-fusion protein under the control of a tetracycline-regulated system, we have shown that this chimeric activator can directly regulate Itpr2.
Kim
J H
JH
Yale University, Department of Pathology, New Haven, Connecticut 06520-8023, USA.
Hui
P
P
Yue
D
D
Aycock
J
J
Leclerc
C
C
Bjoring
A R
AR
Perkins
A S
AS
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015220:Calcium Channels / Q000235:genetics*; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology*; D004926:Escherichia coli; D005091:Exons; D015698:Genomic Library; D006238:Haploidy; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D000074008:MDS1 and EVI1 Complex Locus Protein; D051379:Mice; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000502:physiology*; D011519:Proto-Oncogenes; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors / Q000502:physiology*; D014407:Tumor Cells, Cultured; D016335:Zinc Fingers / Q000502:physiology*
D015220:Calcium Channels; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; C506926:ITPR1 protein, human; C506927:ITPR2 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D000074008:MDS1 and EVI1 Complex Locus Protein; C063575:MECOM protein, human; C499305:Mecom protein, mouse; D015513:Oncogene Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors