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Identification of candidate target genes for EVI-1, a zinc finger oncoprotein, using a novel selection strategy
1Yale University, Department of Pathology, New Haven, Connecticut 06520-8023, USA.
Abstract:
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.
Insights
Researchers identified EVI-1 (Ecotropic Viral Integration 1) target genes in mice, estimating ~4300 binding sites. They isolated novel genes, including Itpr2, crucial for myelopoiesis, and demonstrated EVI-1
Area of Science:
- Molecular Biology
- Genomics
- Cancer Research
Background:
- The zinc finger protein Ecotropic Viral Integration 1 (EVI-1) is implicated in myelogenous leukemia development in both mice and humans.
- Identifying EVI-1 target genes is crucial for understanding its role in leukemogenesis.
Purpose of the Study:
- To identify and isolate target genes regulated by the EVI-1 protein.
- To estimate the number of EVI-1 binding sites in the mouse genome.
Main Methods:
- A two-step selection process was employed: genomic fragment selection for high-affinity EVI-1 binding, followed by cDNA hybrid selection.
- Construction of a sublibrary of genomic fragments enriched for EVI-1 binding sites.
- Utilizing a tetracycline-regulated system to control a chimeric EVI-1-VP16-fusion protein for gene regulation studies.
Main Results:
- An estimated 4300 EVI-1 binding sites per haploid mouse genome were identified.
- A sublibrary of genomic fragments containing a significant fraction of EVI-1 binding sites was constructed.
- Novel genes, including Itpr2 (inositol trisphosphate type two receptor), were isolated and found to be transcriptionally regulated during myelopoiesis.
- The chimeric EVI-1-VP16 activator directly regulated Itpr2 expression.
Conclusions:
- The study successfully identified target genes for the EVI-1 protein using a novel genomic selection strategy.
- Itpr2 is a novel EVI-1 target gene transcriptionally regulated during myelopoiesis.
- These findings provide insights into the molecular mechanisms of EVI-1 in leukemogenesis and offer potential targets for therapeutic intervention.