Some cases of acute promyelocytic leukemia are characterized by a t(11;17) translocation which fuses the retinoic acid receptor-alpha (RARa) to a novel zinc finger gene, promyelocytic leukemia zinc finger gene (PLZF). The PLZF gene encodes a putative transcription factor that is expressed in myeloid progenitor cells and is down-regulated during in vitro differentiation. This suggests that PLZF may have a role in regulating normal hematopoiesis. Little is known about the molecular mechanisms by which PLZF normally regulates hematopoietic differentiation, or contributes to the transformation of progenitor cells when fused to RARa. In this proposal the role of PLZF in regulating myeloid-lineage hematopoiesis will be defined by 1) Identifying the target genes regulated by PLZF. Recombinant PLZF protein containing the nine C2H2 zinc fingers will be used to affinity select (by gel shift assays or chromatography) DNA binding sites from a degenerate oligonucleotide library and a genomic DNA fragment library. The binding site will be used to identify potential target genes that contain the PLZF binding sites within their promoters and are differentially expressed during hematopoiesis. 2) Analyzing the transcriptional regulation of target gene expression by PLZF. PLZF expression vectors will be cotransfected with chloramphenicol acetyl transferase (CAT) reporter genes containing PLZF binding sites into mouse fibroblasts and myeloid progenitor cell lines. Transcriptional regulation of target genes by PLZF will be measured by CAT assays and RNAse protection. The domains of PLZF that are necessary and sufficient for transcriptional regulation will be determined through site-directed mutagenesis. 3) Assessing the oncogenic potential of the PLZF/RARa fusion proteins in myeloid progenitor cells. The biochemical functions of the PLZF/RAR fusion proteins will be assessed by measuring the ability of the fusion proteins to form dimers, bind to the PLZF and RARa DNA binding sites, and to transcriptionally regulate expression of target genes. The transformation potential of the PLZF/RARa fusion proteins will be assessed by transfecting the mouse fibroblast cell line NIH 3T3. The effect of the PLZF/RARa proteins on the proliferation and differentiation of myeloid progenitor cell lines will be determined by establishing cell lines that. either constitutively or inducibly express the PLZF/RARa fusion proteins and determining the effect of PLZF/RARa expression on cell growth, survival and differentiation. The biochemical characterization of PLZF and the chimeric PLZF/RARa proteins will define the role of PLZF in normal hematopoiesis and leukemogenesis. These biochemical functions will provide the basis for the future development of sensitive diagnostic assays, novel treatments, targets for gene therapy, and assays to monitor residual disease.
