Beta-hemogloblinopathies, such as sickle cell anemia, and beta-thalassemia cause considerable morbidity and mortality worldwide. A long-standing goal in the field of hematology has been to find means to re-activate fetal globin production in these patients. Rationally designed approaches first require a firm understanding of the molecular mechanisms controlling normal developmental globin gene expression. Prior work has delineated key cis-acting DNA regulatory elements in the human beta-globin locus control region (LCR) and gamma and beta-globin gene promoters. Clustered within these regions are binding sites for GATA, NF-E2 transcription factors and sequences containing a core """"""""CACCC"""""""" motif. A subset of these """"""""CACCC""""""""-like sequences play important roles in globin gene switching. Yet, which factors bind these sequences under physiologic conditions has not been fully established. In preliminary studies, we purified GATA-1 containing multiprotein complexes from induced mouse erythroleukemia (MEL) cells and identified the Kruppel-type zinc finger transcription factor zfp148 as a novel associated protein. This factor recognizes the consensus sequence CC(A/T)CCCCC and acts as either a transcriptional activator or represser. Recently, Groudine and his colleagues independently identified zfp148 as a factor enriched in NF-E2/mafK complexes in induced MEL cells. Our chimeric mouse studies using zfp148 genetrap ES cells (zfp148 gt/gt) show that they contribute poorly to mature adult erythrocytes. The main objectives of this proposal are to further examine the role of zfp148 in normal erythroid development in vivo, and test the hypothesis that zfp148 plays a role in human globin gene switching.
Specific aims i nclude (1) generation and analysis of erythroid-specific conditional murine zfp148 knock-out mice (zfp148+/- chimeric mice are infertile) (2) Examination of in vivo zfp148 occupancy of the previously identified CACCC sequences by chromatin immunoprecipitation (ChIP) assays. This will be performed on erythroid progenitor cells of transgenic mice harboring a human beta-globin locus yeast artificial chromosome (YAC);(3) determination of whether zfp148 is required for human gamma- to beta-globin gene switching by manipulation of expression levels by lentiviral SiRNA or retroviral overexpression followed by measurement of human gamma- to beta-globin expression ratios in the human beta-globin YAC transgenic mice (with Dr. Stuart Orkin (Project 2);and (4) Identifcation of additional zfp148 direct target genes by ChiP followed by mouse promoter array hybridization (""""""""ChIP on Chip""""""""), and comparison to GATA-1, FOG-1, stat5, and FoxO3a target genes (with Dr. Harvey Lodish (Project 1). The results of these studies should provide important information about a newly recognized erythroid transcription factor that may serve as a valuable target for pharmacologic manipulation in the treatment of beta-hemoglobinopathies and beta-thalassemia.
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