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