Beta-hemogloblinopathies, such as sickle cell anemia, and beta-thalassemia cause considerable morbidity andmortality worldwide. A long-standing goal in the field of hematology has been to find means to re-activatefetal globin production in these patients. Rationally designed approaches first require a firm understandingof the molecular mechanisms controlling normal developmental globin gene expression. Prior work hasdelineated 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-E2transcription factors and sequences containing a core 'CACCC' motif. A subset of these 'CACCC'-likesequences play important roles in globin gene switching. Yet, which factors bind these sequences underphysiologic conditions has not been fully established. In preliminary studies, we purified GATA-1 containingmultiprotein complexes from induced mouse erythroleukemia (MEL) cells and identified the Kruppel-type zincfinger transcription factor zfp148 as a novel associated protein. This factor recognizes the consensussequence CC(A/T)CCCCC and acts as either a transcriptional activator or represser. Recently, Groudineand his colleagues independently identified zfp148 as a factor enriched in NF-E2/mafK complexes in inducedMEL cells. Our chimeric mouse studies using zfp148 genetrap ES cells (zfp148 gt/gt) show that theycontribute poorly to mature adult erythrocytes. The main objectives of this proposal are to further examinethe role of zfp148 in normal erythroid development in vivo, and test the hypothesis that zfp148 plays a role inhuman globin gene switching.
Specific aims i nclude (1) generation and analysis of erythroid-specificconditional murine zfp148 knock-out mice (zfp148+/- chimeric mice are infertile) (2) Examination of in vivozfp148 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-globinlocus 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 overexpressionfollowed by measurement of human gamma- to beta-globin expression ratios in the human beta-globin YAC transgenicmice (with Dr. Stuart Orkin (Project 2); and (4) Identifcation of additional zfp148 direct target genes by ChiPfollowed 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 provideimportant information about a newly recognized erythroid transcription factor that may serve as a valuabletarget for pharmacologic manipulation in the treatment of beta-hemoglobinopathies and beta-thalassemia.
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