Over the last year, our laboratory has studied the negative regulation of the human beta-globin gene. Previous results have shown that the promoter region of adult beta-globin gene includes two upstream silencer sequences, and a common effector, termed beta protein 1 (BP1), which binds to both of these sequences. The BP1 gene belongs to the Distal-less (DLX) family of homeobox genes, which are expressed during early development. This gene shares extensive identity with DNA sequence of human DLX4 and appears to be a splice variant of DLX4. Our current research focuses on the mechanism(s) by which the silencer II region, along with its cognate binding protein BP1, negatively regulates beta-globin transcription. To investigate this mechanism in detail, we developed in vitro and in vivo models of analysis of the negative regulation of beta-globin gene. In the course of recent in vitro studies, we indicated that an inhibition of BP1 by siRNA results in an increase in endogenous beta-globin, and this increase is not directly associated with up-regulation of EKLF or GATA-1. Last year, our results were published in Experimental Hematology. To better understand the mechanism of the negative regulation of beta-globin expression through the silencer II, and to confirm our in vitro observations at the level of the whole organism, we also have developed a transgenic mouse model. Specifically, we introduced a mutated BP1 binding site (silencer II) into the distal promoter of beta-globin gene sequence of 35 kb cosmid construct containing the micro-LCR (locus control region) and other essential elements of human beta-globin gene cluster. This construct and appropriate controls have been microinjected into the single cell mouse embryos. We have alsoestablished a colony of three transgenic mouse lines containing human beta-globin gene locus with mutated silencer II sequence. Currently, we are finishing the analysis of the mechanism(s) of regulation of beta-globin expression by Dlx4 gene which is murine analog of BP1. Both human BP1 and a murine Dlx4 mRNAs are highly homologous: their homeobox sequences encode almost identical protein DNA-binding domains. BP1 and murine Dlx4 are predominantly expressed during early stages of development. Therefore, to detect the differences in developmental regulation of the human beta-globin gene expression in the transgenic mice, we study yolk sac derived embryonic blood at embryonic day 10.5 (E10.5) and fetal liver of mouse embryos at E13.5. In a parallel study, we also analyze adult erythroid cells. Blood collected from E10.5 embryos predominantly contain cells from primitive erythropoiesis. The fetal liver samples from E13.5 embryos and adult blood predominantly contain cells from definitive erythropoiesis. To minimize experimental error, samples from individual animals of three transgenic lines are quantified independently while multiple measurements are performed using real-time PCR assays. Levels of expression of murine alpha-globin RNA are used as internal controls. We found that the expression of human beta globin in transgenic mice containing mutated silencer II was higher at all stages of erythroid cells development compare with control transgenic mice bearing cosmid construct with wild type of silencer II sequence. Particularly, we detected up to 20-fold increase in human beta-globin expression in embryonic blood at E10.5, 3-fold increase in fetal livers of transgenic mice at E13.5, and up to 1.4-fold increase in adult reticulocytes. We also found that increase in human beta-globin expression was correlated with expression pattern of murine Dlx4 which mRNA was predominantly expressed in embryonic blood at E10.5. Thus, our results indicate that transgenic mice bearing human beta-globin gene with mutated BP1 site have significantly higher human beta-globin transcripts levels in blood cells from primitive erythropoesis than control mice.
| Zoueva, Olga P; Rodgers, Griffin P (2004) Inhibition of beta protein 1 expression enhances beta-globin promoter activity and beta-globin mRNA levels in the human erythroleukemia (K562) cell line. Exp Hematol 32:700-8 |
| Drew, L R; Tang, D C; Berg, P E et al. (2000) The role of trans-acting factors and DNA-bending in the silencing of human beta-globin gene expression. Nucleic Acids Res 28:2823-30 |
