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. . To better understand the mechanism(s) of the negative regulation of ?-globin expression through silencer II at the level of the whole organism, we have developed a transgenic mice expressing the human ?-globin gene mutated at the BP1 binding site. Because the silencer II element does not interfere with High Mobility Group protein binding sites as silencer I does, we mutated the silencer II BP1 binding site in a cosmid construct that we subsequently used to create the transgenic mice. To better understand the mechanism of the negative regulation of ?-globin expression by BP1 we have developed transgenic mice bearing the human ?-globin locus-containing cosmid. Specifically, we introduced a mutated BP1 binding site (mtBP1) into the promoter of ?-globin gene sequence of this cosmid construct. We detected up to 20-fold increase in human ?-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 compare to control mice bearing the human ?-globin gene with wild type BP1 binding site sequence (wtBP1). Our in vivo observations support the contention that BP1 binding site at ?-globin promoter plays an important role in the regulation of transcription of adult ?-globin gene. 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 |
