Developmental regulation of expression of globin genes is achieved through a precise balance of negative and positive regulation. The adult beta-globin gene includes two upstream silencer regions, and a common effector, termed beta protein 1 (BP1), binds to both of these regions. BP1 binds within silencer I at ?530 bp and within silencer II at ?300 bp relative to the cap site (+1). At both silencers, architectural chromatin proteins of the high mobility group (HMG) bind to and bend the DNA, possibly providing access for BP1 and other repressor proteins. HMG-I (Y) bends DNA at or near the BP1 binding site in silencer I but not in silencer II; HMG1 bends DNA near the silencer II. 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. In our earlier studies of BP1 function in K562 cells, an erythroleukemia cell line with an embryonic-fetal phenotype, we have shown that introduction of multiple mutations in two BP1 binding sites (silencer I and silencer II) results in enhanced beta-globin promoter activity; thus, BP1 downregulates beta-globin transcription when the silencers are intact. In the present study we explored the mechanisms by which BP1 negatively regulates beta-globin gene expression. We investigated whether multiple mutations near but not at silencer II enhanced the activity of the beta-globin promoter, as do mutations in the silencer II BP1 binding site. We also probed the role of the BP1 protein itself: we suppressed BP1 protein synthesis in K562 cells by transfecting a small RNA duplex corresponding to 21 bases of the BP1 mRNA, then examined its effect on beta-globin promoter activity, beta-globin mRNA levels, and expression of GATA-1 and EKLF. We constructed three DNA clones containing the beta-globin promoter and upstream promoter elements that harbor two BP1 binding sites. We prepared constructs containing the wild-type silencer II sequence, a mutated silencer II sequence, or a mutated control sequence in a 690-bp promoter insert, which in turn was linked to an enhanced green fluorescent protein (EGFP) reporter gene. These EGFP constructs were transfected into K562 erythroleukemia cells. Flow cytometry and fluorescence intensity measurements on digital images showed a 3-fold increase in the beta-globin promoter activity of the mutated silencer II construct. Introduction of a small double-stranded RNA complementary to BP1 into the cells caused a 75% decrease in BP1 expression and a simultaneous ~40% increase in beta-globin promoter activity and beta-globin mRNA levels, as compared to controls. We did not find any association between GATA-1 and EKLF expression in K562 cells and BP1 inhibition. Our results suggest the involvement of BP1 in the negative regulation of beta-globin transcription and pinpoint a novel target for therapy of the severe beta-hemoglobinopathies. These data were presented at the 32th Annual Scientific Meeting of International Society for Experimental Hematology (ISEH) in July, 2003. We have produced transgenic mice (in collaboration with Dr. Bodine) to establish a transgenic mouse model for specific repression of adult beta-globin gene expression by the BP1 protein. The murine Dlx4 (also known as Dlx7) is homologue of human BP1. To alter developmental specificity of the human beta-globin gene, 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. The use of the transgenic approach should permit the in vivo analysis of the role of BP1 silencing motif during the embryonic-fetal developmental stages
| 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 |
