ABSTRACT
The binding of the transcription factor BP1 (beta protein 1) to its site on the promoter of the adult beta-globin gene has a silencing effect on beta-globin transcription in vitro. To better understand the mechanism of BP1's negative regulation of beta-globin expression, we developed transgenic mice bearing a human beta-globin locus-containing cosmid. Specifically, we introduced a mutated BP1 binding site (mtBP1) into the promoter of the beta-globin gene sequence of this cosmid construct. In the mtBP1 mice, we detected a more than a 20-fold increase in human beta-globin expression in the yolk sac-derived blood at E10.5, a 3-fold increase in fetal livers at E13.5, and an approximately 1.4-fold increase in adult reticulocytes compared with control mice bearing the human beta-globin gene with the wild-type BP1 binding site sequence (wtBP1). Our in vivo observations support the contention that the BP1 binding site of the beta-globin promoter plays an important role in the regulation of transcription of the adult beta-globin gene.
Subject(s)
Erythropoiesis , Gene Expression Regulation, Developmental , Homeodomain Proteins/metabolism , Mice, Transgenic/metabolism , Promoter Regions, Genetic , Transcription Factors/metabolism , beta-Globins/genetics , Animals , Base Sequence , Binding Sites , Cell Line , Cosmids/genetics , Humans , Mice , Mice, Transgenic/embryology , Mice, Transgenic/genetics , Polymerase Chain Reaction , Protein Structure, Tertiary , Transfection , beta-Globins/metabolismABSTRACT
OBJECTIVE: In this paper, we report new observations related to the mechanism of the negative regulation of the important adult beta-globin gene in the erythroid cells at the embryonic-fetal stage of their development. We focused on the role of the silencer II region located upstream of the beta-globin gene, which along with its cognate binding protein BP1, negatively regulates beta-globin transcription. MATERIALS AND METHODS: We prepared plasmid constructs containing the wild-type silencer II sequence, a mutated silencer II sequence, or a mutated control sequence in the beta-globin promoter 690-bp insert, which in turn was linked to an enhanced green fluorescent protein (EGFP) reporter gene. A human erythroleukemia cell line (K562) with embryonic-fetal phenotype was transfected with these EGFP constructs. RESULTS: Flow cytometry and fluorescence digital imaging showed about threefold increase in the beta-globin promoter activity of the mutated silencer II construct. Introduction of a small interfering RNA (siRNA) complementary to BP1 into the cells caused a 75% decrease in BP1 expression and a simultaneous approximately 40% elevation of beta-globin promoter activity as well as an increase in beta-globin mRNA levels, as compared with controls. We detected no changes in the mRNA levels of positive regulators of hemoglobin transcription such as EKLF and GATA-1. CONCLUSION: Our results support the involvement of BP1 in the mechanism of the negative regulation of beta-globin transcription. A better understanding of this mechanism may lay the groundwork for novel gene therapy approaches to inhibit the expression of abnormal structural variants of adult beta globin, such as sickle hemoglobin.
Subject(s)
Globins/genetics , Homeodomain Proteins/physiology , Promoter Regions, Genetic , RNA, Messenger/analysis , Repressor Proteins/physiology , Transcription Factors/physiology , DNA-Binding Proteins/genetics , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , Gene Silencing , Genetic Therapy , Humans , K562 Cells , Kruppel-Like Transcription Factors , RNA Interference , Transcription Factors/geneticsABSTRACT
Uncoating of influenza occurs in endosomes where the acid environment is instrumental in membrane fusion and the dissociation of the ribonucleoprotein (RNP) from matrix protein by the action of the hemagglutinin and M2 protein ion channels, respectively. Earlier studies have shown that low pH treatment results in the release of M1 protein from RNP. To obtain RNP free of M1 protein, we attempted to isolate RNP by velocity sedimentation on pH 5 glycerol gradients; however, the RNP sedimented as pellets under centrifugation conditions that had previously resolved RNP on neutral gradients. The increase in sedimentation rate occurred between pH 5.6 and 6.0 and was reversible for a portion of the RNP on raising the pH to neutrality. RNP isolated from infected cells or virions sedimented on acidification and was seen to form clumps visible by electron microscopy. If acidification preceded NP40 detergent lysis, virion RNP appeared to be released as genomic complexes. The pH threshold for viral membrane fusion was 5.8 indicating that the same pH condition also resulted in aggregation of RNP. Because exposure of virions to pH 5 occurs during uncoating in endosomes and is essential for infectivity, it is possible that low pH-induced RNP aggregation may facilitate aspects of viral uncoating such as dissociation of RNP from M1 or transport of genomes to the nucleus.
