ABSTRACT
The human alpha-globin gene complex includes three functional globin genes (5'-zeta2-alpha2-alpha1-3') regulated by a common positive regulatory element named HS-40 displaying strong erythroid-specific enhancer activity. How this enhancer activity can be shared between different promoters present at different positions in the same complex is poorly understood. To address this question, we used homologous recombination to target the insertion of marker genes driven by cytomegalovirus or long terminal repeat promoters in both possible orientations either upstream or downstream from the HS-40 region into the single human alpha-globin gene locus present in hybrid mouse erythroleukemia cells. We also used CRE recombinase-mediated cassette exchange to target the insertion of a tagged alpha-globin gene at the same position downstream from HS-40. All these insertions led to a similar decrease in the HS-40-dependent transcription of downstream human alpha-globin genes in differentiated cells. Interestingly, this decrease is associated with the strong activation of the proximal newly inserted alpha-globin gene, whereas in marked contrast, the transcription of the non-erythroid marker genes remains insensitive to HS-40. Taken together, these results indicate that the enhancer activity of HS-40 can be trapped by non-erythroid promoters in both upstream and downstream directions without necessarily leading to their own activation.
Subject(s)
Enhancer Elements, Genetic , Globins/genetics , Globins/metabolism , Animals , Cell Nucleus/metabolism , Chromosomes, Human, Pair 16 , Cytomegalovirus/genetics , DNA Nucleotidyltransferases/metabolism , Humans , Mice , Models, Genetic , Mutagenesis, Insertional , Plasmids/metabolism , Promoter Regions, Genetic , Recombination, Genetic , Ribonucleases/metabolism , Terminal Repeat Sequences , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
We developed a reverse-transcription polymerase chain reaction assay, performed on single isolated cells, to demonstrate the coexpression of human alpha1- and alpha2-globin mRNA in induced mouse erythroleukemic cells containing a single human alpha-globin locus. These results indicate that both alpha1 and alpha2 genes are activated from the same alpha-globin gene locus implying that HS-40-dependent transcriptional activation is mediated, either by a simultaneous interaction of HS-40 with both a alpha1 and alpha2-globin gene promoters, or by a dynamic process characterized by alternative, but short-lived, interactions with each alpha-globin gene promoter.
