DEFINITION OF THE MINIMAL REQUIREMENTS WITHIN THE HUMAN BETA-GLOBIN GENE AND THE DOMINANT CONTROL REGION FOR HIGH-LEVEL EXPRESSION

The human β-globin dominant control region (DCR) was previously identified as a region from the 5' end of the human β-globin locus which directs high level, site of integration-independent, copy number-dependent expression on a linked human β-globin gene in transgenic mice and stably transfected mouse erythroleukaemia (MEL) cells. We have now analysed the elements comprising the DCR by systematic deletion mutagenesis in stable MEL transfectants. We have identified two independent elements within the DNase I hypersensitive sites 2 and 3, containing fragments which direct strong transcriptional inducibility of a β-globin gene. Whilst the remaining two hypersensitive sites do not direct significant transcriptional induction, our data suggest that all four sites may be necessary for the fully regulated expression conferred by the DCR. We have also tested a number of β-globin minigene constructs under the control of the DCR to assess if any of the local sequences from the gene may be removed without loss of expression. We find that the 3' enhancer may be removed without affecting expression, but there is an absolute requirement for the presence of the second intron, not related to the enhancer present in that intron.