Cis and trans control of erythroid cell-specific gene expression during erythropoiesis
Authors: Harrison P. R. , Plumb M. , Frampton J. , Chambers I. , Llewellyn D. , Chester J. , Macleod K. , Fleming J. , O’prey J. , Walker M. , Wainwright H. 
Published: May 2013

The overall aim of our group’s work is to investigate the molecular mechanisms regulating erythroid cell-specific gene expression during erythroid cell differentiation. We have been successful in cloning two non-globin genes of interest: the first encodes the rabbit red cell-specific lipoxygenase (LOX), which has a role in degrading mitochondrial lipids during maturation of the reticulocyte to the erythrocyte; and the second, mouse glutathione peroxidase (GSHPX), an important seleno-enzyme responsible for protection against peroxide-damage. Characterization of the GSHPX gene revealed that the seleno-cysteine residue in the active site of the enzyme is encoded by UGA, which usually functions as a translation-termination codon. This novel finding has important implications regarding the role of mRNA sequence context effects in codon recognition.

In contrast with the βglobin locus, very little is known about the mechanisms responsible for the erythroid-specific expression of the α-globin genes. By a combination of functional transfection assays and studies of the interactions of nuclear sequence-specific DNA-binding proteins with promoter sequences in vitro, we have recently defined two regions upstream of the mouse α-globin gene involved in its erythroid-specific expression: one contains a sequence motif (GATAAG) that binds to a species-conserved and erythroid-specific factor both in vitro and in vivo. Interestingly, GATAAG motifs binding the same factor are found also in the mouse and chicken adult β-globin gene promoters, the erythroid-specific promoter of the haem pathway enzyme, porphobilinogen (PBG) deaminase and the chicken β-globin 3′ enhancer. We are now commencing purification of this erythroid-specific GATAAG-binding factor, investigating in more detail how it functions in relation to other globin gene control regions and determining whether GATAAG-like regions have a functional role in the erythroid-specific expression of other genes.

We have begun to investigate the regulation of the GSHPX and red cell LOX genes. The presence of tissue-specific 3′ DNase I-hypersensitive sites (DHSS) suggests that different 3′ flanking regions of the GSHPX gene may be important in its regulation in the various cell types in which it is highly expressed, i.e. erythroid cells, liver and kidney. As far as the RBC LOX gene is concerned, its transcription unit has been defined and 5′ and 3′ flanking regions are being investigated for erythroid-specific regulatory elements: the available 2·7kb of 5′ LOX gene flanking sequence gives increased expression of a linked chloramphenicol acetyltransferase (CAT) gene when transfected into a mouse erythroid cell line compared to non-erythroid cell lines; but we have not yet located the regions responsible, except that they are upstream of −154 nucleotides. The limits of the LOX gene chromosomal domain, and regions involved in its activation in erythroid cells, are also being investigated by DNase I digestion studies.