Changes in ninor transcripts from the al and 3 mAi globin and glutathione peroxiddase genes during erythropoiesis

We have analysed the transcriptional regulation of the zbrine al and 0maj globin genes and the glutathione peroxidase (GSHPx) gene, which are all highly expressed during erythropoiesis. The levels of minor RNAs compared to the major message were monitored throughout differentiation within the erythroid lineage. For each gene, upstream transcripts arise from distinct clusters of sites which are regulated differently during differentiation: some occur only during early erythropoiesis, some occur early and persist to the terminal stages, while others accunulate later and roughly in parallel with the main RNA transcript. In addition, opposite strand transcripts from the GSHPx gene were found in increasing amounts during later stages of erythropoiesis. The initiation sites for specific subsets of these minor transcripts lie close to sequences kncown to be involved in globin gene regulation (i.e. the TATA, CAAT and the CACCCT boxes) or other conserved sequences; others lie close to developmentally regulated DNase I hypersensitive sites around the globin and GSHPx genes. INTRODUCTION Murine erythropoiesis is a differentiation process which lends itself to analysis of the molecular mechanisms involved in the co-expression of genes. Much work has centered on control elements around the globin genes especially with regard to l globin gene transcriptional activity (1), inducibility of the 6 globin gene in erythroleukaemic cells (2-6), and minimun sequence requirements for erythroid-specific expression of ( globin genes in transgenic mice (7,8). However, little attention has been directed towards erythroid-specific control elements in a globin genes or non-globin genes. The best characterised mouse red blood cell non-globin proteins for which cloned genes are available are the carbonic anhydra8e II (CAII) isoenzyme (P.Curtiss, personal communication; (9)) and glutathione peroxidase (GSHPx) (10). The GSHPx mRNA is interesting in that it is highly expressed in red cells and in a limited nunber of other tissues (notably liver and kidney) in contrast to its low level in other cell types (10,11). © I RL Press Limited, Oxford, England. 3671 Nucleic Acids Research The upstream flanking sequence of the gene exhibits features characteristic of many constitutive genes (10). However, the GSHPx gene's pattern of expression poses an interesting question in terms of how it comes to be co-expressed with various groups of other genes in different tissues. Transcription occuring from sites other than the normal cap position has been docunented for a number of genes (12-15) including the globin genes (16-21). Generally the transcripts arise from upstream positions, are of the same sense as the major transcript, and are polyadenylated. However, there are examples of minor transcripts occuring from the opposite-sense strand (14,15), and lacking a polyadenylic acid tail (22). Often the ratiQs of minor RNAs to the major transcript from a particular gene are found to vary either in a developmental-specific manner (13,23), or in response to stimuli (14). Studies of sequences close to such minor transcription start sites, particularly from the human E globin gene (24,25), have suggested a regulatory role for these regions. Moreover, there is a close correlation between the positions of E globin gene upstream transcripts and tissue-specific nuclease hypersensitive sites which are thought to be involved in transcriptional activation (26). A further close correlation between minor transcripts and regulatory regions is seen in some viral promoters where minor RNA start sites occur within functionally defined transcriptional enhancer sequences (12,13). In this report, the GSHPx gene is compared to the al and 0 globin genes in terms of changes in minor transcripts throughout erythroid differentiation from haemopoietic stem cells through to terminally differentiated reticulocytes. The pattern of expression of the various minor transcripts implies that they are involved in, or are reflecting, developmentally specific events. MATERIALS AND METHODS Cell lines and tissues Where possible all cells were derived from Balb/c mice in order to minimise the possibility of small regions of non homology between RNAs and the Balb/c DNA probes and primers. Balb/c Friend cells (clone FSD 2/3, a gift from W. Ostertag, Hamburg (unpublished)) were grown in Eagles MEM with double the concentrations of amino acids and vitamins and with 10% foetal calf serum. Induction of differentiation was achieved5by the addition of 5mM HMBA to exponentially growing cells set up at 2x10 /ml, cells being collected 0-2 d later. DBA/2 Friend cells (clone 707B10/1) and the mouse