The Behaviour of Human Globin Gene Recombinants in Mammalian Cells

The human epsilon globin gene has multiple sites of RNA initiation. Of these, the most abundant are the transcripts originating from the -200 promoter giving a cap: -200 ratio of 20:1 in RNA prepared from the erythro- leukaemic cell line K562 and 5-10 week embryos. A series of epsilon globin gene recombinants were introduced into a range of cell lines to compare the transcriptional profile with that obtained in the K562 cell and to identify putative regulatory regions. This approach led to the following observations. 1) Epsilon globin gene activity in stably and acutely transformed non-erythroid and MELtk- cells display a distinct tendency towards greater use of the -200 promoter. Even recombinants with up to 6 kb of 5' flanking DNA, cap: -200 ratios varied from 4:1 in mouse LAtk-, 1:1 in BHKtk- and 0:1 in MELtk- cells. 2) The major epsilon globin promoter (5'- -100/CCAAT/ ATA-cap-3') is not sufficient to support optimal levels of cap site expression in stably transformed BHKtk- cells. 3) However when this structure was extended by the cis -linkage of 350 bp's of endogenous DNA, optimal cap site expression was restored. 4) When this recombinant was further extended by 1468 bp's, the cap: -200 ratio dropped from 4:1 to 1:20 in acutely transformed mouse LAtk- cells. Interestingly, this fragment contains two upstream promoters at -900 and -1480 bp's. Using a functional assay system another putative regulatory region has been identified over 2 kb upstream of the major promoter. A 6.67 kb Eco RI fragment which contains a potential Z-DNA element and the most distal promoter at -4500 bp's markedly increases the expression of the bacterial Tn5 aph(II) gene from the epsilon globin promoter. Additionally, several lines of evidence has been presented which suggest that the major and -200 promoters can be regulated independently by both exogenous cis linked sequences (SV40 origin and enhancer) and viral trans acting factors. Based on these findings it is proposed that these minor promoters particularly the -200 are transcriptionally active during early embryonic erythropoietic devlopment. During this period canonical cap site activity may in part be repressed by some sort of transcriptional interference mechanism. The switch from the upstream promoters occurs in later development mediated by an embryonic erythroid trans acting factor as experimentally demonstrated by the switch in cap: -200 ratio from 1:20 in non-erythroid cells to 20:1 in K562 cells transformed with a mutated epsilon globin gene recombinant to distinguish it from the endogenous gene