Nucleic Acids Research, 2003, Vol. 31, No. 15 4582-4596
© 2003 Oxford University Press
The ERV-9 LTR enhancer is not blocked by the HS5 insulator and synthesizes through the HS5 site non-coding, long RNAs that regulate LTR enhancer function
Department of Biochemistry and Molecular Biology, School of Medicine, Medical College of Georgia, Augusta, GA 30912, USA
*To whom correspondence should be addressed. Tel: +1 706 721 0272; Fax: +1 706 721 6608; Email: dtuanlo{at}mail.mcg.edu
A solitary long terminal repeat (LTR) of ERV-9 human endogenous retrovirus is located upstream of the HS5 site in the human ß-globin locus control region and possesses unique enhancer activity in erythroid K562 cells. In cells transfected with plasmid LTR-HS5-
p-GFP, the LTR enhancer activates the GFP reporter gene and is not blocked by the interposed HS5 site, which has been reported to have insulator function. The LTR enhancer initiates synthesis of long RNAs from the LTR promoter through the intervening HS5 site into the -globin promoter and the GFP gene. Synthesis of the sense, long LTR RNAs is correlated with high level synthesis of GFP mRNA from the -globin promoter. Mutations of the LTR promoter and/or the -globin promoter show that (i) the LTR enhancer can autonomously initiate synthesis of LTR RNAs independent of the promoters and (ii) the LTR RNAs are not processed into GFP mRNA or translated into GFP. However, reversing the orientation of the LTR in plasmid (LTR)rev-HS5-p-GFP, thus reversing the direction of synthesis of LTR RNAs in the antisense direction away from the -globin promoter and GFP gene drastically reduces the level of GFP mRNA and thus LTR enhancer function. The results suggest that the LTR-assembled transcription machinery in synthesizing non-coding, LTR RNAs can reach the downstream -globin promoter to activate transcription of the GFP gene.
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