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

Abstract

A solitary long terminal repeat (LTR) of ERV-9 human endogenous retrovirus is located upstream of the HS5 site in the human beta-globin locus control region and possesses unique enhancer activity in erythroid K562 cells. In cells transfected with plasmid LTR-HS5-epsilonp-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 epsilon-globin promoter and the GFP gene. Synthesis of the sense, long LTR RNAs is correlated with high level synthesis of GFP mRNA from the epsilon-globin promoter. Mutations of the LTR promoter and/or the epsilon-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-epsilonp-GFP, thus reversing the direction of synthesis of LTR RNAs in the antisense direction away from the epsilon-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 epsilon-globin promoter to activate transcription of the GFP gene.

Hypermethylated LTR retrotransposon exhibits enhancer activity

“…23 The ERV-9 LTR enhancer complex is active in stem/progenitor cells including erythroid progenitor cells; 34,35 it initiates synthesis of ERV-9 lncRNAs from the 5 0 end of the R region through the U5 region into the downstream genomic DNA to activate transcription of the globin genes by a long-range tracking and transcription mechanism. 36,37 In the current study, we found that the transcriptionally active ERV-9 LTR was >90% hypermethylated in erythroleukemia K562 cell line and primary human erythroblasts cultured from peripheral blood CD34C cells. Deletion of the ERV-9 LTR in K562 cells by CRISPR-Cas9 demonstrated that the hypermethylated ERV-9 LTR possessed in vivo function and activated transcription of the downstream globin genes.…”

“…Although ERV-9 LTR enhancer function has been extensively characterized, 23,[35][36][37] the function of the neighboring CGI at the 5 0 border of the LTR remained unknown. It has been shown that the DNase hypersensitive HS4 site located at the 5 0 border of the chicken b-globin gene locus binds USF1 and CTCF and possesses strong insulator and barrier functions to protect the globin gene locus from epigenetic and transcriptional interference of flanking gene domains.…”

“…For each transfection, 2 £ 10 4 cells were analyzed for the percentage of fluorescent cells and the mean fluorescence intensities of the gated cells, calculated statistically by the analysis software from hundreds to thousands of gated cells. 34,36 The enhancer activity of the transfected plasmids was calculated as the product of the percentage of gated green fluorescent cells times the mean fluorescence intensities of the gated cells as described. 34,36 For BS-Sseq and oxBS-seq, the transfected K562 cells were sorted with FACSAria II SORP cell sorter (BD Bioscience); the sorted green fluorescent cells were used for DNA isolation and processed as described above.…”

“…ERV-9 lncRNA and angled arrow: Long, noncoding RNA initiated by the LTR enhancer and promoter from the 5 0 end of the R region into the downstream globin gene locus. 34,36 …”

The Long Terminal Repeat (LTR) of ERV-9 Human Endogenous Retrovirus Binds to NF-Y in the Assembly of an Active LTR Enhancer Complex NF-Y/MZF1/GATA-2

“…Subsequent studies showed that this 5Ј HS5 ERV-9 LTR possesses prominent enhancer activity in embryonic and erythroid cell lines (16) and in oocytes and progenitor cells, including the erythroid progenitor cells, but not in differentiated somatic tissues in transgenic Zebrafish and humans (34). Unlike the HS2 and HS3 enhancers of the ␤-LCR, whose enhancer activities are blocked by the HS5 site with reported insulator properties when HS5 is interposed between the enhancer and the cis-linked promoter (35,36), the ERV-9 LTR enhancer is not blocked by the interposed HS5 site (37).…”