Long-range function of an intergenic retrotransposon

Abstract

Retrotransposons including endogenous retroviruses and their solitary long terminal repeats (LTRs) compose >40% of the human genome. Many of them are located in intergenic regions far from genes. Whether these intergenic retrotransposons serve beneficial host functions is not known. Here we show that an LTR retrotransposon of ERV-9 human endogenous retrovirus located 40-70 kb upstream of the human fetal γ-and adult β-globin genes serves a long-range, host function. The ERV-9 LTR contains multiple CCAAT and GATA motifs and competitively recruits a high concentration of NF-Y and GATA-2 present in low abundance in adult erythroid cells to assemble an LTR/RNA polymerase II complex. The LTR complex transcribes intergenic RNAs unidirectionally through the intervening DNA to loop with and modulate transcription factor occupancies at the far downstream globin promoters, thereby modulating globin gene switching by a competitive mechanism.

Enhancer turnover and conserved regulatory function in vertebrate evolution

“…Although there are well-characterized examples of cis-acting regulatory regions restricted to particular mammalian lineages (e.g. [28,29]), several diseaseassociated mutations mapping to non-coding regions in humans have turned out to reside within phylogenetically conserved enhancers [6,30 -36].…”

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.…”

“…In CGI-1 probe, the CCAAT motif did not bind NF-Y, potentially because the 3 0 flanking bases of the CCAAT core were not conducive to NF-Y binding 37,61 ; for yet unknown reasons, the E box motif (CACTTG) did not bind any of the E box binding TFs (USF1, 2, c-Myc, and c-Myb) (Fig. 5d, lanes 2-4, 7-8).…”

“…Thus, hypermethylated ERV-9 LTR possessed long-range enhancer function in activating transcription of the downstream globin genes, in agreement with our earlier findings that deletion of ERV-9 LTR affects occupancies of key TFs in the b-globin locus, impairs interaction/looping of the LCR with the globin genes, and dysregulates transcription of the globin genes. 37 It has been shown that mCpGs-with methylcytosines (5mCs)-bind to methyl DNA binding proteins MeCP2 and MBD1, 2, and 4 to assemble repressor complexes that suppress transcription of the resident genes. 47 In contrast, hmCpGswith hydroxymethyl cytosines (5hmCs)-bind MeCP2 and the MBDs at orders of magnitude lower affinities and do not assemble the repressor complexes to suppress transcription of linked genes.…”

“…2a). 37 After 2 rounds of transfections with small guide RNAs (sgRNAs) and donor DNA, 16 clonal lines expanded from single cells were analyzed by PCR with primers P1-3 to screen for clonal lines with homozygous LTR deletion (Fig. 2b, left panel).…”