GREM, a technique for genome-wide isolation and quantitative analysis of promoter active repeats

Abstract

We developed a technique called GREM (Genomic Repeat Expression Monitor) that can be applied to genome-wide isolation and quantitative analysis of any kind of transcriptionally active repetitive elements. Briefly, the technique includes three major stages: (i) generation of a transcriptome wide library of cDNA 5' terminal fragments, (ii) selective amplification of repeat-flanking genomic loci and (iii) hybridization of the cDNA library (i) to the amplicon (ii) with subsequent selective amplification and cloning of the cDNA-genome hybrids. The sequences obtained serve as 'tags' for promoter active repetitive elements. The advantage of GREM is an unambiguous mapping of individual promoter active repeats at a genome-wide level. We applied GREM for genome-wide experimental identification of human-specific endogenous retroviruses and their solitary long terminal repeats (LTRs) acting in vivo as promoters. Importantly, GREM tag frequencies linearly correlated with the corresponding LTR-driven transcript levels found using RT-PCR. The GREM technique enabled us to identify 54 new functional human promoters created by retroviral LTRs.

Figure 1

Schematic representation of solitary (left) and proviral (right) LTRs expression. The transcription driven from 5′ proviral LTRs results in mRNAs of viral genes, whereas the expression of either solitary or 3′ proviral LTRs results in the transcription of host genomic sequences, flanking the 3′ ends of the retroelements.

Figure 2

Schematic representation of the GREM technique (for details, see text). The procedure includes three major stages: (Stage 1) genome-wide amplification of the genomic DNA flanking the 3′ ends of target repetitive elements (here, HS LTRs). Treatment of the resulting amplicon with ExoIII generates 5′ protruding ends to be used at the third stage. (Stage 2) A double-stranded oligo d(T)-primed cDNA library is synthesized for tissues where expression of repetitive elements is to be studied. At this stage cDNAs are tagged by a linker oligonucleotide (CS) at the RNA transcription start sites using the ‘cap-switch’ effect. cDNAs are then digested with AluI restriction endonuclease that has no recognition sites within HS LTRs. This step precludes amplification of LTR sequences read-through in the sense orientation. (Stage 3) Finally, the genomic DNA amplicon (Stage 1) is hybridized to the 5′ tagged cDNAs (Stage 2). The protruding DNA ends are filled in with DNA polymerase, and the hybrids obtained (ELTs) are nested PCR amplified with primers specific to the flanking genomic DNA adapter and cDNA 5′ terminal tag sequence, respectively.