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. 2014 Apr;42(7):4546-62.
doi: 10.1093/nar/gku091. Epub 2014 Feb 3.

An antisense promoter in mouse L1 retrotransposon open reading frame-1 initiates expression of diverse fusion transcripts and limits retrotransposition

Jingfeng Li  1 , Manoj Kannan, Anna L Trivett, Hongling Liao, Xiaolin Wu, Keiko Akagi, David E Symer
Affiliations

An antisense promoter in mouse L1 retrotransposon open reading frame-1 initiates expression of diverse fusion transcripts and limits retrotransposition

Jingfeng Li et al. Nucleic Acids Res. 2014 Apr.

Abstract

Between 6 and 30% of human and mouse transcripts are initiated from transposable elements. However, the promoters driving such transcriptional activity are mostly unknown. We experimentally characterized an antisense (AS) promoter in mouse L1 retrotransposons for the first time, oriented antiparallel to the coding strand of L1 open reading frame-1. We found that AS transcription is mediated by RNA polymerase II. Rapid amplification of cDNA ends cloning mapped transcription start sites adjacent to the AS promoter. We identified >100 novel fusion transcripts, of which many were conserved across divergent mouse lineages, suggesting conservation of potential functions. To evaluate whether AS L1 transcription could regulate L1 retrotransposition, we replaced portions of native open reading frame-1 in donor elements by synonymously recoded sequences. The resulting L1 elements lacked AS promoter activity and retrotransposed more frequently than endogenous L1s. Overexpression of AS L1 transcripts also reduced L1 retrotransposition. This suppression of retrotransposition was largely independent of Dicer. Our experiments shed new light on how AS fusion transcripts are initiated from endogenous L1 elements across the mouse genome. Such AS transcription can contribute substantially both to natural transcriptional variation and to endogenous regulation of L1 retrotransposition.

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Figures

Figure 1.
Figure 1.
Mapping an active AS promoter within L1 ORF1. (A) Schematic representation of an L1 TF subfamily retrotransposon, L1spa, with coordinates indicated as used throughout this article. L1spa was identified in GenBank accession no. AF016099. Below: Probes for phage cDNA library hybridization against ORF2 (2858–3269 nt) and ORF1 (1814–2101 nt). (B) Various DNA fragments were directionally engineered upstream of a promoterless reporter gene, i.e. β-lactamase TEM1. (C) Linearized DNAs containing various candidate promoter-reporter cassettes were transfected into HeLa cells. Functional beta-lactamase protein expression was measured by staining cells with CCF2-AM, whose fluorescence emission shifts from green to blue on increased enzymatic cleavage (48). Cells expressing (left) or not expressing (right) β-lactamase were evaluated both by flow cytometry (top), which measured quantitative blue/green emission ratios (49), and by fluorescence microscopy (bottom). (D) Fragments derived from various L1 positions and subclasses were numbered and directionally oriented as indicated (Supplementary Figure S1 and Supplementary Table S1). Their promoter strengths were assayed as described in part B. Key: colors and thicknesses indicate promoter activity scores for each fragment assayed. The highest scores (>50, red, thick line) indicate strongest promoter activities. (E) TEM1 transcript levels were measured using qRT-PCR (arrows: primer binding sites) to assess the candidate fragments’ promoter activities. (F) The ratio of TEM1 to beta-actin transcript concentrations was calculated (y-axis) after correction for amplification of contaminating plasmid or genomic DNA. As a positive control, SV40 early promoter was engineered upstream of the TEM1 reporter, and as negative controls, no promoter was included or no plasmid was transfected. The AS L1 promoter activity (fragment 6) is half that of the sense-stranded mouse L1 5′ UTR promoter (fragment 1). Fragments are numbered as in (D).
Figure 2.
Figure 2.
RNA polymerase II transcribes AS L1 fusion transcripts (A) Chromatin immunoprecipitation (ChIP) with anti-RNA polymerase II (left) and anti-RNA polymerase III (right) antibodies, followed by PCR amplification of target L1 or SINE B2 genomic sequences as indicated (right), showed specific enrichment (pulldown) of pol II at the AS L1 promoter in mouse testis (asterisks, L1 ORF1 sequences). Coordinates from L1spa reference are shown (right, cf. Figure 1A). RNA pol II also immunoprecipitated proximal L1 sequences, i.e. templates for transcribed AS fusion transcripts. As a control, both pol II and pol III pulled down SINE B2 elements genome-wide (bottom) as expected (54). (B) Mouse spermatocytes were treated with α-amanitin (RNA polII inhibitor) as indicated (top). Total RNAs were isolated, and reverse transcriptase was added as indicated (+ or −; top) before PCR amplification of various cDNAs as indicated (right). As a negative control, U6 transcripts (RNA pol III, not inhibited by α-amanitin) were amplified (bottom).
Figure 3.
Figure 3.
Contribution of an AS L1 RIFT to overall Arhgap15 gene expression in various mouse strains. (A) Schematic representation of Arhgap15 exons, including a polymorphic AS L1 integrant in the B6 reference genome but not in other strains. (B) AS L1 RIFT expression at Arhgap15 was detected in B6 mice, using the novel RIFT assay where we performed RT-PCR using AS L1 and oligo-d(T) primers, followed by hybridization of resulting cDNA products to an Affymetrix mouse exon microarray. We required five consecutive exon probes to be strongly positive to call RIFTs. Shown are genomic positions of probes within exons (x-axis) and hybridization signal intensities on a log scale (y-axis). Legend, inset: five mouse strains, different symbol colors and shapes. (C) Conventional assay for Arhgap15 expression in total RNAs (see legend, B). The AS L1 RIFT in B6 mice affects total RNA expression levels at the 3′ exons downstream of the polymorphic, initiating L1 integrant (see corresponding positions, A).
Figure 4.
Figure 4.
Comparison of AS L1 RIFTs expressed in various mouse tissues and strains. Distinct AS L1 RIFTs were counted in Venn diagrams depicting shared (overlapping) and unique (distinct) RIFTs expressed in different mouse strains and tissues. Numbers indicate unique RIFTs in each group. (A) AS L1 RIFTs expressed in B6 testis (n = 71, blue), brain (n = 9, red) and kidney (n = 8, green); (B) AS L1 RIFTs expressed in testis of five mouse strains: 129S1 (n = 70, blue), 129X1 (n = 66, red), A/J (n = 63, green), B6 (n = 71, purple) and DBA/2 J (n = 62, orange).
Figure 5.
Figure 5.
AS transcription start sites found by 5′ RACE in multiple tissues. (A) A 5′ RACE was performed by PCR for 5′ ends of AS L1 RIFTs, using total RNAs from testis, kidney and brain. Products were separated by agarose gel electrophoresis. Individual cloned 5′ ends were sequenced from these pools. (B) The cumulative positions of TSS for AS L1-gene RIFTs are plotted by summing individual transcripts’ 5′ ends, mapped against coordinates from L1spa. We analyzed 19 5′ RACE clones from testis (red), 35 from kidney (blue) and 54 from brain (green). Also superimposed here are the cumulative positions of 5′ ends from 24 RIKEN clones that align well with L1spa, although these formally are not ends determined by 5′ RACE cloning (Supplementary Table S2) (45).
Figure 6.
Figure 6.
AS L1 transcription helps to limit retrotransposition. (A) Cis effects. Native L1 ORF1 sequences in L1spa (black) were replaced either with a synonymously recoded fragment from smL1 with its markedly reduced A/T content (47) (pink), or a new recoded fragment that preserves A/T content more similar to that found in endogenous L1s (blue). Resulting marked L1 donors, i.e. pJL3 and pMK28, were assayed for retrotransposition by transfecting human HeLa cells. As controls, native L1spa (in pTN201) (50), smL1 (in the same pCEP4 donor plasmid backbone and marked with NeoR/AI) and an empty donor plasmid (pCEP4) were transfected in parallel. Following selection on hygromycin for 47 d, 1 million HygroR cells were plated per flask, and new L1 integrants were selected for NeoR, followed by staining of colonies. Retrotransposition frequencies are indicated relative to L1spa in pTN201 (right). (B) Trans effects. To measure overexpressed AS smL1 RIFTs' suppressive effects on retrotransposition by smL1, first we directionally cloned four AS fragments from smL1, i.e. coordinates 2119-1120 (PCR amplicons DES3820 × DES3818, Supplementary Table S1); 2800–1120 (DES3821 × DES3818); 2119–1812 (DES3820 × DES3819); and 2800–1812 (DES3821 × DES3819) into pCEP4 downstream of its strong CMV promoter. Each cloned construct was co-transfected into HeLa cells with the smL1 retrotransposition donor plasmid, pCEP4/smL1/Neo. As positive and negative controls, smL1 donor alone and pCEP4 alone were transfected into HeLa cells, respectively. After transfection, cells were plated at various dilutions, selected on G418 for 2 weeks and NeoR colonies were stained and counted (see Supplementary Figure S7). The mean and range of duplicate counts were determined, and retrotransposition frequencies were normalized relative to that of the smL1 positive control (defined as 100%). Asterisks: significantly different from control retrotransposition frequency (two-tailed t-test, P < 0.05 in all pairwise comparisons).
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