Alu expression in human cell lines and their retrotranspositional potential
- Andrew J Oler†2, 4,
- Stephen Traina-Dorge1,
- Rebecca S Derbes1,
- Donatella Canella3,
- Brad R Cairns2 and
- Astrid M Roy-Engel1Email author
© Oler et al.; licensee BioMed Central Ltd. 2012
Received: 8 February 2012
Accepted: 20 June 2012
Published: 20 June 2012
The vast majority of the 1.1 million Alu elements are retrotranspositionally inactive, where only a few loci referred to as ‘source elements’ can generate new Alu insertions. The first step in identifying the active Alu sources is to determine the loci transcribed by RNA polymerase III (pol III). Previous genome-wide analyses from normal and transformed cell lines identified multiple Alu loci occupied by pol III factors, making them candidate source elements.
Analysis of the data from these genome-wide studies determined that the majority of pol III-bound Alus belonged to the older subfamilies Alu S and Alu J, which varied between cell lines from 62.5% to 98.7% of the identified loci. The pol III-bound Alus were further scored for estimated retrotransposition potential (ERP) based on the absence or presence of selected sequence features associated with Alu retrotransposition capability. Our analyses indicate that most of the pol III-bound Alu loci candidates identified lack the sequence characteristics important for retrotransposition.
These data suggest that Alu expression likely varies by cell type, growth conditions and transformation state. This variation could extend to where the same cell lines in different laboratories present different Alu expression patterns. The vast majority of Alu loci potentially transcribed by RNA pol III lack important sequence features for retrotransposition and the majority of potentially active Alu loci in the genome (scored high ERP) belong to young Alu subfamilies. Our observations suggest that in an in vivo scenario, the contribution of Alu activity on somatic genetic damage may significantly vary between individuals and tissues.
KeywordsAlu source elements Alu expression RT-PCR ChIP-seq Retrotransposition SINE
Alu elements are major contributors to genomic instability  and genetic disease  due to their ability to generate new copies that randomly insert throughout the genome and to induce non-homologous recombination between different copies. When comparing copy numbers, Alu has been vastly more successful than other non-autonomous elements, such as the retropseudogenes and even the autonomous L1 element . Alu-induced mutagenesis is responsible for the majority of the documented instances of human retroelement insertion-induced disease  and presents a retrotransposition rate estimated up to ten-fold higher than L1 [4, 5]. The human genome contains over one million Alu inserts , which can be divided into subfamilies based on specific diagnostic nucleotides and their evolutionary period of activity [6, 7]. About 80% of Alu elements belong to the older previously active Alu J and Alu S subfamilies . Germline derived evidence supports the current activity of only the subsets of the younger Alu Y subfamilies (such asY, Ya, and Yb), although recent data appear to indicate that Alu retrotransposition in germline and somatic tissues may show different distributions .
Data Source of Alu loci
Canella et al. 
ChIP-seqa for detection of sites bound by POLR3D
(RPC4), TFIIIB subunits BDP1 and BRF1
Oler et al. 
ChIP-seqa and ChIP-arrayb for detection of sites bound by Pol III (RPC32 subunit), TFIIIC63 subunit, BRF1, BRF2
HeLa, Jurkat, HEK, 293 T
Moqtaderi et al. 
ChIP-seqa for detection of sites bound by TFIIIC-110
subunit, TFIIIB subunits BDP1 and BRF1, Pol III (RPC155 subunit) and BRF2
Alu subfamily distribution of Alu elements bound by RNA polymerase III factors
# of disease
% Alu loci
# Pol III
full Alus (530,850)
cases due to ade novo insertion
bound by pol III
bound Alu enrichment
bound Alu with ERP
S + J
In addition to the ability to be transcribed, specific sequence features of Alu elements can influence retrotransposition efficiency . Therefore, we proceeded to evaluate the individual pol III-bound Alu loci using our own designed dichotomous key based on the previously identified criteria known to affect retrotransposition rates: 1) sequence divergence from the consensus (loss of retrotransposition efficiency with higher divergence [22, 16]); 2) A-tail length (a minimum length is required ); 3) length of the unique sequence (loss of efficiency with longer sequences ); and 4) A-tail homogeneity (loss of efficiency with higher % disruptions ). Our results are schematically represented in Figure 2A (details in Additional file 1: Table S1). We selected limits for our criteria parameters that have been shown to significantly reduce retrotransposition levels. We also separately assigned a numerical value of the impact on retrotransposition (‘R’) for each Alu feature variant relative to an Alu reference (Additional file 1: Tables S12-15) to roughly calculate the ERP of each individual Alu (Additional file 1: Table S1, column T). However, the ERP should not be taken as the sole defining criteria for in vivo predictions, as it is based on a limited amount of data generated from engineered Alus in a tissue culture system and does not include transcription status. This scoring system was applied to the pol III-bound subset as well as all Alus genome-wide using an algorithm that incorporates each of the scoring criteria (implemented in Perl, score_alus.pl; available upon request). As expected, young Alu elements had a higher score genome-wide than Alu J + S elements (median values of 0.0042 and 0.000001 for Y and J + S, respectively; P = 2.2e-16 in Wilcoxon test). While pol III-bound Alus had a higher ERP score in general than Alus not bound by pol III (median 0.000229 and 0.000001, respectively; P = 0.013 in Wilcoxon test), the ERP score for the vast majority of the pol III-bound Alus was considerably lower than the arbitrarily selected minimal threshold for retrotransposition competency of 0.20. Of the 162 pol III-bound Alu sequences only one AluYb8 (Canella 37 from IMR90 cells) was highly conserved relative to the consensus sequence, met the rest of the criteria and scored 0.20 ERP (an ‘ideal’ Alu will have a score of 1.00). In addition, it scored low in the pol III ChIP assay  and Canella 37 AluYb8 transcripts were undetectable in HeLa and IMR90 cells by northern blot probing with end-labeled oligonucleotides complementary to the unique sequence (Figure 2B). We opted not to use an RT-PCR approach, as it is unable to differentiate between RNA pol II and pol III transcripts (Figure 1). In contrast, when using a low ERP threshold to evaluate the reference genome, several thousands of Alus genome-wide were identified (6,103 and 1,818 Alus at ERP threshold of 0.10 or 0.20, respectively; Additional file 1: Table S16). Furthermore, a more conservative threshold (ERP scores of ≥0.50) yields only 163 of genome-wide Alus (all young elements), corroborating the previously proposed Alu source model that only a small portion of Alus in the genome are likely active .
The next ‘best’ candidates identified only partially met the criteria, corresponding to three Alu loci belonging to older S subfamilies: Moq 13 h (HeLa), Moq 11 h (HeLa) and Moq 28 k (K562) with 5.3%, 7.8% and 9.3% sequence divergence from consensus, respectively. Some of the sequence changes were within the RNA pol III A box and in the sequences predicted to bind the SRP9 and SRP14 proteins. Lower binding of SRP9/14 would likely reduce the retrotransposition capability of these elements, but further testing is required. Moq 28 k shows a very low ERP of 0.04. Interestingly, Moq 11 h and Moq 13 h present acceptable A-tail length with marginal ERP values of 0.10 and 0.14, respectively. Moq 13 h showed an A-tail with high % A-tail disruption (24.3%), which is not observed in de novo inserts . The published work on Moq 11 h showed significant pol III binding by ChIP-seq . If expressed, Moq 11 h could prove retrotranspositionally competent. However, the RNA-seq data showed only three sequence reads in HeLa and none in the K562 and a non-detectable transcript by northern blot analysis (Figure 2B). Evaluation of expression from five other randomly selected Alu loci, Moq 19 k (Figure 2B) and Canella 2 and 28, Oler 38 h, and 3c, (data not shown) by northern blot analysis also proved unsuccessful in the detection of pol III Alu transcripts. Due to the sensitivity limitation of our assay, we are unable to unambiguously confirm that these identified Alu candidate loci with the best retrotransposition potential (Canella 37 and Moq 11 h) are transcriptionally silent. Thus, we cannot eliminate the possibility that very low amounts of expression may occur, resulting in retrotransposition. Alternatively, these or other identified Alu loci may be more efficiently expressed in other cell types, tissues or under other conditions such as heat shock known to increase Alu expression .
Presently, we are unable to rule out that any of the other identified pol III-bound Alu candidates that partially fulfill our criteria or contain borderline attributes may undergo retrotransposition at very low rates. However, the limits of the criteria are based on the results using a tissue culture system  that significantly favors Alu activity through the overexpression of both a tagged Alu transcript and the enzymatic machinery required for retrotransposition(L1 ORF2 protein). This opens the possibility that an Alu locus identified as potentially active by the selected parameters may not be able to retrotranspose under natural cellular conditions. Thus, it is unlikely that the ‘less perfect’ Alu candidate elements (those with low ERP scores) contribute to retrotransposition in any significant manner.
Our findings indicate that up to now, most cells analyzed may support RNA pol III expression of a collection of Alu elements, although the vast majority lack sequence features associated with retrotransposition competence (Table 2). A striking observation is the overall low number of detected Alu loci (162), and even lower when considering retrotransposition potential (only three loci from all cell lines combined had ERPs above 0.10). So why is there little to no evidence of expression by pol III of the active younger Alu elements? Although speculative, these data could be indicative of a general mechanism, such as DNA methylation, that selectively limits Alu transcription of the retrotranspositionally competent elements. Also, it could be a reflection that younger, less mutated retroelements still maintain most of their CpGs making them good substrates for regulation by methylation . In addition, the inability to detect transcripts from the candidates identified may reflect variability in Alu expression, where the same cell lines in different laboratories have different expression patterns. It is possible that Alu expression varies by cell type, growth conditions, epigenetic signals and transformation state. Our observations support the hypothesis that in an in vivo scenario, the contribution of Alu activity on somatic genetic damage may significantly vary between individuals and tissues.
This publication was made possible by Grants Number GM45668 (PD) and P20GM103518/P20RR020152, plus R01GM079709A (to AMR-E) from the National Institutes of Health (NIH) and the Howard Hughes Medical Institute (AJO). An allocation of resources from the Center for High Performance Computing at the University of Utah is gratefully acknowledged. Huntsman Cancer Institute Biostatistics Core Facility supported by grant P30 CA042014 also participated in this work. The contents are solely the responsibility of the authors and do not necessarily represent the official views of NCRR or NIH. We would like to thank Prescott Deininger for his helpful comments on the manuscript.
- Xing J, Witherspoon DJ, Ray DA, Batzer MA, Jorde LB: Mobile DNA elements in primate and human evolution. Am J Phys Anthropol. 2007, 45 (Suppl): 2-19.View ArticlePubMedGoogle Scholar
- Belancio VP, Hedges DJ, Deininger P: Mammalian non-LTR retrotransposons: for better or worse, in sickness and in health. Genome Res. 2008, 18: 343-358. 10.1101/gr.5558208.View ArticlePubMedGoogle Scholar
- Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McKernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Raymond C, Rosetti M, Santos R, Sheridan A, Sougnez C: Initial sequencing and analysis of the human genome. Nature. 2001, 409: 860-921. 10.1038/35057062.View ArticlePubMedGoogle Scholar
- Xing J, Zhang Y, Han K, Salem AH, Sen SK, Huff CD, Zhou Q, Kirkness EF, Levy S, Batzer MA, Jorde LB: Mobile elements create structural variation: analysis of a complete human genome. Genome Res. 2009, 19: 1516-1526. 10.1101/gr.091827.109.PubMed CentralView ArticlePubMedGoogle Scholar
- Iskow RC, McCabe MT, Mills RE, Torene S, Pittard WS, Neuwald AF, Van Meir EG, Vertino PM, Devine SE: Natural mutagenesis of human genomes by endogenous retrotransposons. Cell. 2010, 141: 1253-1261. 10.1016/j.cell.2010.05.020.PubMed CentralView ArticlePubMedGoogle Scholar
- Shen MR, Batzer MA, Deininger PL: Evolution of the master Alu gene(s). J Mol Evol. 1991, 33: 311-320. 10.1007/BF02102862.View ArticlePubMedGoogle Scholar
- Batzer MA, Schmid CW, Deininger PL: Evolutionary analyses of repetitive DNA sequences. Methods Enzymol. 1993, 224: 213-232.View ArticlePubMedGoogle Scholar
- Stewart C, Kural D, Stromberg MP, Walker JA, Konkel MK, Stutz AM, Urban AE, Grubert F, Lam HY, Lee WP, Busby M, Indap AR, Garrison E, Huff C, Xing J, Snyder MP, Jorde LB, Batzer MA, Korbel JO, Marth GT: 1000 Genomes Project: A comprehensive map of mobile element insertion polymorphisms in humans. PLoS Genet. 2011, 7: e1002236-10.1371/journal.pgen.1002236.PubMed CentralView ArticlePubMedGoogle Scholar
- Baillie JK, Barnett MW, Upton KR, Gerhardt DJ, Richmond TA, De Sapio F, Brennan PM, Rizzu P, Smith S, Fell M, Talbot RT, Gustincich S, Freeman TC, Mattick JS, Hume DA, Heutink P, Carninci P, Jeddeloh JA, Faulkner GJ: Somatic retrotransposition alters the genetic landscape of the human brain. Nature. 2011, 479: 534-537. 10.1038/nature10531.PubMed CentralView ArticlePubMedGoogle Scholar
- Paulson KE, Schmid CW: Transcriptional inactivity of Alu repeats in HeLa cells. Nucleic Acids Res. 1986, 14: 6145-6158. 10.1093/nar/14.15.6145.PubMed CentralView ArticlePubMedGoogle Scholar
- Shaikh TH, Roy AM, Kim J, Batzer MA, Deininger PL: cDNAs derived from primary and small cytoplasmic Alu (scAlu) transcripts. J Mol Biol. 1997, 271: 222-234. 10.1006/jmbi.1997.1161.View ArticlePubMedGoogle Scholar
- Oler AJ, Alla RK, Roberts DN, Wong A, Hollenhorst PC, Chandler KJ, Cassiday PA, Nelson CA, Hagedorn CH, Graves BJ, Cairns BR: Human RNA polymerase III transcriptomes and relationships to Pol II promoter chromatin and enhancer-binding factors. Nat Struct Mol Biol. 2010, 17: 620-628. 10.1038/nsmb.1801.PubMed CentralView ArticlePubMedGoogle Scholar
- Canella D, Praz V, Reina JH, Cousin P, Hernandez N: Defining the RNA polymerase III transcriptome: genome-wide localization of the RNA polymerase III transcription machinery in human cells. Genome Res. 2010, 20: 710-721. 10.1101/gr.101337.109.PubMed CentralView ArticlePubMedGoogle Scholar
- Moqtaderi Z, Wang J, Raha D, White RJ, Snyder M, Weng Z, Struhl K: Genomic binding profiles of functionally distinct RNA polymerase III transcription complexes in human cells. Nat Struct Mol Biol. 2010, 17: 635-640. 10.1038/nsmb.1794.PubMed CentralView ArticlePubMedGoogle Scholar
- Comeaux MS, Roy-Engel AM, Hedges DJ, Deininger PL: Diverse cis factors controlling Alu retrotransposition: what causes Alu elements to die?. Genome Res. 2009, 19: 545-555. 10.1101/gr.089789.108.PubMed CentralView ArticlePubMedGoogle Scholar
- Bennett EA, Keller H, Mills RE, Schmidt S, Moran JV, Weichenrieder O, Devine SE: Active Alu retrotransposons in the human genome. Genome Res. 2008, 18: 1875-1883. 10.1101/gr.081737.108.PubMed CentralView ArticlePubMedGoogle Scholar
- Dewannieux M, Heidmann T: Role of poly(A) tail length in Alu retrotransposition. Genomics. 2005, 86: 378-381. 10.1016/j.ygeno.2005.05.009.View ArticlePubMedGoogle Scholar
- Deininger PL, Batzer MA, Hutchison CA, Edgell MH: Master genes in mammalian repetitive DNA amplification. Trends Genet. 1992, 8: 307-311.View ArticlePubMedGoogle Scholar
- Roy-Engel AM, Salem AH, Oyeniran OO, Deininger L, Hedges DJ, Kilroy GE: Active alu element "A-Tails": size does matter. Genome Res. 2002, 12: 1333-1344. 10.1101/gr.384802.PubMed CentralView ArticlePubMedGoogle Scholar
- Kim C, Rubin CM, Schmid CW: Genome-wide chromatin remodeling modulates the Alu heat shock response. Gene. 2001, 276: 127-133. 10.1016/S0378-1119(01)00639-4.View ArticlePubMedGoogle Scholar
- Dewannieux M, Esnault C, Heidmann T: LINE-mediated retrotransposition of marked Alu sequences. Nat Genet. 2003, 35: 41-48. 10.1038/ng1223.View ArticlePubMedGoogle Scholar
- Szpakowski S, Sun X, Lage JM, Dyer A, Rubinstein J, Kowalski D, Sasaki C, Costa J, Lizardi PM: Loss of epigenetic silencing in tumors preferentially affects primate-specific retroelements. Gene. 2009, 448: 151-167. 10.1016/j.gene.2009.08.006.PubMed CentralView ArticlePubMedGoogle Scholar
- Wimmer K, Callens T, Wernstedt A, Messiaen L: The NF1 gene contains hotspots for L1 endonuclease-dependent de novo insertion. PLoS Genet. 2011, 7: e1002371-10.1371/journal.pgen.1002371.PubMed CentralView ArticlePubMedGoogle Scholar
- Roy AM, West NC, Rao A, Adhikari P, Alemán C, Barnes AP, Deininger PL: Upstream flanking sequences and transcription of SINEs. J Mol Biol. 2000, 302: 17-25. 10.1006/jmbi.2000.4027.View ArticlePubMedGoogle Scholar
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