title: Establishment of a genome-wide and quantitative protocol for assessment of transcriptional activity at human retrotransposon L1 antisense promoters.
abstract: Long interspersed element 1 (L1) retrotransposon sequences are widespread in the human genome, occupying ~500,000 locations. The majority of L1s have lost their retrotransposition capability, although a significant population of human L1s maintains bidirectional transcriptional activity from the internal promoter. While the sense promoter drives transcription of the entire L1 mRNA and leads to L1 retrotransposition, the antisense promoter (ASP) transcribes L1-gene chimeric RNAs that include neighboring exon sequences. Activation mechanisms and functional impacts of L1ASP transcription are thought to vary at every L1ASP location. To explore the locus-specific regulation and function of L1ASP transcription, quantitative methodology is necessary for identifying the genomic positions of highly active L1ASPs on a genome-wide scale. Here, we employed deep-sequencing techniques and built a 3' RACE-based experimental and bioinformatics protocol, named the L1 antisense transcriptome protocol (LATRAP). In LATRAP, the PCR primer and the read mapping scheme were designed to reduce false positives and negatives, which may have been included as hits in previous cloning studies. LATRAP was here applied to the A549 human lung cancer cell line, and 313 L1ASP loci were detected to have transcriptional activity but differed in the number of mapped reads by four orders of magnitude. This indicates that transcriptional activities of the individual L1ASPs can vary greatly and that only a small population of L1ASP loci is active within individual nuclei. LATRAP is the first experimental method for ranking L1ASPs according to their transcriptional activity and will thus open a new avenue to unveiling the locus-specific biology of L1ASPs.
authors.affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology.
authors.forename: Koichi
authors.initials: K
authors.lastname: Ishiguro
authors.affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology.
authors.forename: Saneyuki
authors.initials: S
authors.lastname: Higashino
authors.affiliation: Kazusa DNA Research Institute.
authors.forename: Hideki
authors.initials: H
authors.lastname: Hirakawa
authors.affiliation: Kazusa DNA Research Institute.
authors.forename: Shusei
authors.initials: S
authors.lastname: Sato
authors.affiliation: Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology.|Center for Biological Resources and Informatics, Tokyo Institute of Technology.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Aizawa
mesh_terms: D000072283:A549 Cells; D016373:DNA, Antisense; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011401:Promoter Regions, Genetic; D018626:Retroelements; D014158:Transcription, Genetic
chemical_list: D016373:DNA, Antisense; D018626:Retroelements
keywords: antisense promoter; cancer; deep sequencing; human L1; retrotransposon