Interchromosomal translocation in neural progenitor cells exposed to L1 retrotransposition

LINE-1 (L1) elements are a class of transposons, comprising approximately 19% and 21% of the mouse and human genomes, respectively. L1 retrotransposons can reverse transcribe their own RNA sequence into a de novo DNA copy integrated into a new genomic location. This activity, known as retrotransposition, may induce genomic alterations, such as insertions and deletions. Interestingly, L1s can retrotranspose and generate more de novo L1 copies in brains than in other somatic tissues. Here, we describe for the first time interchromosomal translocation triggered by ectopic L1 retrotransposition in neural progenitor cells. Such an observation adds to the studies in neurological and psychiatric diseases that exhibited variation in L1 activity between diseased brains compared with controls, suggesting that L1 activity could be detrimental when de-regulated.

Keywords:
L1 retrotransposition; neural progenitor cells; translocations; brain mosaicism

Authorship

Alysson R. Muotri

designed, performed and interpreted

University of California San Diego, Department of Pediatrics, La Jolla, CA, USA.University of California San DiegoUSALa Jolla, CA, USAUniversity of California San Diego, Department of Pediatrics, La Jolla, CA, USA.

University of California San Diego, Department of Cellular & Molecular Medicine, La Jolla, CA , USA.University of California San DiegoUSALa Jolla, CA, USAUniversity of California San Diego, Department of Cellular & Molecular Medicine, La Jolla, CA , USA.

University of California San Diego, Center for Academic Research and Training in Anthropogeny, Kavli Institute for Brain and Mind, Archealization Center, La Jolla, CA , USA. University of California San DiegoUSALa Jolla, CA, USAUniversity of California San Diego, Center for Academic Research and Training in Anthropogeny, Kavli Institute for Brain and Mind, Archealization Center, La Jolla, CA , USA.

http://orcid.org/0000-0003-0867-2875

Associate Editor: Carlos F. M. Menck

Send correspondence to Alysson R. Muotri. Sanford Consortium for Regenerative Medicine. 2880 Torrey Pines Scenic Drive, La Jolla, CA 92037, USA. E-mail: muotri@ucsd.edu.

Conflict of Interest

ARM is a co-founder and has an equity interest in TISMOO, a company dedicated to genetic analysis and human brain organogenesis, focusing on therapeutic applications customized for autism spectrum disorders and other neurological disorders origin genetics. The terms of this arrangement have been reviewed and approved by the University of California, San Diego, following its conflict of interest policies.

Author Contribution

All the experiments were designed, performed, and interpreted by ARM.

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Figures

Figures (4)

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Figure 1 -
Isolation of clone C6+ by L1-EGFP retrotransposition. a, The retrotransposition-competent human L1 (L1_RP) contains a 5’ untranslated region (UTR) that harbors an internal promoter, two open reading frames (ORF1 and ORF2; not drawn to scale), and a 3’ UTR that ends in a poly (A) tail. ORF2 contains an endonuclease (EN) and reverse transcriptase (RT) domains, as well as a cystiyne-rich 3’ end (C). The EGFP retrotransposition indicator cassette consists of a backward copy of the EGFP gene whose expression is controlled by the human cytomegalovirus major immediate early promoter (pCMV) and the herpes simplex virus thymidine kinase polyadenylation sequence (pA). This arrangement ensures that EGFP expression will only become activated upon L1 retrotransposition. The black arrows indicate PCR primers flanking the intron present in the EGFP gene. b, Bright field and UV images are shown of clone C6+ cells containing an L1 retrotransposition event derived from a single cell. Bar = 20 mm. c, PCR analysis of genomic DNA isolated from the clone C6+. PCR was conducted using the primers shown in panel a. The 1243-bp PCR product corresponds to the original L1 vector harboring the intron-containing EGFP indicator cassette. The 343-bp PCR product, diagnostic for the loss of the intron, is indicative of a retrotransposition event. Sequencing of the 343-bp PCR product confirmed the precise splicing of the intron (data not shown).

Thumbnail

Figure 2 -
Neural differentiation for the clone C6+. After a mixed differentiation protocol using retinoic acid and serum (see ^{Supplementary Material file Methods} Methods. ), cells were stained and quantified for neuronal (Map2ab), astrocytes (GFAP), or oligodendrocytes (RIP) markers. These experiments were performed in triplicates (n=3) and the bars represent standard deviation.

Thumbnail

Figure 3 -
Schematic representation of the Clone 6+ L1 insertion. Inverse PCR indicated that the L1 was inserted in the antisense orientation of the dystrophin major muscle isoform (chromosome Xq).

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Figure 4 -
Inverse PCR putative interchromosomal translocation confirmation in Clone C6+ cells. Isolated cellular chromosomal spread in metaphase hybridized with X paint probe (arrowheads) and a BAC probe control for chromosome 3 (arrows) illustrates the extra fluorescence signal observed in the Clone C6+ but absence in the HCN parental cell. Bottom: spectral analysis of chromosomal spreads in the Clone C6+ revealed a translocation between chromosome X (brown) and chromosome 10 (blue).

Figure 1 - Isolation of clone C6+ by L1-EGFP retrotransposition. a, The retrotransposition-competent human L1 (L1_RP) contains a 5’ untranslated region (UTR) that harbors an internal promoter, two open reading frames (ORF1 and ORF2; not drawn to scale), and a 3’ UTR that ends in a poly (A) tail. ORF2 contains an endonuclease (EN) and reverse transcriptase (RT) domains, as well as a cystiyne-rich 3’ end (C). The EGFP retrotransposition indicator cassette consists of a backward copy of the EGFP gene whose expression is controlled by the human cytomegalovirus major immediate early promoter (pCMV) and the herpes simplex virus thymidine kinase polyadenylation sequence (pA). This arrangement ensures that EGFP expression will only become activated upon L1 retrotransposition. The black arrows indicate PCR primers flanking the intron present in the EGFP gene. b, Bright field and UV images are shown of clone C6+ cells containing an L1 retrotransposition event derived from a single cell. Bar = 20 mm. c, PCR analysis of genomic DNA isolated from the clone C6+. PCR was conducted using the primers shown in panel a. The 1243-bp PCR product corresponds to the original L1 vector harboring the intron-containing EGFP indicator cassette. The 343-bp PCR product, diagnostic for the loss of the intron, is indicative of a retrotransposition event. Sequencing of the 343-bp PCR product confirmed the precise splicing of the intron (data not shown).

Figure 2 - Neural differentiation for the clone C6+. After a mixed differentiation protocol using retinoic acid and serum (see ^{Supplementary Material file Methods} Methods. ), cells were stained and quantified for neuronal (Map2ab), astrocytes (GFAP), or oligodendrocytes (RIP) markers. These experiments were performed in triplicates (n=3) and the bars represent standard deviation.

Figure 3 - Schematic representation of the Clone 6+ L1 insertion. Inverse PCR indicated that the L1 was inserted in the antisense orientation of the dystrophin major muscle isoform (chromosome Xq).

Figure 4 - Inverse PCR putative interchromosomal translocation confirmation in Clone C6+ cells. Isolated cellular chromosomal spread in metaphase hybridized with X paint probe (arrowheads) and a BAC probe control for chromosome 3 (arrows) illustrates the extra fluorescence signal observed in the Clone C6+ but absence in the HCN parental cell. Bottom: spectral analysis of chromosomal spreads in the Clone C6+ revealed a translocation between chromosome X (brown) and chromosome 10 (blue).

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[1] Associate Editor: Carlos F. M. Menck