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Differential L1 regulation in pluripotent stem cells of humans and apes

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Abstract

Identifying cellular and molecular differences between human and non-human primates (NHPs) is essential to the basic understanding of the evolution and diversity of our own species. Until now, preserved tissues have been the main source for most comparative studies between humans, chimpanzees (Pan troglodytes) and bonobos (Pan paniscus)1,2. However, these tissue samples do not fairly represent the distinctive traits of live cell behaviour and are not amenable to genetic manipulation. We propose that induced pluripotent stem (iPS) cells could be a unique biological resource to determine relevant phenotypical differences between human and NHPs, and that those differences could have potential adaptation and speciation value. Here we describe the generation and initial characterization of iPS cells from chimpanzees and bonobos as new tools to explore factors that may have contributed to great ape evolution. Comparative gene expression analysis of human and NHP iPS cells revealed differences in the regulation of long interspersed element-1 (L1, also known as LINE-1) transposons. A force of change in mammalian evolution, L1 elements are retrotransposons that have remained active during primate evolution3,4,5. Decreased levels of L1-restricting factors APOBEC3B (also known as A3B)6 and PIWIL2 (ref. 7) in NHP iPS cells correlated with increased L1 mobility and endogenous L1 messenger RNA levels. Moreover, results from the manipulation of A3B and PIWIL2 levels in iPS cells supported a causal inverse relationship between levels of these proteins and L1 retrotransposition. Finally, we found increased copy numbers of species-specific L1 elements in the genome of chimpanzees compared to humans, supporting the idea that increased L1 mobility in NHPs is not limited to iPS cells in culture and may have also occurred in the germ line or embryonic cells developmentally upstream to germline specification during primate evolution. We propose that differences in L1 mobility may have differentially shaped the genomes of humans and NHPs and could have continuing adaptive significance.

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Figure 1: Characterization of iPS cells derived from the three primate species.
Figure 2: RNA-seq profiling of human and NHP iPS cells.
Figure 3: Reduced levels of A3B and PIWIL2 and increased L1 mobility in NHP iPS cells.
Figure 4: Species-specific L1 elements are more abundant in chimpanzee genomes than in human genomes, correlating with decreased levels of A3B and PIWIL2.

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Accessions

Gene Expression Omnibus

Data deposits

RNA-seq and small RNA-seq data have been deposited in the Gene Expression Omnibus under accession number GSE47626.

Change history

  • 27 November 2013

    An image credit was added to the Figure 1 legend.

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Acknowledgements

The work was supported by funds from the National Institutes of Health (NIH) (TR01: MH095741 and Eureka: MH08848 to F.H.G.), the Mathers Foundation and the Helmsley Foundation. This work was also partially supported by funds from the NIH to A.R.M. (MH094753), M.D.W. (AI074967) and G.W.Y. (NS075449, HG004659 and GM084317). G.W.Y. is a recipient of the Alfred P. Sloan Research Fellowship. We thank J. V. Moran and W. An for reagents. We would like to thank A. Varki, P. Gagneux, L. Fourgeaud and I. Guimont for discussions, N. Varki for help with teratoma analysis, R. Keithley, I. Gallina and Y. Nunez for technical assistance, and M. L. Gage for editorial comments.

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Authors and Affiliations

Authors

Contributions

M.C.N.M. and I.N. are the leading authors. M.C.N.M., I.N. and A.M.D. contributed to the concept, designed and performed the experiments, and analysed the data. M.C.N.M. reprogrammed NHP fibroblasts and performed iPS cell cultures and transduction assays. I.N. and M.C.N.M. performed L1 assays. I.N. designed and performed biochemical experiments. A.M.D., C.B. and I.N. designed and performed comparative analysis of L1 insertions in the human and NHP genomes. T.A.L. produced lentiviruses and provided tissue culture assistance. I.N. and K.N.D. generated the chimp-L1 reporter plasmid. C.B., A.C.M.P. and R.H.H performed bioinformatics analysis. I.N., C.B. and J.L.N. contributed to the generation of libraries and analysis of RNA-seq data. M.D.W., G.W.Y. and A.R.M. contributed to concept and financial support. F.H.G. is the senior author. He contributed to the concept, analysed the data, revised the manuscript and provided financial support. I.N., M.C.N.M., A.M.D. and F.H.G wrote the manuscript. All the authors read and approved the final manuscript.

Corresponding author

Correspondence to Fred H. Gage.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Cell lines used, number of mapped reads per sample in RNA-seq and gene ontology enrichment analysis for differentially expressed genes.

a, Origin of iPS cells used or generated in this study. b, Total number of mapped reads per sample in RNA-seq. c, d, Gene ontology (GO) enrichment analysis of differentially expressed genes. c, Top 10 enriched GO terms for genes with higher expression in human versus NHP iPS cells. d, Top 10 enriched GO terms for genes highly expressed in NHP versus human iPS cells. GO analysis was restricted to differentially expressed protein-coding genes (FDR < 0.05 and fold change > 2). GO enrichment for biological processes (level 2) was performed using DAVID. Figure shows GO term, number of genes (count), and P values for EASE score and Benjamini adjustment.

Extended Data Figure 2 Amino acid alignment of A3B and PIWIL2.

a, b, Protein sequences of human, chimp and bonobo A3B (a) or PIWIL2 (b) were aligned using ClustalW. a, Alignment of A3B showing >93% identity between human and NHP proteins. b, Alignment of PIWIL2 showing >98% identity between human and NHP proteins.

Extended Data Figure 3 mRNA levels of APOBEC3 and PIWI-like protein family members in iPS cells.

a, Comparative analysis of PIWIL2 mRNA levels. qRT–PCR analysis of PIWIL2 mRNA levels in human testis, human iPS cell lines, and available fibroblasts from which the iPS cell lines were derived. mRNA levels were normalized to GAPDH and shown relative to human testis (mean ± s.e.m.; n = 3 biological replicates). Compared to testis, PIWIL2 levels are 20–40 fold lower in iPS cells and 1,100-fold lower in fibroblasts. b, c, Quantification of mRNA levels of APOBEC3 and PIWI-like family members in human and NHP iPS cells by RNA-seq. Increased mRNA levels in human iPS cells are restricted for APOBEC3B and PIWIL2. y axes in b and c denote the reads per kilobase per million mapped reads (RPKM).

Extended Data Figure 4 L1 reporter activity in iPS cells.

a, L1 retrotransposition reporter system. The L1-reporter plasmid contains a retrotransposition-competent human L1 element and carries either an eGFP or a luciferase reporter construct in its 3′ UTR region. The reporter gene is interrupted by an intron in the same transcriptional orientation as the L1 transcript. This arrangement ensures that eGFP/luciferase-positive cells will arise only when a transcript initiated from the promoter driving L1 expression is spliced, reverse transcribed, and integrated into chromosomal DNA, thereby allowing expression of the reporter gene from a heterologous promoter. bf, Efficient A3B knockdown in human ES and iPS cells. b, Stable shRNA-mediated knockdown of A3B in human ES cells (HUES6) using lentivirus expressing different shRNAs against A3B (shA3B-1 and shA3B-2) or scrambled control (shScr). Levels of A3B expression were normalized to GAPDH and shown relative to shScr (mean ± s.e.m.; n = 3 biological replicates). c, Western blot confirming stable A3B knockdown in human ES cells. df, shRNA-mediated knockdown in human ES cells (HUES6) and iPS cell lines 1 and 2 (WT-33 and ADRC-40, respectively) was specific for A3B. gh, qRT–PCR analysis of plasmid expression in iPS cell lines transfected with L1–eGFP plasmid. Total RNA samples were obtained 60–72 h after transfection. L1 plasmid expression was normalized to GAPDH (g) or puromycin (h). L1–eGFP contains a puromycin expression cassette under PGK promoter control. Thus, puromycin expression was used as normalizer for transfection. iPS cells from two different individuals per species were transfected, and eGFP levels are shown as relative to human iPS cells. No significant differences were observed for L1 plasmid expression between human and NHP iPS cell lines (mean ± s.e.m.; n = 3 biological replicates). i, Relative L1 5′ UTR promoter activity. Human and chimp L1 promoters (L1 5′ UTR) controlling firefly luciferase were transfected into human and NHP iPS cell lines. Renilla luciferase was co-transfected as control. Luciferase activity was quantified as firefly luciferase units relative to Renilla luciferase units. Results are shown as normalized to human L1 5′ UTR activity in human iPS cell. iPS cells from two different individuals per species were transfected. No significant differences were observed for L1 promoter activities between human and NHP iPS cell lines (mean ± s.e.m.; n = 4 biological replicates).

Extended Data Figure 5 Nucleic acid alignment of human and chimpanzee L1 elements.

Sequence of the chimpanzee L1Pt element cloned and used to generate the chimpanzee L1–eGFP tagged reporter plasmid (L1IN71) (top sequence). LRE3: human L1 (bottom sequence).

Extended Data Figure 6 Immunoprecipitation of piRNAs associated with PIWIL2 in human iPS cells and annotated piRNAs mapping to consensus L1Hs in iPS cells.

a, Immunoprecipitation of PIWIL2 RNPs using Flag-tag antibodies from Tet-inducible Flag-tagged PIWIL2 human iPS cells after addition of doxyclycine to the culture media. HA-tag antibody was used as control. b, [γ-32P]ATP end-labelling of RNAs associated with Flag–PIWIL2 RNPs. Signal in the piRNAs size range is detected only in anti-Flag but not in control antibody anti-HA immunoprecipitates. c, Size distribution of RNA reads detected by small RNA-seq from small RNAs samples extracted from human iPS cell lines. d, Number of mapped reads per sample in small RNA-seq. e, Number of annotated piRNAs (piRNAbank) detected by RNA-seq in human iPS cells 1 and 2. f, Characterization of 5′ end of piRNAs detected in human iPS cells relative to annotated piRNAs. Read count distribution relative to piRNA 5′ ends (piRNAbank). g, Sequences of annotated piRNAs (piRNAbank) mapping to consensus L1Hs detected in human iPS cells 1 and 2. The 26–33-nucleotide RNA reads from human iPS cell lines 1 and 2 characterized by RNA-seq are aligned to annotated piRNAs mapping to the consensus L1Hs sequence. Analysis of mapping sequences was performed allowing two mismatches.

Extended Data Figure 7 Mapping of 26–33-nucleotide RNAs in human iPS cells to consensus L1Hs.

a, Mapping of annotated piRNAs (piRNAbank) detected by RNA-seq from human iPS cell lines to the consensus sequence for L1Hs (from Repbase). All annotated piRNAs (piRNAbank) complementary to L1Hs are indicated (black bars). b, Total 26–33-nucleotide RNA reads characterized by small RNA-seq mapped to L1Hs. c, Similar analysis as in b of ENCODE data for small RNAs from H1 cells. Positive and negative values indicate sense (+) and antisense (−) piRNAs, respectively. Schematic representation of the L1Hs element is shown (top). y axes represent read counts normalized to 107 reads per experiment.

Extended Data Figure 8 Higher levels of endogenous L1 RNA and recent species-specific L1 elements in chimpanzee.

a, Scheme of amplicons mapped to the L1Hs consensus sequence. Six primer pairs (two per region) were used for quantification of 5′ UTR, ORF1 and ORF2. The primers were designed to recognize both species-specific and common families. b, Positions of the amplicons in L1Hs consensus sequence and the number of in silico PCR hits on the human and chimp genomes. c, qRT–PCR analysis using primers for different regions of L1 element show higher levels of L1 RNA in NHP iPS cells regardless of the L1 region tested: 5′ UTR, ORF1 and ORF2 (mean ± s.e.m.; n = 3 biological replicates; *P < 0.01 between indicated groups, t-test). dg, Quantification of L1 elements in human and chimpanzee genomes using a population divergence model. Number of L1 elements found in the human and chimpanzee genomes for families: L1PA4 (d), L1PA3 (e), L1PA2 (f) and L1Pt and L1Hs (g) plotted as a histogram relative to their divergence (number of mutations relative to the canonical element). The standard deviation describes the differences in L1 density based on the sampling of different genomic regions and represents the variability of L1 coverage across the genomes (see Methods).

Extended Data Figure 9 Relative A3B and PIWIL2 mRNA levels in iPS cells and fibroblasts.

Relative expression of A3B (a) and PIWIL2 (b) in human and NHP iPS cell lines, and the available source fibroblasts from which iPS cells were derived. mRNA levels were normalized to GAPDH and shown relative to human iPS cell line 1.

Extended Data Table 1 List of primers used in this study

Supplementary information

Supplementary Table 1

This file shows microRNAs detected in human iPSCs by small RNA-seq. (XLSX 176 kb)

Supplementary Table 2

This file shows annotated piRNAs detected in human iPSCs by small RNAseq. (XLSX 1622 kb)

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Marchetto, M., Narvaiza, I., Denli, A. et al. Differential L1 regulation in pluripotent stem cells of humans and apes. Nature 503, 525–529 (2013). https://doi.org/10.1038/nature12686

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