Differential l1 regulation in pluripotent stem cells of humans and apes

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

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THE ORIGINS OF ANIMAL STEM CELLS Article Open access 14 November 2024 ACCESSION CODES ACCESSIONS GENE EXPRESSION OMNIBUS * GSE47626 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. _ REFERENCES * The Chimpanzee

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molecules enable highly efficient neuronal conversion of human fibroblasts. _Nature Methods_ 9, 575–578 (2012) Article  CAS  Google Scholar  Download references 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. AUTHOR INFORMATION Author notes * Maria C. N. Marchetto and Iñigo Narvaiza: These

authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California

92037, USA, Maria C. N. Marchetto, Iñigo Narvaiza, Ahmet M. Denli, Christopher Benner, Thomas A. Lazzarini, Apuã C. M. Paquola & Fred H. Gage * Department of Cellular and Molecular

Medicine, University of California San Diego, Stem Cell Program, Institute for Genomic Medicine, Sanford Consortium for Regenerative Medicine, 2880 Torrey Pines Scenic Drive, La Jolla,

California 92037, USA, Jason L. Nathanson & Gene W. Yeo * Division of Biological Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA, Keval

N. Desai * Department of Pediatrics/Rady Children’s Hospital San Diego, Department of Cellular & Molecular Medicine, University of California San Diego, School of Medicine, Stem Cell

Program, Sanford Consortium, MC 0695, 2880 Torrey Pines Scenic Drive, La Jolla, California 92093, USA, Roberto H. Herai & Alysson R. Muotri * Department of Pathology and Laboratory

Medicine, University of Pennsylvania Perelman School of Medicine and Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, 19104-4318,

Pennsylvania, USA Matthew D. Weitzman * Center for Academic Research and Training in Anthropogeny (CARTA), 9500 Gilman Drive, La Jolla, 92093, California, USA Alysson R. Muotri & Fred H.

Gage Authors * Maria C. N. Marchetto View author publications You can also search for this author inPubMed Google Scholar * Iñigo Narvaiza View author publications You can also search for

this author inPubMed Google Scholar * Ahmet M. Denli View author publications You can also search for this author inPubMed Google Scholar * Christopher Benner View author publications You

can also search for this author inPubMed Google Scholar * Thomas A. Lazzarini View author publications You can also search for this author inPubMed Google Scholar * Jason L. Nathanson View

author publications You can also search for this author inPubMed Google Scholar * Apuã C. M. Paquola View author publications You can also search for this author inPubMed Google Scholar *

Keval N. Desai View author publications You can also search for this author inPubMed Google Scholar * Roberto H. Herai View author publications You can also search for this author inPubMed 

Google Scholar * Matthew D. Weitzman View author publications You can also search for this author inPubMed Google Scholar * Gene W. Yeo View author publications You can also search for this

author inPubMed Google Scholar * Alysson R. Muotri View author publications You can also search for this author inPubMed Google Scholar * Fred H. Gage View author publications You can also

search for this author inPubMed Google Scholar 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. ETHICS DECLARATIONS COMPETING INTERESTS 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. B–F,

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. D–F, 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_. G–H, 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). D–G,

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. 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) POWERPOINT SLIDES

POWERPOINT SLIDE FOR FIG. 1 POWERPOINT SLIDE FOR FIG. 2 POWERPOINT SLIDE FOR FIG. 3 POWERPOINT SLIDE FOR FIG. 4 RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS

ARTICLE Marchetto, M., Narvaiza, I., Denli, A. _et al._ Differential L1 regulation in pluripotent stem cells of humans and apes. _Nature_ 503, 525–529 (2013).

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