The importance of microRNAs and long noncoding RNAs in the regulation of pluripotency has been documented; however, the noncoding components of stem cell gene networks remain largely unknown. Here we investigate the role of noncoding RNAs in the pluripotent state, with particular emphasis on nuclear and retrotransposon-derived transcripts. We have performed deep profiling of the nuclear and cytoplasmic transcriptomes of human and mouse stem cells, identifying a class of previously undetected stem cell–specific transcripts. We show that long terminal repeat (LTR)-derived transcripts contribute extensively to the complexity of the stem cell nuclear transcriptome. Some LTR-derived transcripts are associated with enhancer regions and are likely to be involved in the maintenance of pluripotency.
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The authors thank the RIKEN GeNAS sequencing platform for sequencing of the libraries. This work was supported by a grant to P.C. from the Japan Society for the Promotion of Science (JSPS) through the Funding Program for Next-Generation World-Leading Researchers (NEXT) initiated by the Council for Science and Technology Policy (CSTP), by a grand-in-aid for scientific research from JSPS to P.C. and A.F., and by a research grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) to the RIKEN Center for Life Science Technologies. FANTOM5 was made possible by a research grant for the RIKEN Omics Science Center from MEXT Japan to Y. Hayashizaki and by a grant for Innovative Cell Biology by Innovative Technology (Cell Innovation Program) from MEXT to Y. Hayashizaki. A.F. was supported by a JSPS long-term fellowship (P10782) and by a Swiss National Science Foundation Fellowship for Advanced Researchers (PA00P3_142122). K.H. was supported by European Union Framework Programme 7 (MODHEP project) for P.C. A.B. was supported by the Sigrid Juselius Foundation Fellowship. D.Y. and H.K. were supported by the Japan Science and Technology Agency CREST. R.A. and A. Sandelin were supported by funds from FP7/2007-2013/ERC grant agreement 204135, the Novo Nordisk Foundation, the Lundbeck Foundation and the Danish Cancer Society.
The authors declare no competing financial interests.
A full list of members and affiliations appear in the Supplementary Note.
Integrated supplementary information
(a,b) Normalized CAGE expression levels (tpm, tags per million) for stem cell marker genes in hiPS.F (a) and miPS.T (b). Expression levels for ESCs (blue, n = 3) and somatic cell types (purple) used for iPSC derivation are shown. (c,d) Immunofluorescence analyses for the expression of Ssea1, Oct4 and Nanog in miPS.T (c) and TRA-1-60, TRA-1-81 and SSEA4 in hiPS.F (d). ESCs are used as controls. (e) Histological sections of teratomas, formed 4 weeks after subcutaneous injection of hiPS.F cells into nude mice, hematoxylin and eosin staining. Three representative germ layers (mesoderm, ectoderm and endoderm) developed from hiPS.F cells. (f) Chimeric mouse derived from miPS.T cells and germline transmission.
Number of stem cell samples in which CAGE tag clusters are found expressed in human (a) and mouse (b). A value of 0 corresponds to differentiated cell type samples. (c,d) Hierarchical clustering based on Spearman coefficients calculated from CAGE tag cluster expression values for human (c) and mouse (d).
Numbers and cellular distribution of transcripts identified from RNA-seq assemblies for human (a) and mouse (b) data sets.
(a–d) M-Aplots of differentially expressed CAGE clusters (edgeR27, FDR < 0.01 indicated in red) for the mouse (a,b) and human (c,d) nuclear and cytoplasmic data sets. (e) Proportion of CAGE clusters significantly upregulated in stem cells (Up-Stem) at FDR < 0.01.
(a–d) Number of stem cell samples that expressed NASTs for human (a,b) and mouse (c,d) data sets. Panels show NASTs identified in the nuclear (Nu), cytoplasmic (Cy) compartments or both (Nu/Cy). (e) Percentage of CAGE clusters overlapped by CAGE-scan 5' tags. (f) Number of tissues and differentiated cell type samples from the FANTOM5 expression atlas29 in which annotated CAGE clusters overexpressed in stem cells are expressed. Bin width = 1. (g) Nuclear (x axis) and cytoplasmic (y axis) normalized expression (tpm, tags per million) for the human CAGE clusters overexpressed in stem cells. Similar plots are shown in h,i for a set of mouse (h) and human (i) nuclear (red) and cytoplasmic (blue) transcripts. (j,k) GRO-Seq30,31 signal enrichment at human (j) and mouse (k) NAST positions.
NASTs were classified as enhancers, promoters or others based on specific combinations of histone marks (Online Methods), using ChIP-seq signal from the ENCODE Project5 for the mouse ES-Bruce4 and ES-E14 cell lines as well as for the human H1-ES cell line. Normalized tag frequencies for all histone mars are plotted for each category and cell line.
(a,b) CAGE-based normalized expression (tpm, tags per million) for human (a) and mouse (b) NASTs (red) and annotated CAGE tag clusters (blue), identified in the nucleus (Nu) or cytoplasm (Cy) or in both cellular compartments (Nu/Cy). (c) Ct values for five NASTs and Gapdh are shown together with spiked firefly RNAs, used as a reference to evaluate copy number per cell. n = 3; error bars, s.d. (d) Transcript length as defined by RNA-seq assembly for NASTs and annotated genes compared for three expression groups (≥5, 1–5 and 0.1–1 tpm). n, number of clusters or transcripts per group. P values for two-sided Wilcoxon and Mann-Whitney tests are shown. (e) Fraction of NASTs and annotated CAGE clusters, as defined by CAGE-scan, overlapping short RNA-seq (15- to 40-bp fraction) clusters, grouped by expression levels.
Supplementary Figure 8 Histone marks and transcription factor binding at repeat-associated NAST loci.
(a,b) Normalized expression (tpm, tags per million) is plotted for mouse (a) and human (b) annotated genes and NASTs carrying promoter-associated histone marks. P values for Wilcoxon and Mann-Whitney tests are shown. n, number of CAGE clusters per group. (c) Frequency plots of normalized ChIP-seq tag counts (ENCODE data5) for H3K4me3 (promoter) and H3K9me3 (repressive) marks at NAST-associated and non-expressed (N.Exp.) MaLR elements. (d–f) ChIP-seq normalized tag counts for stem cell–specific transcription factors at NAST-associated and non-expressed (N.Exp.) mouse ERVK (d), mouse MaLR (e) and human ERV1 (f) elements. Values for non-expressed elements are shown in gray (dotted lines).
(a) Repeat family normalized expression values (tpm, tags per million) are plotted for human ESCs, iPSCs and differentiated cells (Dif.). Error bars, s.d. (b–d) Mouse ERVK (b) and MaLR (c) as well as human ERV1 (d) normalized nuclear expression is plotted for ESCs, iPSCs and differentiated cells (Dif). N, number of CAGE tag clusters carrying promoter-associated histone marks. (e) Normalized expression for selected human subfamily repeats are plotted against associated FDR (calculated with edgeR27). (f) The number of repeat elements with at least five CAGE tags is plotted against copy number found in the genome for human LTRs.
(a,b) Relative CAGE tag distributions along mouse ERVK-RLTR9E (a) and human ERV1-LTR7 and HERVH-int (b) elements. Gray bars mark the 5' and 3' extremities of each repeat element. Green and purple bars indicate CAGE tags mapping to the plus and minus strands, respectively. (c) Density plot for directionality scores at loci showing divergent transcription overlapping intergenic LTRs (red) and from annotated TSSs (blue). (d–f) Density plots of normalized tag counts for human DNase I footprints40 (d) and ChIP-seq5,41 (e,f) at loci presenting divergent transcription patterns and overlapping LTRs. (f) Promoters, NASTs associated with LTRs and classified as promoters in Figure 2b; enhancers, loci presenting divergent transcription patterns and overlapping LTRs. (g) Number of tissue and differentiated cell type samples from the FANTOM5 expression atlas29 in which LTR enhancer-associated CAGE tag clusters are expressed. Enlarged plots are shown for the first five bins. Bin width = 1 sample. (h) Frequency distribution of the distances between interacting loci identified by ChIA-PET.
Supplementary Figure 11 Multiple negative controls for knockdown experiments in Nanog-GFP iPS_MEF-Ng-20D17 cells.
The normalized Nanog-GFP–positive population, adjusted to the mock control (black bar), quantified by flow cytometry analysis 48 h after siRNA transfections is shown for 12 negative control siRNAs: 2 scrambled sequences, 1 siRNA targeting the luciferase transcript and 7 siRNAs targeting LTR, LINE and SINE elements not expressed in our data set, as well as 2 siRNAs targeting mRNAs originating from genes (Sdr16c6, Wfdc6a) with promoters overlapping LTR elements. Positive control siRNAs (green bars) targeting Nanog and Sox2 are shown for comparison. n = 3; error bars, s.d.
Supplementary Figures 1–11 and Supplementary Tables 1–5 (PDF 7729 kb)
Human stem cell transcriptome profiling data sets. (ZIP 28724 kb)
Mouse stem cell transcriptome profiling data sets. (ZIP 22435 kb)
Supplementary Note (XLS 54 kb)
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Fort, A., Hashimoto, K., Yamada, D. et al. Deep transcriptome profiling of mammalian stem cells supports a regulatory role for retrotransposons in pluripotency maintenance. Nat Genet 46, 558–566 (2014) doi:10.1038/ng.2965
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