Direct reprogramming of fibroblasts converts both protein-coding gene and lincRNA expression to a puripotent cell-specific profile. A, C) Unsupervised hierarchical clustering of protein-coding gene expression (A) and lincRNA expression (C) segregates fibroblasts (red) from ESCs and fibroblast-derived iPSCs (blue). B, D) Supervised hierarchical clustering analysis identifies 6865 protein-coding genes (B) and 237 lincRNAs (D) that are differentially expressed between ESCs/iPSCs and fibroblasts (genes: >2fold, P<0.05; lincRNAs: >2fold, FWER<0.05). Expression values are represented in shades of red and blue relative to being above (red) or below (blue) the median expression value across all samples (log scale 2, from -3 to +3). hFib2 fibroblasts are represented as two replicates (hFib2 and hFib2a). E) Examples of reprogrammed lincRNAs; left: lincRNA expressed in all fibroblasts is repressed in all pluripotent cells, right: a pluripotent cell-specific lincRNA that becomes activated during reprogramming. Expression values for each tiled probe (x-axis) are displayed as normalized hybridization intensity (y-axis). F) Correlation analysis of lincRNAs and neighboring genes. Density plot of multiple testing corrected p-values (x-axis) for lincRNAs that are positively (blue) or negatively (red) correlated with their protein-coding gene neighbors.

Several lincRNAs show enriched expression in iPSCs compared with ESCs. A) Heatmap of 28 and 52 lincRNAs that are more highly expressed in fibroblast-derived iPSCs (left) and CD34⁺-derived iPSCs (right), respectively, compared with ESCs (>2fold, FWER<0.05). Expression values are represented in shades of red and blue relative to being above (red) or below (blue) the median expression value across all samples (log scale 2, from -3 to +3). B) Top: Venn Diagram shows 10 lincRNAs that are commonly enriched in fibroblast- and CD34⁺-derived iPSCs. Bottom: qRT-PCR validation of the 10 commonly enriched lincRNAs (named according to their 3’ protein-coding gene neighbor) across three human ESC lines (H1, H9, BG01), fibroblasts (MRC5, MSC, hFib2) and CD34⁺ cells, and their derivative iPSC lines. Expression values are represented relative to the RNA levels in H9 ESCs.

Transcriptional regulation of iPSC-enriched lincRNAs. A) iPSC-enriched lincRNA loci are bound by pluripotency transcription factors. Top: lincRNA loci demarcated by domains enriched in histone H3K4me3 indicating RNA polymerase II promoters and H3K36me3 indicating regions of transcriptional elongation10,29 in human ESCs (green and blue, respectively). Bottom: ChIP in hFib2-iPS5 cells followed by quantitative PCR analysis detects binding of OCT4, SOX2, and NANOG within lincRNA-SFMBT2, lincRNA-VLDLR, and lincRNA-ST8SIA3 regions close to lincRNA promoter regions (peaks of H3K4me). ChIP enrichment values are displayed normalized to a control region (chr12: 7,839,777- 7,839,966; hg18); anti-GFP ChIP was used as a negative control. Positions of ChIP-PCR fragments are indicated by black lines. B) Changes in iPSC-enriched lincRNA levels upon siRNA-mediated knock-down of OCT4 in iPSC. Top: qRT-PCR of OCT4, NANOG and LMNA transcript levels upon depletion of OCT4. Bottom: qRT-PCR of iPSC-enriched lincRNA levels upon depletion of OCT4. Transcript levels are displayed relative to non-targeting control siRNAs (ctrl siRNA) (n=3, error bars +/-s.e.m). C) iPSC-enriched lincRNA expression during EB differentiation. Top: qRT-PCR analysis monitoring transcript levels of pluripotency markers (OCT4 and NANOG) and the differentiation marker LMNA over a ten day differentiation time-course; bottom: qRT-PCR analysis of iPSC-enriched lincRNAs. RNA levels are depicted relative to undifferentiated cells on day 0 (n=3, error bars +/-s.e.m).

LincRNA-ST8SIA3 expression modulates reprogramming. A) qRT-PCR verifies lincRNA-ST8SIA3 knock-down with Linc-sh1 and Linc-sh2 in hFib2-iPS5 cells relative to a non-targeting shRNA control (n=2, error bar: +/-s.e.m). B) Quantification of Tra-1-60⁺ iPSC colonies upon knock-down of lincRNA-ST8SIA3 relative to the control (day 21; n=4, error bar: +/-s.e.m). C) Quantification of cell numbers on day 6-7 of reprogramming in lincRNA-ST8SIA3 shRNA samples relative to the control (n=4, error bar: +/-s.e.m). D) Images showing quarters of Tra-1-60 stained reprogramming plates upon infection of a non-targeting control and two lincRNA-ST8SIA3 targeting shRNAs. Arrowheads mark Tra-1-60+ iPSC colonies. E) Structure of the lincRNA-ST8SIA3 locus. Green, Blue: Demarcation of the H3K4me-H3K36me domain in ESCs. Red: Structure of lincRNA-ST8SIA3 RNA; asterisk marks position of OCT4/SOX2/NANOG binding (see Figure 3A). Right: Northern hybridization of lincRNA-ST8SIA3 detects a 2.6kb transcript in hFib2-iSP5, but not dH1f (full-length blot see Supplementary Figure 12). F) qRT-PCR verifies lincRNA-ST8SIA3 overexpression from a retroviral vector (pBabe-lincRNA-ST8SIA3) compared with pBabe-puro and pBabe-puro-GFP vectors in dH1f relative to the levels in H9 ESCs and hFib2-iPS5 (n=2, error bars: +/-s.e.m). G) Quantification of Tra-1-60⁺ iPSC colonies upon overexpression of lincRNA-ST8SIA3 compared to pBabe and pBabe-GFP controls (n=5, error bar: +/-s.e.m.). H) Quantification of cell numbers on day 6-7 in lincRNA-ST8SIA3 overexpressing cells and controls. Cell numbers are relative to the pBabe control (day 28 +/-2; n=5, error bar: +/- s.e.m.). I) Image of quarter-plates of Tra-1-60 stained colonies (arrowheads) in pBabe, pBabe-GFP, and pBabe-lincRNA-ST8SIA3 infected samples. Statistical analysis was performed using Student’s t-test.