Figure 1: Reprogramming-induced expression of the L1 retrotransposition machinery is abrogated during embryoid body formation.
(a) Schematic of organization and expression of a functional human L1 element. Binding sites of TaqMan primer/probe combinations (small convergent arrows) on L1 cDNA used for qRT–PCR analyses and of the 1,299-bp [α-32P]dCTP-labelled PCR product in the 5′UTR region (black bar) used for northern analysis are shown. Methylation status of the CpG island (position number 232–491 of the L1.3 reference sequence) was analysed. Open circles, CpG residues. (b) Relative full-length L1 (FL-L1) mRNA transcript levels were assessed by qRT–PCR from early passage (until p24) HFF-1-derived (hFF-iPS4, hiPS-SB4 and hiPS-SB5) and hCBEC-derived (hCBiPS1 and hCBiPS1) hiPSC lines (left panel), and after differentiation of hFF-iPS4 (p50) and hiPS-SB4 (p98) lines into embryoid bodies (EBs) (middle panel) (*P<0.05, **P<0.01, ***P<0.001). hiPS-SB5.1 cells (p10) were differentiated into EBs. L1 transcript levels were quantified on day 0 before initiation of differentiation, and after 2, 4, 6 and 8 days of differentiation by qRT–PCR (right panel; ***P<0.001, linear regression t-test). Bars represent arithmetic means±s.d. from experiments performed as technical duplicates of biological triplicates, or, in the case of hCBEC, hCBiPS1 and hCBiPS2 (green bars), arithmetic means of technical duplicates of one biological sample. (c) Northern analysis of cytoplasmic poly-A+ mRNA with a 1,299-bp L1 5′UTR-specific probe confirmed exceeding activation of FL-L1 transcription during hiPSC cultivation. β-Actin mRNA (1.8 kb, lower panel) served as loading control. (d) Endogenous L1 promoter sequences are significantly hypomethylated in hiPSC lines relative to their parental HFF-1 and hCBEC cells. Overall percentage methylation of 5′UTR CpG islands in HFF-1 and hCBEC cells (n=29 CpG islands; blue bar) and in five derived hiPSC lines (n=95 CpG islands; red bar), respectively, is presented. Error bars indicate s.e.m.***P<0.001; χ2 test. (e) Immunoblot analysis of cell lysates from HFF-1 and hCBEC cells and their respective derived hiPSC lines measures L1 ORF1p (40 kDa) and Oct-3/4 expression (A isoform, 45 kDa; B isoform 33 kDa). Shorter (exp.1) and longer exposures (exp.2) of the αOct-3/4 immunoblot are provided. Lysates from hESC lines HES-3 (left panel) and H1 (right panel) served as positive control for L1 ORF1p and Oct-3/4 expression. β-Actin (42 kDa) served as loading control.
