Fig. 6: Heterochromatin positioning by the lamins and LBR is essential for EpiLC survival.
From: The nuclear periphery confers repression on H3K9me2-marked genes and transposons to shape cell fate
a, Diagram of naive (inner cell mass/mES cell) to primed (epiblast/EpiLC) developmental transition in vivo and its approximation in vitro. b, Normalized cell numbers after 2 days of culture in EpiLC differentiation conditions (normalized to WT; n = 4 replicates per genotype). The columns indicate the mean and error bars indicate s.d. ****Padj < 0.0001 for WT versus LBR KO and WT versus QKO; **Padj = 0.0058 for WT versus TKO by one-way ANOVA followed by Dunnett’s multiple comparisons test. c, The percentage Otx2-positive cells after 2 days of culture in EpiLC differentiation conditions (n = 3 replicates per genotype). The columns indicate the mean and the error bars indicate s.d. All EpiLC conditions n.s.; WT versus LBR KO EpiLC, P = 0.0867; WT versus TKO, P = 0.9935; WT versus QKO, P = 0.5942. d, Normalized cell numbers after 2 days of culture in EpiLC differentiation conditions (normalized to TKO; n = 6 replicates per genotype). The columns indicate the mean, error bars indicate s.d. ****Padj < 0.0001 for TKO versus QKO and TKO versus QKO + Halo-NLS by one-way ANOVA followed by Dunnett’s multiple comparisons test. n.s. indicates P = 0.6244. e, Normalized cell numbers of lamin TKO mES cells after 2 days of culture in EpiLC differentiation with (+) or without (−) co-induction of LUC or LBR miR-E. The columns indicate the mean and error bars indicate s.d. n = 6 replicates from 2 independent clones shown. *P = 0.011 and ****P < 0.0001 by unpaired two-tailed t-test. Dox, doxycycline. f–i, Immunofluorescence of H3K9me2 localization compared with the INM protein LAP2β in WT (f–f′), LBR KO (g–g′), lamin TKO (h–h′) and lamin + LBR QKO (i–i′) cells; f–i show EpiLC samples while f′–i′ show mES cell samples stained and imaged in parallel. Central z slices (xy) are shown. Scale bar, 5 μm. j, Radial intensity analysis of H3K9me2 position versus LAP2β in WT, TKO, LBR KO and QKO EpiLCs. WT versus QKO, shell 12, P = 0.0022; shell 13, P = 0.0002; shells 14–19, P < 0.0001; shell 20, 0.0029; shell 22, 0.0097; shell 23, 0.0007; shells 24–25, P < 0.0001 by unpaired t-test (two tailed). k, H3K9me2 total fluorescence intensity per nucleus in WT mES cells (n = 17) versus WT EpiLCs (n = 26); **P = 0.0015. TKO mES cells (n = 25) versus TKO EpiLCs (n = 18); ****P < 0.0001. LBR KO mES cells (n = 28) versus LBR KO EpiLCs (n = 41); ****P < 0.0001. QKO mES cells (n = 19) versus QKO EpiLCs (n = 20); ****P < 0.0001 by one-way ANOVA followed by Dunnett’s multiple comparisons test. The bars indicate mean and error bars indicate s.d. l,m, TEM images showing a 1.3 μm by 2.6 μm section of the nuclear periphery in WT (l) and lamin + LBR QKO (m) EpiLCs. C, cytoplasm; Hc, heterochromatin; N, nucleus; NE, nuclear envelope. Scale bar, 0.5 μm.
