Fig. 6: LIN28A interaction with PRC2 is RNA-dependent.

A Western blot analysis of LIN28A immunoprecipitates with and without RNase treatment. Immunoprecipitation was performed using anti-FLAG beads in ESCs transfected with a LIN28A-Flag expressing vector or an empty vector. B Western blot analysis after immunoprecipitation using an anti-EZH2 antibody in WT and KO ESCs. Lysates were treated or not with RNase before immunoprecipitation, as indicated. Black arrow indicates the unspecific band of IgG light chains. C RNA immunoprecipitation followed by qPCR analysis to evaluate the binding of ncRNA Neat1 to LIN28A. The 3’UTR of Lin28b was used as negative control. The graph shows the mean of the relative levels ±SD of four biological replicates, *p < 0.05 (Student’s t test). D Proximity ligation assay (PLA) to assess the interaction between LIN28A and the PRC2 subunit EED upon silencing of the ncRNA Neat1. ESCs were analyzed by PLA coupling the anti-LIN28A with anti-EED antibodies. Quantification of positive PLA spots/nucleus (for >300 cells) ± SD from three independent biological replicates is shown in the graph. Two-tailed, unpaired Student’s t test: ****p ≤ 0.00001. E qPCR analysis showing the mean of the relative levels ± SD of Neat1 RNA upon silencing with a control or a specific siRNA for the experiments reported in (D). n = 3 biological replicates, *p ≤ 0.05 (Student’s t test). F The proposed model for LIN28A involvement in epigenetic regulation. When differentiation is induced EZH2 is redistributed in WT cells allowing for the timely activation of developmental genes. By contrast, Lin28a KO ESCs exhibit restricted EZH2 occupancy under pluripotent conditions, likely due to a disrupted eviction/recruitment dynamic. During differentiation, this imbalance leads to persistent EZH2 binding and widespread recruitment to developmental gene promoters, resulting in their repression and ultimately impairing proper cell differentiation. Adapted from BioRender.com.