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. 2019 Jun 6;74(5):951–965.e13. doi: 10.1016/j.molcel.2019.03.041

Figure 4.

Figure 4

TDP-43 Regulates APA of Genes Important for Pluripotency, Including Sox2

(A) Scatterplot displaying relative changes of pA sites upon differentiation of hESCs (Figures S3A and S3B) and changes accruing by knocking down TDP-43 in undifferentiated hESCs for 48 h (n = 4 control short hairpin [shCTRL] and n = 8 shTDP-43 using 2 TDP-43-targeting short hairpin RNAs [shRNAs] with 4 replicates for each; adjusted p < 0.05, Fisher’s exact test). Linear regression (gray line) and the 90% confidence interval region (light blue) are shown (Pearson’s correlation coefficient [r] = 0.62). Increased use of the proximal pA site has positive values, and decreased use has negative values, based on genes passing filtering and statistical analysis as outlined in Figures S3C–S3E.

(B and C) Non-redundant Gene Ontology (GO) terms (B) and ground-state and general pluripotency factors (C), characterized by (Kalkan et al., 2017), of genes exhibiting APA upon cTdp-43 KO in mESCs (n = 10 independent sample replicates per group; genes passing filtering and statistical analysis as outlined in Figure S3C–S3E).

(D) Representative diagrams displaying the transcript isoforms of Sox2 upon cTdp-43 KO in mESCs and frequencies of the pA sites in Sox2 and SOX2 upon cTdp-43 KO in mESCs or KD of TDP-43 and 72 h CHIR99021 treatment of hESCs; samples as above.

(E) The positions of TDP-43 crosslinking (red bars) in Sox2 transcript from mESCs, as defined by iCLIP, surrounding the proximal and distal pA sites; n = 2 biological replicates (Figure S5C) combined into a single track.

(F) Western blot analysis of SOX2, TDP-43, and histone H3 following tamoxifen treatment (3 days) of cTdp-43 KO mESCs; independent replicates are shown.

(G) An illustration of the EGFP-SOX2 3′ UTR reporter minigene displaying endogenous positions of the proximal and distal pA sites and the miR-21 binding site.

(H–J) Representative flow cytometry analyses of HEK293T cells with the doxycycline-inducible TDP-43 gene cassette co-transfected with miR-21 (transfected cells were gated using tdTomato as described in STAR Methods), and the respective EGFP-SOX2 3′ UTR constructs: unmodified (H), harboring deletion of the proximal pA site (I), or with deletion of the miR-21 binding site (J). Control (CTR) indicates cells that were not treated with doxycycline.

(K) Boxplot depicting relative levels of miR-21 in undifferentiated mESCs and in primitive streak (PS)-like progenitors generated by 3 d CHIR99021 treatment (n = 6 independent replicates, two sided t test; p value ∗∗∗ ≤ 0.001).

(L) Boxplot analyses depicting levels of the Sox2 transcript in WT mESCs and cells lacking the endogenous miR-21 binding site in Sox2 (Sox2ΔmiR21) under pluripotency conditions (top) and upon PS differentiation (bottom) (n = 5 and 6 independent replicates, two-sided t test; ∗p ≤ 0.01).