Fig 1: miR-9 overexpression in Area X downregulates the expression of FoxP1 and FoxP2 and disrupts dopamine signaling.(A) Expression of FoxP1 and FoxP2 mRNAs in Area X measured 4 weeks after injection with the lenti-miR-9 virus by qRT-PCR. p = 0.006, t(12) = 4.608 for FoxP1; p < 0.0001, t(12) = 7.062 for FoxP2, unpaired t-test. n = 7 for Area X; n = 4 for adjacent tissue. (B) Western blot showing expression of FoxP1 and FoxP2 proteins in Area X 4 weeks after injection with the lenti-miR-9 virus. p = 0.0007, t(6) = 6.313 for FoxP1; p = 0.0037, t(6) = 4.608 for FoxP2, unpaired t-test; n = 4. (C) Expression of dopamine receptors D1R and D2R mRNAs in Area X 4 weeks after injection with the lenti-miR-9 virus measured by qRT-PCR. For D1R, p < 0.0001, t(12) = 6.441; for D2R, p = 0.3384, t(12) = 0.9971, unpaired t-test; n = 7 for Area X. (D) DARPP-32 protein expression in Area X 4 weeks after injection with lenti-miR-9 virus. p = 0.0022, t(6) = 5.104, unpaired t-test; n = 4. Data are presented as mean ± SEM. Lenti-control and lenti-miR-9 viruses were injected into Area X of contralateral hemispheres, #40, #45, #49 and #52 in western blot images are IDs of animals.
Fig 2: Upper layers are predominantly affected by inhibiting FGF signaling.pCAG-EGFP plus either pCAG-sFGFR3 or pCAG control vector was electroporated at E33, and the brains were dissected at P16. Coronal sections were subjected to Hoechst 33342 staining plus either immunohistochemistry or in situ hybridization. (A) FoxP2 immunohistochemistry. (B) Ctip2 immunohistochemistry. (C) Rorb in situ hybridization (white). (D) Cux1 in situ hybridization (white). The images within the boxes in the upper panels were magnified and are shown in the lower panels. Note that sFGFR3 did not affect the layer structure of the cerebral cortex while cortical folding was inhibited (sFGFR3, EP, arrows). Scale bars = 1 mm. (E–H) Quantification of the thicknesses of layer 2/3 (E), layer 4 (F), layer 5 (G) and layer 6 (H). The ratios of the thickness of the electroporated side relative to that of the non-electroporated side are shown. Note that the thickness ratio of layer 2/3 was significantly reduced by sFGFR3, while those of layer 4, layer 5 and layer 6 were not. n = 3 animals for each condition. Bars present mean ± SD. *p<0.05; ns, not significant; Student's t-test. (I) A model of the mechanisms underlying the formation of cortical folds in the gyrencephalic brain.
Fig 3: miR-9 overexpression alters expression levels of numerous FoxP1 and FoxP2 downstream target genes in Area X.Blue bars, upregulated genes; red bars, downregulated genes. Genes are grouped into five functional groups: (A) neurotransmitter receptors and channels; (B) proteins involved in dendritic growth and synapse formation; (C) transcription factors; (D) signaling molecules; and (E) proteins for protein processing and degradation. Reverse transcription and qRT-PCR were performed twice, and qRT-PCR was performed in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired t-test; n = 7 for all genes, except for BDNF, CTNNA3, NRTK, and STX1A, n = 4. Data are presented as mean ± SEM.
Fig 4: Loss of PRC2 activity drives increased FOXP2 expression in normal and malignant lung cells.a Relative expression of Foxp2 mRNA in mouse primary 2D tumor cells and 3D tumor spheroids with the indicated Ezh2 genotypes graphed as mean values +/- SEM. * indicates P < 0.043 by one-way ANOVA with multiple comparisons and Holm–Šídák’s post hoc test between genotypes and two-tailed t test between conditions. n = 3 individual mouse tumor-derived cultures per genotype/culture system. b FOXP2 protein expression in 2D cells and 3D tumoroids of the indicated Ezh2 genotypes was examined by immunoblot. Total histone H3 is the same as Fig. 2e. ? indicates the non-specific band seen with FOXP2 antibody from Cell Signaling Technologies. c Immunoblot and d the normalized abundance of the indicated proteins in Ezh2 WT, Ezh2 null, Ezh2 null with WT Ezh2 re-expression (WT), and Ezh2 null with F667I-mutant Ezh2 re-expression (F6771). Quantification data are plotted as mean values +/- SEM. * indicates P < 0.0261 by one-way ANOVA with multiple comparisons and Holm–Šídák’s post hoc test. n = 3 individual blotting experiments from two cell cultures. e Percentages of positively stained tumor nuclei for FOXP2 in the tumors of the indicated genotypes plotted as box-and-whisker plots, error bars are min–max, box bounds are 25th and 75th percentiles and center line is median. * indicates P = 0.0233 by one-way ANOVA with multiple comparisons and Holm–Šídák’s post hoc test for both comparisons. n = 29, 38, 23 for Ezh2 WT, heterozygous, null individual mice, respectively. f Visualization of H3K27me3 ChIP-seq peaks enriched in the enhancer and promoter regions of Foxp2 in the indicated 3D tumor spheroid samples. g Expression of FOXP2 mRNA in human lines HBEC3KT, BEAS2B, BEAS-KP, H460, H2030, A549, and H2009 with or without 5 µM EPZ6438 treatment for 10 days plotted as mean values +/- SEM. * indicates P < 0.043, ** indicates P < 0.009, *** indicates P = 0.0003 by two-tailed t test. n = 3 individual cell cultures per cell line/condition. h Immunoblot and i the normalized abundance of the indicated proteins in human lines HBEC3KT, BEAS2B, BEAS-KP, H460, H2030, A549, and H2009 with or without 5 µM EPZ6438 treatment for 10 days. Top FOXP2 panel is SIGMA antibody, bottom FOXP2 panel is Cell Signaling Technologies antibody. Quantification data are plotted as mean values +/- SEM. * indicates P < 0.032, *** indicates P = 0.000778 with two-tailed t test. n = 4 with two individual blotting experiments derived from one cell culture per cell line/condition each probed with two distinct FOXP2 antibodies. Genotypes are signified as: WT, +/+; heterozygous, ?/+; null, ?/?. Source Data are provided for this figure.
Fig 5: Decoupling of EZH2 from PRC2 activity may explain co-expression of EZH2 and FOXP2 in patient samples.a Positively stained tumor nuclei in ADC, ADSCC, poorly differentiated tumor and SCC specimens for the markers EZH2, H3K27me3 and FOXP2 plotted as box-and-whisker plots, error bars are min–max, box bounds are 25th and 75th percentiles and center line is median. * indicates P = 0.0164, ** indicates P < 0.0084, *** indicates P < 0.0006 by one-way ANOVA with multiple comparisons and Holm–Šídák’s post hoc test. n = 92, 24, 17, 104 for ADC, ADSCC, poorly differentiated and SCC individual tumors, respectively. b The percentage of EZH2 and H3K27me3 positive nuclei in lung adenocarcinoma and poorly differentiated cancer specimens. * indicates P < 0.0425, ** indicates P < 0.0086 by one-way ANOVA with multiple comparisons and Holm–Šídák’s post hoc test. n = 13, 51, 97 for well, moderately, poorly differentiated individual tumors, respectively. c Representative images of lung adenocarcinoma specimens defined as well, moderately, and poorly differentiated stained for the indicated markers, scale bar = 100 µm. The number of tumors stained are summarized in (b). d Correlation between EZH2 and FOXP2 based on the percentage of positively stained tumor nuclei in primary lung cancer specimens. Pearson’s correlation coefficients and p values are shown. n = 161 individual tumors. e Kaplan–Meier relapse-free survival curves for FOXP2-high and FOXP2-low tumors as measured by RNA-sequencing 250 months post diagnosis; positive and negative groups were determined by best cut-off. The P value was calculated by log-rank test. n = 366 individual tumors. f, g Immunoblot in 2D (f) H460, A549, BEAS-KP, and g Ezh2 WT UK803 cells treated with or without 500 µM SAM for 6 days. h Normalized protein abundance in H460, A549, and BEAS-KP cells treated with or without 500 µM SAM for 6 days plotted as mean values +/- SEM. * indicates P = 0.012, ** indicates P = 0.009 for EZH2 and P = 0.0045 for FOXP2 by two-tailed t test. n = 3 different cell lines. Genotypes are signified as: WT, +/+; heterozygous, ?/+; null, ?/?. SAM, S-adenosyl methionine. Source Data are provided for this figure.
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