Fig. 6: Analysis of FOXJ3 binding, target gene identification, and expression regulation.

A Overlapping binding regions shared between FOXJ3 clone #1 peaks and FOXJ3 clone #2 peaks. B Overlapping binding regions shared between FOXJ3-overlapping peaks and H3K27ac peaks. C GO enrichment analysis of potential FOXJ3 target genes was performed using DAVID, with terms ranked by -log(p-value). D scRNA-seq data were used to identify potential FOXJ3 downstream target genes based on expression patterns similar to Foxj3. Identified genes include Pten, Tsc1, Pafah1b1, Cep120, Dicer1, Nde1, Wdr62 and Mcph1. E Enriched peaks for the candidate genes Pten, Tsc1, Pafah1b1, Cep120, Dicer1, Nde1, Wdr62, and Mcph1 were co-localized with FOXJ3 #1, FOXJ3 #2, H3K27ac, and RNA Pol-II. DNA input was used as the control. F Bar graph showing significant upregulation of Pten, Tsc1, and Pafah1b1 mRNA levels in cortical tissue overexpressing human FOXJ3. In contrast, overexpression of the p.N351S variant failed to upregulate these downstream target genes, indicating a loss-of-function effect. Error bars represent S.E.M. **: p < 0.01 ***: p < 0.001 (n = 3 independent experiments; one-way ANOVA with post-hoc Fisher’s LSD test; p = 0.0065, 0.0025, 0.0007, 0.0007, 0.002, 0.0004). G qPCR analysis of transcriptional changes in downstream genes Pten and Tsc1 upon FOXJ3 expression. CRISPR/Cas9-mediated Foxj3 knockout in mouse embryonic stem (mES) cells resulted in decreased expression of Pten but not Tsc1. Error bars represent S.E.M. *: p < 0.05, (n = 3 independent experiments; one-way ANOVA with post-hoc Fisher’s LSD test; p = 0.0123, 0.0143, 0.0294, 0.0172, 0.0309, 0.0406). H UMAP clustering from scRNA-seq from the human fetal brain dataset⁵⁷ visualized cell clusters based on cell type, with expression patterns of FOXJ3, PTEN, and TSC1 mapped and visualized.