Fig. 5: KDM3A regulates Wnt/β-catenin signaling at the transcriptional level.

A Bar charts illustrate the distribution of Kdm3a, H3K9me2, and Ctnnb1 peaks as determined by ChIP-seq analysis. B Gene ontology analysis of ChIP-seq data indicates that Wnt/β-catenin signaling is enriched for Kdm3a binding, and H3K9me2 binding is increased in KO cells. C Enriched peaks of Kdm3a, H3K9me2, and Ctnnb1 on Wnt target genes in WT and KO NSPCs were identified using IGV software. D ChIP-PCR assays demonstrate that the loss of KDM3A results in increased binding of H3K9me2 and decreased binding of Ctnnb1 on the promoters or introns of Ctnnb1, Wnt7a, Ccnd1 and Fzd5. Experiments were repeated at least three times, with quantification presented as mean ± SEM. Statistical significance was assessed using Student’s t test (*** for p < 0.001). E WT and KO NSPCs were treated with varying concentrations of IOX1 (10, 50 µM) for 48 h, followed by RNA extraction. Real-time PCR analysis shows that IOX1 significantly downregulates the mRNA expression levels of Wnt target genes Ccnd1 and c-myc in WT NSPCs but not in KO NSPCs. Experiments were repeated at least three times, with quantification represented as mean ± SEM. Statistical significance was determined by Student’s t test (* and ** for p < 0.05 and 0.01, respectively). F Luciferase assays indicate that overexpression of WT Kdm3a enhances Topflash activity in both the presence and absence of WNT3A. This effect is diminished in Kdm3a(H1122A)-transfected cells. Experiments were repeated at least three times, with quantification as mean ± SEM. Statistical significance was assessed using one-way ANOVA followed by Tukey’s post-hoc test (* and ** for p < 0.05 and 0.01, respectively). G Gene ontology analysis of ChIP-seq data reveals differences in neurogenesis genes enriched for Kdm3a, H3K9me2, and Ctnnb1 binding between WT and KO NSCs. H Enriched peaks of Kdm3a, H3K9me2, and Ctnnb1 on neurogenesis genes in WT and KO NSCs were identified using IGV. I ChIP-PCR assays indicate that the loss of KDM3A increases the binding of H3K9me2 on the promoter and introns of Neurod1 and Dcx. The experiments were repeated at least three times, with quantification presented as mean ± SEM (n = 3). Statistical significance was determined by Student’s t test (*** for p < 0.001).