Fig 2: ZNF292 deficiency alters cIN migration, morphology, and expression of mature cIN markers(A and B) Neurosphere outgrowth assay, with representative images in (A) and quantification of average length of neuron migration from the periphery of the neurosphere in (B) (n = 3).(C–F) Morphometric analysis of interneurons from the control and KD models at D23, with quantification of (D) cell soma size and number of cINs with (E) >3 primary neurites and (F) >2 secondary neurites. In (C) (boxes), examples of neurite branching are highlighted with white lines in representative images.(G–I) Fraction of cells expressing the mature neuronal and cIN markers MAP2 and CALB1 at D30, relative to all cells (marked by DAPI expression) is shown in representative images in (G) and quantified in (H) and (I) under control and KD conditions (n = 3).(J) Principal-component analysis (PCA) plot of cIN RNA-seq data, with segregated clustering of the control versus KD samples.(K) Heatmap visualization of relative expression of cIN DEGs in the control versus KD samples.(L) GO term enrichment analysis of KD-up- and -downregulated DEGs in cINs.Data were analyzed using Student’s t test and are represented as percentage mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Scale bars, 100 μm.

Fig 3: Putative ZNF292 direct targets in NPCs defined by ZNF292 association and differential expression upon KD(A and B) (A) Relative expression of ZNF292-bound and -regulated DEGs in control and KD NPCs (n = 4) and (B) top GO terms.(C–L) For bound and KD-upregulated DEGs in NPCs, (C) hierarchical clustering of D0–D60 expression changes, (D) genes of interest under denoted GO terms, and (E–L) browser tracks for selected genes (E, G, I, and K) and accompanying transcriptomic data (F, H, J, and L; bar plots visualize RNA-seq reads per kilobase of transcript per million mapped reads (RPKM) mean ± SEM values; *p < 0.05, **p < 0.01, and ****p < 0.0001).

Fig 4: Effect of KD upon ZNF292 genome-wide occupancy in NPCs(A) Deeptool plots depicting ZNF292 genome-wide occupancy in NPCs, including shared (top) and “control unique” (bottom) ZNF292-bound peak centers under control and KD conditions.(B) Unique and shared peak numbers under control and KD (G1/G2) conditions; highlighted peaks bound in control and lost upon G1 and/or G2 KD were further analyzed.(C–E) For control unique peak fraction (C) mapping to genomic features is shown (see STAR Methods), (D) top GO terms, and (E) TFBS enrichment.(F) Intersection of peaks with D0–D60 chromatin state.(G and H) TFBS enrichment analysis of ZNF292-bound active and bivalent peaks.

Fig 5: ZNF292 KD alters cortical interneuron function(A–C) (A) Whole-cell inward and outward currents evoked by voltage step from —80 to 0 mV. KD (G1/G2) cINs exhibit significantly less inward (B) sodium current and (C) current density normalized by cell capacitance versus controls (WT/KRAB).(D) Reversal potential for steady-state current (Vm for 0 K current) was also significantly less negative in KD cINs versus control.(E) Voltage responses to hyperpolarizing and depolarizing current injections.(F–I) (F) Whole-cell currents evoked by GABA (blue bar) and kainate (red bar). Compared to control, KD cINs displayed (G and I) larger currents evoked by GABA and (H and I) smaller currents evoked by kainate.(J–L) Spontaneous inhibitory synaptic currents (sIPSCs; J) were (K) more frequent and (L) larger in amplitude in KD neurons versus controls.p < 0.05 was defined by Mann-Whitney rank-sum test, bars plot mean ± SEM, circles show the data values for each neuron, and thick lines show the median value.