Fig 2: RFX3 exhibits dosage sensitive binding near synaptic genesA. Aggregate plot of RFX3 CUT&RUN reads within RFX3 binding peaks called in WT neurons (n=4,240 peaks). Trace shows mean +/− SEM of n=7 WT replicates, n=6 HET replicates, n=6 KO replicates.B. RFX3 binding motif enriched within RFX3 binding peaks.C. SIX1 binding motif enrichment near RFX3 binding motifs in RFX3 binding peaks. Primary (RFX3) and secondary (SIX1) motif alignment. CRE binding motif enrichment near RFX3 binding motifs in RFX3 binding peaks. Primary (RFX3) and secondary (CRE) motif alignment.D. Gene Ontology (GO) enrichment analysis of genes with an RFX3 peak in the promoter (promoter_peaks) or distal regions (distal_peaks).E. Log2 fold-changes in expression levels of all genes or RFX3 target genes in RFX3 HET or KO neurons compared to WT. *p<0.05, t-test with Bonferroni correction.F. Aggregate plot of RFX3 CUT&RUN reads within RFX3 binding peaks significantly decreased in RFX3 HET neurons (n=947 peaks, FDR<0.2). Trace shows mean +/− SEM of n=7 WT replicates, n=6 HET replicates, n=6 KO replicates.G. Gene Ontology (GO) enrichment analysis of RFX3 target genes with significantly decreased peak and expression in RFX3 HET neurons. Dashed line indicates significance threshold FDR 0.05.H. Peak width and RFX3 CUT&RUN signal among RFX3 dosage sensitive, RFX3 dosage insensitive, ciliary, non-ciliary, and synaptic regions. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, t-test.I. ATAC-seq signal in Ngn2 neurons in different sets of RFX3 binding regions. Data represent mean +/− 95% CI. *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001, one-way t-test with Bonferroni correction.

Fig 3: Decreased synchronized neural networks in RFX3 deficient neuronsA. Quantification and representative images of SYN1 and PSD95 colocalized puncta on neurites in day 14 neuronal cultures. n=32 wells per genotype. White arrows mark representative colocalized puncta. Scale bar=100 um. *p<0.05, one-way t-test with Bonferroni correction.B. Mean firing rate (Hz) of neuronal cultures from day 7 to day 62.C. Average number of spikes per network burst of neuronal cultures from day 7 to day 62.D. Area under cross-correlation (measure of synchrony) of neuronal cultures from day 7 to day 62.E. Neural activity score of neuronal cultures from day 7 to day 62. (B-E) Data normalized to the number of covered electrodes per well. Data represented as mean +/− SEM. n=32 wells per genotype from two independent MEA plates. *p<0.05, one-sided t-test, red * indicates KO vs. WT, yellow * indicates HET vs. WT.

Fig 4: RFX3 promotes CREB binding in unstimulated neuronsA. Aggregate plot of CREB CUT&RUN reads within CREB binding peaks called in WT neurons (n=3095 peaks). Trace shows mean +/− SEM of n=2 WT replicates, n=2 RFX3 HET replicates.B. CREB and RFX3 binding motifs enriched within CREB binding regions in WT neurons.C. Overlap between CREB-bound and RFX3-bound regions in WT neurons.D. CREB CUT&RUN signal at all, RFX3 unbound, and RFX3-bound CREB binding sites in WT neurons. ****p<0.001, ns not significant, one-way t-test with Bonferroni correction.E. Aggregate plot of CREB CUT&RUN reads within CREB and RFX3 co-bound sites (n=567 peaks). Trace shows mean +/− SEM of n=2 WT replicates, n=2 RFX3 HET replicates.F. Average observed log2 fold-change in CREB peak intensity at regions co-bound by RFX3 in RFX3 HET neurons compared to a permuted distribution (n permutations=1,000, 90 percentile interval). *left-tailed p <0.05, permutation test.G. CREB binding peaks near FOSB, and JUNB in RFX3 WT and HET neurons. RFX3 binding peaks near FOSB and JUNB in RFX3 WT neurons.

Fig 5: RFX3 modulates CREB-dependent activity-dependent transcriptional responsesA. RFX3 binding peaks in the promoter regions of FOSB and JUNB in RFX3 WT, HET, and KO day 14 neurons.B. Schematic of neuronal stimulation with KCl.C. Volcano plot of differentially expressed genes in 1.5 hour KCl stimulated HET vs. WT or KO vs. WT neurons at day 14 in culture. Genes significantly downregulated are in blue and genes significantly upregulated are in red (|log2FC|>1, FDR<0.05). Select immediate early genes are labeled.D. Fold-induction of all immediate early genes, FOSB, and JUNB at 1.5, 4, and 6 hours post KCl depolarization compared to unstimulated neurons. Data represented as mean and 95% CI, *p<0.05 t-test, n=3 independent neuron differentiations per genotype.E. Fold-induction of FOSB, FOSL2, JUNB, NR4A2 at 1.5 hours post KCl depolarization or H89 pretreatment and KCl depolarization assessed by RT-qPCR. Data represented as mean +/− SEM, n=3–4 independent wells per condition. *p<0.05, **p<0.01, ***p<0.005, ns not significant, one-way t-test with Bonferroni correction.