Fig 2: BCL7A loss alters neuronal activity and mouse behavioral performance ASimplified ClueGO pathway analysis of genes with reduced SMARCA4/BRG1 occupancy at promoter regions in BCL7A KO vs. wt eNPCs.B, CRepresentative Ca2+ measurements in (B) wt vs. BCL7A KO cortical neurons (CN, 10 days in vitro) and in (C) smNPC-derived parental and BCL7A KO neurons. Ca2+ changes were detected using Fluo-4 and upon treatment with (B) 50 µM of glutamate (final concentration) and (C) 100 µM of glutamate (final concentration). Upper right panels report Ca2+ peaks (F) relative to baseline (F0). In (B) wt: n = 18 cells; ko: n = 23 cells (n = 2 independent experiments). In (C) P: n = 27 cells; KO1: n = 20 cells; KO2: n = 19 cells (n = 2 independent experiments).DImmunoblot analysis of hippocampal tissues. Densitometries are shown on the right (ctr: n = 4–5 mice; ko: n = 3–4 mice).ERT-PCR analysis of FOS expression upon glutamate (100 µM for 10 min) stimulation in smNPC-derived parental and BCL7A KO neurons (n = 5–6 biological replicates).FImmunofluorescence staining of cFos in the dentate gyrus of adult Bcl7a fl/fl ; Baf53b-Cre tg/wt (ko) mice and control (ctr) littermates. Mice were either kept in standard housing conditions (upper images; ctr: n = 4 mice; ko: n = 4 mice) or exposed to an enriched environment (lower images; ctr: n = 6 mice; ko: n = 5 mice). Quantification of cFos+ cells are shown below. Scale bar = 30 µm.G–KBehavioral characterization of Bcl7a fl/fl ; Nestin-Cre tg/wt (ko) mice compared to control (ctr) littermates. Animals were tested for their locomotor activity and coordination in (H) the open field (ctr: n = 13 mice; ko: n = 11 mice) and (I) RotaRod test (ctr: n = 12 mice; ko: n = 7 mice). Cognitive function was assessed in (J) the Y-maze spontaneous alternation task (ctr: n = 13 mice; ko: n = 11 mice) and (K) fear conditioning paradigm (ctr: n = 11 mice; ko: n = 10 mice). The percentage of spontaneous alternations and freezing were used as measures for working and long-term memory function, respectively.L–QBehavioral characterization of adult Bcl7a fl/fl ; Baf53b-Cre tg/wt (nKO) mice compared to control (ctr) littermates. Locomotor activity and coordination were assessed in (M) the open field (ctr: n = 14 mice; nKO: n = 12 mice) and (N) RotaRod paradigm (ctr: n = 16 mice; nKO: n = 12 mice). Cognitive function was tested in (O) the Y-maze (ctr: n = 11 mice; nKO: n = 11 mice) and (P, Q) Barnes maze test (ctr: n = 11 mice; nKO: n = 11 mice). Representative heatmaps of Barnes maze test are reported on the right. The time spent in target (T) and nontarget quadrants during the probe trial are shown on the right. Data information: Data are presented as mean ± SEM. In (B) and (D), Mann–Whitney test was used while in (H), (J), (K), (M), and (O), unpaired two-tailed Student's t test was used. Data in (C) and (E) were analyzed via Kruskal–Wallis test. In (F), (I), (N), (P), and (Q), two-way ANOVA, with repeated measures where appropriate, were done followed by Bonferroni's post hoc test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available online for this figure.

Fig 3: BCL7A KO impairs mitochondrial bioenergetics AHeatmap of significantly dysregulated genes in BCL7A KO compared to wt eNPCs, which were exposed to differentiation medium for 48 h.BIPA of up- and down-regulated genes and relative prediction of the top 10 most significantly overrepresented canonical pathways.CHeatmap of significantly dysregulated mitochondrial genes in BCL7A KO vs. wt eNPCs. Cells were exposed for 48 h to differentiation medium before RNAs were extracted.D(D1) Genome browser screenshots of SMARCA4/BRG1 ChIP-seq signals in eNPCs (left hand panels) and smNPCs (right hand panels). Peaks show the reduced SMARCA4/BRG1 occupancy at representative mitochondrial genes (i.e., Ndufc2, Cox6c, Gls/GLS) in control and BCL7A KO cells. (D2) Overlap between SMARCA4/BRG1-bound genes, DEGs and mitochondrial genes identified in MitoCarta 3.0. (D3-4) Genome browser screenshots of SMARCA4/BRG1 ChIP-seq signals in (D3) eNPCs and (D4) smNPCs. Peaks show the reduced SMARCA4/BRG1 occupancy at transcription factors known to be involved in mitochondrial biogenesis.E, FOCR measurements in in control and BCL7A KO (E) eNPCs and (F) smNPCs. On the right, it is reported the maximal respiration following treatment with the mitochondrial uncoupler FCCP (n = 5 biological replicates).GProximity ligation assay (PLA) for the OXPHOS subunits NDUFB8 and MTCO1 in adult hippocampal sections from Bcl7a fl/fl ; Baf53b-Cre tg/wt (ko; n = 4) and control littermates (ctr; n = 4). Sections were co-stained with TOM20 and NeuN to label mitochondria and neurons, respectively. The total number of PLA dots was normalized to the mitochondrial area (panel on the lower right). Scale bars = 10 µm. Data information: Data in (E–G) are presented as mean ± SEM. In (E–G), unpaired two-tailed Student's t test and one-way ANOVA followed by Bonferroni's post hoc test for multiple comparisons was used. *P < 0.05, **P < 0.01.

Fig 4: BCL7A is upregulated during NPC specification ASchematic representation of stem cell differentiation into neural progenitors and subsequently into neurons.BGraphical summary of SWI/SNF/BAF complex subunits that are part of the ATPase (orange) or core (dark blue) modules. Highlighted in yellow are the subunits that have been assessed by immunoblot analyses.C, DRepresentative western blots and respective densitometries (right panel) for the selected SWI/SNF/BAF complex subunits (n = 3–5 biological replicates). Arrow heads indicate nonspecific bands, while # symbol highlights BAF53B.ESchematic representation of nucleosome-bound SWI/SNF/BAF complex. BCL7A is stably incorporated in the ATPase module, with amino acids residues at the N-terminus of BCL7A interacting with BRM/BRG1 or with H2A/H2B dimers.FRepresentative immunoblots of co-immunoprecipitation (co-IP) experiments in eNPCs (top panels) and smNPCs (bottom panels). Co-IPs were performed with antibodies against BAF170 (top) and BRG1 (bottom). Inputs are homogenates from eNPCs (left) and smNPCs (right). IgG indicates IgG-bound Sepharose beads (as controls), while IP indicates immunoprecipitated samples after elution from the beads. Arrow head indicates BCL7A band at the correct MW.GImmunofluorescence staining of BCL7A (purple) along with actin, nestin, and ß-III tubulin (green) as well as GFAP (white) in iPSCs, smNPCs, and smNPC-derived neurons and astrocytes, respectively. Arrow heads indicate GFAP-positive glial cells with low BCL7A expression. Scale bars = 20 µm.HWestern blot analysis of BCL7A and BAF170 in cortex and hippocampus of embryonic (E17.5), early postnatal (PD0 and PD7), and adult (PD120) mouse brain tissue showing a progressive temporal reduction of protein levels in the analyzed tissues.IImmunofluorescence staining of BCL7A (purple), EdU (green), and NeuN (white) in embryonic (E17.5) and adult (PD120) hippocampal brain sections. EdU was used to label proliferating NPCs, whereas NeuN was used as a neuronal marker. Scale bars = 50 µm (left panels) and 10 µm (right panels). Data information: Data are presented as mean ± SEM. Kruskal–Wallis test was used in (C) and (D): *P < 0.05, **P < 0.01. Source data are available online for this figure.

Fig 5: Lack of BCL7A impairs neurogenesis and reduces neurite complexity of differentiating neurons in vitro and in vivo Immunoblots of spontaneously differentiated wt and BCL7A KO eNPCs. Samples were collected at the indicated differentiation time points. Respective densitometries are shown on the right (n = 6–7 biological replicates).Sholl analysis of ß-III tubulin-positive immature wt and BCL7A KO neurons following 7 days of spontaneous eNPC differentiation (n = 4 biological replicates, each experiment with 10–15 cells per condition). Scale bar = 20 µm.Immunofluorescence staining (central panel) and quantification (right panel) of ß-III tubulin- and GFAP-positive cells as well as RT-PCR analysis (right panels) of spontaneously differentiated iPSC-derived parental and BCL7A KO smNPCs (n = 3–7 biological replicates). Scale bar = 50 µm.Immunofluorescence staining of BCL7A expression in hippocampal sections from adult control and Bcl7a fl/fl ; Nestin-Cre tg/wt mice. Scale bar = 200 µm.EdU experiments in adult mice. Animals were injected with EdU twice daily for 3 days and sacrificed 21 days thereafter. Images show immunofluorescence stainings for EdU (green), doublecortin (DCX, white), and S100ß (red) in hippocampal brain sections from Bcl7a wt/wt ; Nestin-Cre tgt/wt (as control) and Bcl7a fl/fl ; Nestin-Cre tg/wt mice. Representative EdU+ DCX+ (yellow arrow heads) or EdU+ S100ß+ (yellow asterisk) double-labeled cells are indicated in the insets. Scale bar = 50 and 20 µm (for insets).Quantification of EdU+ (left panel), EdU+ DCX+ (middle panel), and EdU+ S100ß+ (right panel) cells within the hippocampal dentate gyrus (DG) of adult control (n = 4) and Bcl7a fl/fl ; Nestin-Cre tg/wt (n = 5) animals.Immunofluorescence staining for DCX+ cells within the DG region of adult Bcl7a fl/fl ; Nestin-Cre wt/wt (as control) and Bcl7a fl/fl ; Nestin-Cre tg/wt animals. Confocal imaging analysis shows a lower number, misalignment, and reduced neuritic complexity of DCX+ labeled cells in BCL7A KO mice compared to controls. Yellow arrows indicate misaligned DCX+ cells. Scale bar = 50 µm.Immunofluorescence staining of BCL7A (red) of hippocampal sections from Bcl7a fl/fl ; Baf53b-Cre wt/wt (as control) and Bcl7a fl/fl ; Baf53b-Cre tg/wt mice. Scale bar = 200 µm.Immunofluorescence staining of BCL7A (red) and DCX (green) in hippocampal sections of control and Bcl7a fl/fl ; Baf53b-Cre tg/wt mice. Yellow arrow indicates dividing cells, whereas yellow asterisks mark differentiating immature neurons. Scale bar = 5 µm.Immunofluorescence staining for DCX (white) within the DG region of adult control and Bcl7a fl/fl ; Baf53b-Cre tg/wt mice. Representative images show no obvious difference in number, morphology or alignment of DCX+ labeled cells. Quantification of DCX+ labeled cells in control and Bcl7a fl/fl ; Baf53b-Cre tg/wt mice is shown on the right (n = 3 per genotype). Scale bar = 50 µm. Data information: Data are presented as mean ± SEM. In (A) and (B), two-way ANOVA and two-way RM ANOVA, respectively, were used followed by Bonferroni's post hoc test for multiple comparisons. One-way ANOVA was used in (C) while in (F) and (J), unpaired two-sided Student's t test was used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available online for this figure.