Fig 2: Mapping cortical projection output in Snf2h cKO. (A) Insertion of lipophilic tracers and diffusion throughout the brain. The left panel shows a wild type mouse brain 1 h after insertion in the pia mater of a single DiA crystal in the right motor cortex area and a single Dil crystal in the right somatosensory cortex area. The right panel shows the same brain 1 week after diffusion of both neuronal tracers. The dorsoventral (d and v) and mediolateral (m and l) orientations are indicated. (B) Representative epifluorescent images of both lipophilic tracers in P7 brains after 4 weeks of incubation. Dil+ (red) synaptic terminals were visualized within control brains in the thalamus, CC, and brainstem (left panels), but these were reduced or almost absent in the mutant brains (right panels). DiA+ (green) projections toward the hypothalamus (top panels) and the projection output from the cerebral cortex (middle panels) were also reduced within the mutant brain. Rightmost panels: Relative orientation of the dorsoventral (d and v) and the anteroposterior (a and p) brain. A schematic representation of the whole brain in a sagittal section is shown to highlight the corpus callosum (CC), thalamus (Th), and caudate putamen (Cp). Scale bars, 50 µm.

Fig 3: A reduced neocortex is observed in Snf2h cKO mice. (A) Adult brains (P40) from Snf2h cKO mice show a gross reduction in the size of the neocortex (Cx) compared to WT littermates, but no apparent difference in the size of the cerebellum (Cb). (A’) Cresyl violet stained midline sagittal sections of P60 WT and Snf2h cKO brains highlight the reduced size of the mutant cortex. Relative thickness differences are shown above the dotted lines that depict the location of dorsal-ventral (D-V) and anterior-posterior (A-P) positions used for analysis. (B) Cresyl violet stained coronal sections from WT and Snf2h cKO brains throughout neurogenesis (E12.5-E15.5) and at birth (P0). Scale bar, 100 µm. (B’) Quantification of cortical thickness at different ages indicates that the reduced size of the Snf2h cKO neocortex becomes apparent at E15.5. (C) Normalized RT-qPCR expression of the ISWI genes (Smarca5 and Smarca1l), and key transcription factors involved in neocortical development (FoxG1, Cux1, Satb2, Ctip2, Tbr1, Tbr2, and Ebf3) from mutant cortical samples relative to WT samples at E15.5. Mean values were used to calculate the fold change differences relative to control levels. *P < 0.05, **P < 0.01, n = 4, Student’s t-test. (D) Immunoblots for cortical layer markers in neocortical extracts prepared from E15.5 brains. Vinculin was used as loading control. Values denote averaged pixel densitometry normalized to Vinculin and relative to control samples, n = 3 mice per genotype.

Fig 4: Nuclear abnormalities in S5KO cells. (A) Cytology of control (left) and S5KO clone #D7 (right), nuclear abnormalities indicated and shown (B) as mean % ± Stdev of control, 400 cells/subclone analyzed. Student’s t-test, p < 0.05 *, p < 0.01 **, p < 0.0001 ****. (C) Immunoblotting of Smarca5: MEF cell lines (Smarca5fl/fl Cre-Esr1: untreated, 4OHT-treated (100 nM, 6 h exposure, 4 days of culture). ß-actin = loading control. (D) IncuCyte cell proliferation analysis; control Smarca5fl/fl (upper panel) vs. Smarca5fl/fl Cre-Esr1 (lower panel) MEFs in absence/presence of 4OHT (100 nM, 6 h exposure), or alternatively, 4OHT was added 72 h prior to IncuCyte monitoring (4OHT—72 h). Y-axis: mean confluency (%) and ± Stdev of at least 16 different regions of the cultivation plate, X-axis: time (h). (E) Flow cytometry analysis of control and Smarca5fl/fl Cre-Esr1 MEF population cell cycle progression using EdU/DAPI double staining (upper dot plots). Black rectangles depict all S-phase and non-S-phase cells with different ploidy (2N-16N). Histograms show percentage of phospho-histone H3 (Ser10) positive mitotic events in experimental cell lines. (D) and (E) represent biological triplicates.

Fig 5: Decreased +1 nucleosome positioning under SNF2H shortage. (A) c-Myc-C and c-Myc-601F mRNA levels in the indicated conditions analyzed by RT-qPCR. Values were normalized as in Figure 2D and c-Myc-C was set to 1. (B) RNAPII ChIP analysis of c-Myc-C (left panel) and c-Myc-601F (right panel) constructs under SNF2H knockdown conditions. Total RPB1 ChIP was performed as described in Figure 2B by using N-20 antibody. (C) Micrococcal nuclease mapping of promoter and 5' region of the c-Myc-C (left panel) and c-Myc-601F (right panel) genes. Data were analyzed as described in Figure 1B. siControl value at position c-Myc-601F +203 was set to 1. (A–C) Average signals and standard deviations from three independent experiments are displayed (*P < 0.05; **P < 0.01, between the indicated samples by student's t-test). Primer sequences are listed in Supplementary Table S1.