Journal: Cell Death & Disease

Article Title: Loss of FAM60A disrupts Sin3/HDAC control of the Hippo signaling and promotes oncogenic YAP1 activation

doi: 10.1038/s41419-026-08778-y

Figure Lengend Snippet: A A schematic of a refined proteomics workflow employing Mass spectrometry for protein identification. B HEK293T cells were transfected with Halo-SIN3A, and the cell lysates were subjected to affinity purification with Halo magnetic beads. Purified protein complexes were identified by Mass Spectrometry and analyzed using QSPEC (QSPEC log2FC ≥ 1, QSPEC FDR < 0.05). The results were then visualized in a differential network using Cytoscape. C Halo-SIN3A or Halo plasmid alone was ectopically expressed in HEK293T cells, followed by Halo affinity purification. The Halo affinity-purified proteins were subjected to immunoblotting with anti-FAM60A, anti-HDAC1, anti-SUDS3, anti-RBBP7, anti-RBBP4, and anti-SIN3A antibodies. Total cell lysates were also analyzed by immunoblotting using the same antibodies and anti-GAPDH as a loading control. D Identified subunits of Sin3/HDAC complex were N-terminally Halo-tagged and ectopically expressed in HEK293 cells, followed by affinity purification and identified using Mass-spectrometry analysis (QSPEC log2FC ≥ 1, FDR < 0.05) and visualized in a heatmap.

Article Snippet: Antibodies used in this study were sourced as follows: RbAp46/RBBP7 (A22274), RBBP4 (A3645), and SIN3A (A21966) from ABclonal, USA; Halo-Tag from Promega, USA (G9211); SAP30 (27679-1-AP) and SUDS3 (25845-1-AP) from Proteintech, USA; FAM60A from MRC PPU, Dundee University, UK (Cat # S381C).

Techniques: Mass Spectrometry, Transfection, Affinity Purification, Magnetic Beads, Purification, Plasmid Preparation, Western Blot, Control

Journal: Cell Death & Disease

Article Title: Loss of FAM60A disrupts Sin3/HDAC control of the Hippo signaling and promotes oncogenic YAP1 activation

doi: 10.1038/s41419-026-08778-y

Figure Lengend Snippet: A Schematic diagram illustrates various deletions in the FAM60A gene. B Halo-tagged FAM60A deletion mutants were transfected into HEK293T cells, labeled with Halo-Tag TMRDirect fluorescent ligand (red), and DNA was stained using Hoechst dye (blue). C Halo-FAM60A deletion mutants were expressed in HEK293T cells, purified, and analyzed via proteomics (QSPEC log2FC ≥ 1, FDR < 0.05). Results were displayed in a heatmap. D The interactions of full-length and truncated FAM60A with Sin3/HDAC complex core-subunits were visualized in a heatmap (grey indicates no interaction). E Heatmap showing FAM60A conserved motif-specific interactions with Sin3/HDAC complex core-subunits (Grey signify no interaction).

Article Snippet: Antibodies used in this study were sourced as follows: RbAp46/RBBP7 (A22274), RBBP4 (A3645), and SIN3A (A21966) from ABclonal, USA; Halo-Tag from Promega, USA (G9211); SAP30 (27679-1-AP) and SUDS3 (25845-1-AP) from Proteintech, USA; FAM60A from MRC PPU, Dundee University, UK (Cat # S381C).

Techniques: Transfection, Labeling, Staining, Purification

Journal: Cell Death & Disease

Article Title: Loss of FAM60A disrupts Sin3/HDAC control of the Hippo signaling and promotes oncogenic YAP1 activation

doi: 10.1038/s41419-026-08778-y

Figure Lengend Snippet: A Workflow for Halo-FAM60A-expressing HEK293 cell extract preparation, Halo-Tag protein purification, size exclusion chromatography, and AP-MS analysis. B Halo-FAM60A affinity-purified proteins were fractionated through size-exclusion chromatography and analyzed by LC-MS and visualized in a heatmap (Red and Black). C HDAC1-Halo or Halo-HDAC2, or Halo-SUDS3 was ectopically expressed in HEK293T cells. Halo affinity purification followed by mass spectrometry analysis showed that FAM60A and SIN3A copurified with HDAC1-Halo and Halo-SUDS3 but not with Halo-HDAC2. D – F HDAC1-Halo or Halo-HDAC2, or Halo-SUDS3 or Halo plasmid alone, was ectopically expressed in HEK293T cells. Halo affinity purification was performed, followed by immunoblot analysis using specific antibodies and GAPDH as a loading control. G Whole-cell lysates from HEK293 cells were subjected to immunoprecipitation using an anti-FAM60A antibody, followed by immunoblotting with antibodies against HDAC1, HDAC2, SUDS3, and Sin3A. H HDAC1 and SUDS3 were individually or combinatorially knocked out using CRISPR/Cas9. Subsequently, either the Halo tag alone or Halo-FAM60A was overexpressed, and protein complexes were purified by affinity purification. These complexes were then subjected to immunoblotting analysis and probed with various antibodies.

Article Snippet: Antibodies used in this study were sourced as follows: RbAp46/RBBP7 (A22274), RBBP4 (A3645), and SIN3A (A21966) from ABclonal, USA; Halo-Tag from Promega, USA (G9211); SAP30 (27679-1-AP) and SUDS3 (25845-1-AP) from Proteintech, USA; FAM60A from MRC PPU, Dundee University, UK (Cat # S381C).

Techniques: Expressing, Protein Purification, Size-exclusion Chromatography, Protein-Protein interactions, Affinity Purification, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Plasmid Preparation, Western Blot, Control, Immunoprecipitation, CRISPR, Purification

Journal: bioRxiv

Article Title: Hierarchical regulation of cerebellar neurogenesis by Sin3A-mediated gene repression

doi: 10.1101/2025.10.17.683101

Figure Lengend Snippet: (A) External views (left) and H&E stained midsagittal sections (right) from control and Sin3A CKO mice at postnatal day 40 (P40), showing a reduction in cerebellar size and foliation. Scale bars indicate 250 μm. (B) Immunofluorescence staining for Cre recombinase at P0 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Scale bar indicates 250 μm. (C) Immunofluorescence staining for Ki67 and NeuN at P7 in the cerebellum of control and Sin3A CKO mice. A series of sections from the lateral to medial cerebellum revealing significant variability in Sin3A CKO across different sagittal planes, attributable to disorganization within the tissue architecture. The dashed lines delineate the boundaries of different layers within the cerebellar cortex. EGL: external granular layer, ML: molecular layer, IGL: internal granular layer, PWM: prospective white matter. Arrows indicate acellular gaps in EGL. Arrowheads indicate ectopic clusters of GCPs. Scale bar indicates 250 μm. (D and E) Quantification of the cross-sectional area of EGL (D) and the number of Ki67 + cells (E) in EGL. Shown are mean ± SEM. Two-tailed t test: ** p ≤ 0.01, *** p ≤ 0.001, n.s. p>0.05. n= 3 mice per group. (F and G) Quantification of the cross-sectional area of IGL (F) and the number of NeuN + cells (G) in IGL. Shown are mean ± SEM. Two-tailed t test: ** p ≤ 0.01, *** p ≤ 0.001, n.s. p>0.05. n= 3 mice per group.

Article Snippet: Primary antibodies used in this study are listed below: GFP (1:1000, Aves Labs, cat#: GFP-1020), Ki67 (1:1000, Thermo Fisher Scientific, cat#: 14-5698-82), Cdkn1b (1:500, Cell Signaling Technology, cat#: 3698), NeuroD1 (1:1000, Cell Signaling Technology, cat#: 62953), Sin3A (1:200, Novus Biologicals, cat#: NB600-1263), Sox2 (1:500, Thermo Fisher Scientific, cat#: 740013T), Ccnd1 (1:300, Cell Signaling Technology, cat#: 55506), Cleaved Caspase-3 Asp175 (1:200, Cell Signaling Technology, cat#: 9664), Cre Recombinase (1:500, Cell Signaling Technology, cat#: 15036), NeuN (1:500, Cell Signaling Technology, cat#: 24307).

Techniques: Staining, Control, Immunofluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Hierarchical regulation of cerebellar neurogenesis by Sin3A-mediated gene repression

doi: 10.1101/2025.10.17.683101

Figure Lengend Snippet: (A) Ki67 and EdU staining in cerebellum of control and Sin3A CKO mice at P0 and P7. Nuclei were counterstained with DAPI. Mice were administrated with EdU 30 minutes before sacrifice. Arrowheads indicate cells positive for both Ki67 and EdU. Scale bars indicate 250 μm. (B) Quantification of the percentage of Ki67 + cells relative to the total number of DAPI + cells in EGL at P0 and P7. Shown are mean ± SEM. Two-tailed t test: ** p ≤ 0.01, n.s. p>0.05. n= 3 mice per group. (C) Quantification of the percentage of EdU + /Ki67 + double-positive cells relative to the total number of Ki67 + cells in EGL at P0 and P7. Shown are mean ± SEM. Two-tailed t test: ** p ≤ 0.01, n.s. p>0.05. n= 3 mice per group. (D) Immunofluorescence staining for p27 at P0 and NeuN at P7 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. The dashed lines delineate the outer margin of the EGL. Scale bars indicate 250 μm. (E) Quantitative analysis showing the percentage of p27 + or NeuN + cells relative to the total cell population in the EGL. Shown are mean ± SEM. Two-tailed t test: * p ≤ 0.05, ** p ≤ 0.01. n= 3 mice per group. (F) Ki67, NeuN and EdU staining in the cerebellum of control and Sin3A CKO mice at P7. Nuclei were counterstained with DAPI. Mice were administrated EdU 48 hour before sacrifice. Arrowheads indicate cells positive for both NeuN and EdU, and arrows indicate cells positive for both Ki67 and EdU. The dashed lines delineate the boundaries of different layers within the cerebellar cortex. Scale bars indicate 250 μm. (G) Quantification of the percentage of EdU + /NeuN + double-positive cells relative to the total number of EdU + cells. Shown are mean ± SEM. Two-tailed t test: *** p ≤ 0.001. n= 3 mice per group. (H) Immunofluorescence analysis for Ki67 at P15 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Scale bars indicate 250 μm. (I) Immunofluorescence staining for cleaved caspase-3 (CP3) and Ki67 at P0 and P7 in the cerebellum of control and Sin3A CKO mice. Arrowheads indicate CP3 + cells. Scale bars indicate 250 μm. (J) Quantification of the CP3 + cells in the EGL. Shown are mean ± SEM. Two-tailed t test: * p ≤ 0.05. n= 3 mice per group.

Article Snippet: Primary antibodies used in this study are listed below: GFP (1:1000, Aves Labs, cat#: GFP-1020), Ki67 (1:1000, Thermo Fisher Scientific, cat#: 14-5698-82), Cdkn1b (1:500, Cell Signaling Technology, cat#: 3698), NeuroD1 (1:1000, Cell Signaling Technology, cat#: 62953), Sin3A (1:200, Novus Biologicals, cat#: NB600-1263), Sox2 (1:500, Thermo Fisher Scientific, cat#: 740013T), Ccnd1 (1:300, Cell Signaling Technology, cat#: 55506), Cleaved Caspase-3 Asp175 (1:200, Cell Signaling Technology, cat#: 9664), Cre Recombinase (1:500, Cell Signaling Technology, cat#: 15036), NeuN (1:500, Cell Signaling Technology, cat#: 24307).

Techniques: Staining, Control, Two Tailed Test, Immunofluorescence

Journal: bioRxiv

Article Title: Hierarchical regulation of cerebellar neurogenesis by Sin3A-mediated gene repression

doi: 10.1101/2025.10.17.683101

Figure Lengend Snippet: (A) Schematic representation of the strategy employed to isolate cells followed by high-throughput sequencing analysis. (B) Uniform Manifold Approximation and Projection (UMAP) visualization of cell populations from the P6 cerebellum of two Sin3A CKO and two control mice. UBC: unipolar brush cells, PC: purkinje cells, Oligo: oligodendrocytes, IN: GABAergic interneurons, Golgi: golgi cells, AC: astrocytes, BG: bergmann glial cells, (C) UMAP representation of the results from Milo differential abundance (DA) testing between Sin3A CKO and control cerebellum with nodes showing cell neighborhoods. Nodes with significant differential abundance are highlighted using a gradient scale of red or blue. (D) Beeswarm plot showing the log-fold change between Sin3A CKO and control cerebellum across groups of nearest-neighbor cells from various cell type clusters. Significant changes are highlighted using a gradient scale of red or blue. (E) Volcano plot illustrating the differential gene expression results between the GCP clusters of control and Sin3A CKO cerebellum. (F) Heatmap and average intensity plots of the Hdac1 ChIP-seq signals across 10 kb from the center of Sin3A peaks throughout the genome the genome from dissociated cerebellar cells of control mice. (G) Heatmap and average intensity plots of the H3K27ac ChIP-seq signals across 10 kb from the center of Sin3A peaks throughout the genome from isolated GCPs of control and Sin3A CKO mice. (H) Pie chart and Venn diagram illustrating the process of screening and identifying target genes of Sin3A. (I) Bar chart showing the results of the functional enrichment analysis for 96 candidate target genes.

Article Snippet: Primary antibodies used in this study are listed below: GFP (1:1000, Aves Labs, cat#: GFP-1020), Ki67 (1:1000, Thermo Fisher Scientific, cat#: 14-5698-82), Cdkn1b (1:500, Cell Signaling Technology, cat#: 3698), NeuroD1 (1:1000, Cell Signaling Technology, cat#: 62953), Sin3A (1:200, Novus Biologicals, cat#: NB600-1263), Sox2 (1:500, Thermo Fisher Scientific, cat#: 740013T), Ccnd1 (1:300, Cell Signaling Technology, cat#: 55506), Cleaved Caspase-3 Asp175 (1:200, Cell Signaling Technology, cat#: 9664), Cre Recombinase (1:500, Cell Signaling Technology, cat#: 15036), NeuN (1:500, Cell Signaling Technology, cat#: 24307).

Techniques: Next-Generation Sequencing, Control, Gene Expression, ChIP-sequencing, Isolation, Functional Assay

Journal: bioRxiv

Article Title: Hierarchical regulation of cerebellar neurogenesis by Sin3A-mediated gene repression

doi: 10.1101/2025.10.17.683101

Figure Lengend Snippet: (A) Immunofluorescence staining for Sox2, Atoh1-GFP, and NeuroD1 at E13.5 in the cerebellum of control and Sin3A CKO mice. Arrowheads indicate cells positive for Sox2 and Atoh1 in the EGL. Scale bar indicates 250 μm. (B) Immunofluorescence staining for Sox2, Atoh1-GFP, and NeuroD1 at E16.5 in the cerebellum of control and Sin3A CKO mice. Arrowheads indicate cells positive for Sox2 and Atoh1, and arrows indicate cells positive for only Sox2. Scale bar indicates 250 μm. (C) Immunofluorescence staining for Sox2 and Atoh1-GFP at P0 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Arrowheads indicate cells positive for Sox2 and Atoh1, and arrows indicate cells positive for only Sox2. Scale bar indicates 250 μm. (D) Immunofluorescence staining for Sox2 and Atoh1-GFP at P7 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Arrowheads indicate cells positive for Sox2 and Atoh1, and arrows indicate cells positive for only Sox2. Scale bar indicates 250 μm. (E) Quantification of the Sox2 + ; Atoh1-GFP + cells relative to the Atoh1-GFP + population in the medial and anterior EGL. Shown are mean ± SEM. Two-tailed t test: * p≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, n.s. p>0.05. n= 3 mice per group. (F) Diagram illustrating the developmental model transitioning from multipotent ECPs to rapidly proliferating GCPs. (G) UMAP displaying the developmental trajectory of GC lineages from E12 to E16 wildtype mice, based on pseudotime analysis. (H) UMAP displaying the Sox2 and Atoh1 expression in the GC lineage cell of E12 to E16 wildtype mice. (I) Quantification of cells counts in the medial and anterior EGL of E16.5, P0 and P7 cerebellum. Shown are mean ± SEM. Two-tailed t test: * p≤ 0.05, ** p ≤ 0.01. n= 3 or 4 mice per group.

Article Snippet: Primary antibodies used in this study are listed below: GFP (1:1000, Aves Labs, cat#: GFP-1020), Ki67 (1:1000, Thermo Fisher Scientific, cat#: 14-5698-82), Cdkn1b (1:500, Cell Signaling Technology, cat#: 3698), NeuroD1 (1:1000, Cell Signaling Technology, cat#: 62953), Sin3A (1:200, Novus Biologicals, cat#: NB600-1263), Sox2 (1:500, Thermo Fisher Scientific, cat#: 740013T), Ccnd1 (1:300, Cell Signaling Technology, cat#: 55506), Cleaved Caspase-3 Asp175 (1:200, Cell Signaling Technology, cat#: 9664), Cre Recombinase (1:500, Cell Signaling Technology, cat#: 15036), NeuN (1:500, Cell Signaling Technology, cat#: 24307).

Techniques: Immunofluorescence, Staining, Control, Two Tailed Test, Expressing

Journal: bioRxiv

Article Title: Hierarchical regulation of cerebellar neurogenesis by Sin3A-mediated gene repression

doi: 10.1101/2025.10.17.683101

Figure Lengend Snippet: (A) Bulk RNA-seq tracks (reads per million values) showing the expression level of Atoh1 in the GCPs of control and Sin3A CKO mice. ChIP-seq tracks (fold changes of ChIP-seq signal relative to IgG) depicting the intensity of Sin3A, Hdac1, and H3K27ac occupation at the Sox2 locus in the dissociated cerebellar cells or isolated GCPs. The italicized text to the left of each track denotes the genotype of the mice used in ChIP-seq experiments; absence of genotype annotation indicates a wild-type or Atoh1 M1GFP/+ animal. (B) Violin plots showing the expression of Sox2 in the GCP population of Sin3A CKO and control cerebellum. (C) Splitted UMAP displaying the Sox2 expression in the sub-clusters of GCPs between Sin3A CKO and control. (D) Sox2, Atoh1-GFP, and EdU staining in the cerebellum of control and Sin3A CKO mice at P0. Mice were administrated with EdU 30 minutes before sacrifice. Arrowheads indicate cells positive for both Sox2 and EdU, and arrows indicate cells positive for both Atoh1-GFP and EdU. Scale bar indicates 250 μm. (E) Quantitative analysis showing the percentage of EdU + cells relative to the Sox2 + ; Atoh1-GFP + or Atoh1-GFP + population in the EGL. Shown are mean ± SEM. Two-tailed t test: * p≤ 0.05, *** p ≤ 0.001. n= 3 mice per group. (F) Schematic representation of the strategy employed to overexpress Sox2 using electroporation. (G) NeuroD1, tdTomato and Sox2 staining in the cerebellum of electroporated mice harvested at P5. Arrowheads indicate tdTomato + cells in the EGL. Scale bar indicates 20 μm. (H) Quantitative analysis showing the percentage of EdU + cells relative to the tdTomato + ; Ki67 + population in the EGL. Shown are mean ± SEM. Two-tailed t test: * p≤ 0.05. n= 3 mice per group. (I) tdTomato and EdU staining in the cerebellum of electroporated mice at P5. Mice were administrated with EdU 30 minutes before sacrifice. Arrowheads indicate tdTomato + cells in the EGL. Scale bar indicates 20 μm.

Article Snippet: Primary antibodies used in this study are listed below: GFP (1:1000, Aves Labs, cat#: GFP-1020), Ki67 (1:1000, Thermo Fisher Scientific, cat#: 14-5698-82), Cdkn1b (1:500, Cell Signaling Technology, cat#: 3698), NeuroD1 (1:1000, Cell Signaling Technology, cat#: 62953), Sin3A (1:200, Novus Biologicals, cat#: NB600-1263), Sox2 (1:500, Thermo Fisher Scientific, cat#: 740013T), Ccnd1 (1:300, Cell Signaling Technology, cat#: 55506), Cleaved Caspase-3 Asp175 (1:200, Cell Signaling Technology, cat#: 9664), Cre Recombinase (1:500, Cell Signaling Technology, cat#: 15036), NeuN (1:500, Cell Signaling Technology, cat#: 24307).

Techniques: RNA Sequencing, Expressing, Control, ChIP-sequencing, Isolation, Staining, Two Tailed Test, Electroporation

Journal: bioRxiv

Article Title: Hierarchical regulation of cerebellar neurogenesis by Sin3A-mediated gene repression

doi: 10.1101/2025.10.17.683101

Figure Lengend Snippet: (A) Bulk RNA-seq tracks (reads per million values) showing the expression level of Atoh1 in the GCPs of control and Sin3A CKO mice. ChIP-seq tracks (fold changes of ChIP-seq signal relative to IgG) depicting the intensity of Sin3A, Hdac1, and H3K27ac occupation at Atoh1 locus in the dissociated cerebellar cells or isolated GCPs. The italicized text to the left of each track denotes the genotype of the mice used in ChIP-seq experiments; absence of genotype annotation indicates a wild-type or Atoh1 M1GFP/+ animal. Enhancers A and B are two critical regulatory elements located downstream of the Atoh1 coding region, identified through reported gene assay and cross-species conservation sequence analysis . (B) Immunofluorescence staining for Atoh1-Cre at P0 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Scale bar indicates 250 μm. (C) Immunofluorescence staining for Atoh1-GFP and Ki67 at P7 and P15 in the cerebellum of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Scale bar indicates 250 μm. (D) Boxplot showing the quantification of the relative fluorescence intensity of GFP or Cre in EGL cells of control and Sin3A CKO mice. Two-tailed t test: **** p ≤ 0.0001. n= 200 cells per group. (E) Flow cytometry analysis of GCPs isolated from Atoh1-Cre; Atoh1 M1GFP/+ and Sin3A CKO ; Atoh1 M1GFP/+ mice. Cells were dissociated from the anterior regions of the cerebellum in P7 mice and subsequently sorted based on endogenous GFP expression. (F) Single-molecule fluorescence in situ hybridization (smFISH) analysis showing the transcription levels of Atoh1 in EGL of control and Sin3A CKO mice. Nuclei were counterstained with DAPI. Scale bar indicates 20 μm. (G) Quantification of the count of Atoh1 transcripts per 100 square micrometers. Shown are mean ± SEM. Two-tailed t test: ** p≤ 0.01. n= 3 mice per group.

Article Snippet: Primary antibodies used in this study are listed below: GFP (1:1000, Aves Labs, cat#: GFP-1020), Ki67 (1:1000, Thermo Fisher Scientific, cat#: 14-5698-82), Cdkn1b (1:500, Cell Signaling Technology, cat#: 3698), NeuroD1 (1:1000, Cell Signaling Technology, cat#: 62953), Sin3A (1:200, Novus Biologicals, cat#: NB600-1263), Sox2 (1:500, Thermo Fisher Scientific, cat#: 740013T), Ccnd1 (1:300, Cell Signaling Technology, cat#: 55506), Cleaved Caspase-3 Asp175 (1:200, Cell Signaling Technology, cat#: 9664), Cre Recombinase (1:500, Cell Signaling Technology, cat#: 15036), NeuN (1:500, Cell Signaling Technology, cat#: 24307).

Techniques: RNA Sequencing, Expressing, Control, ChIP-sequencing, Isolation, Gene Assay, Sequencing, Immunofluorescence, Staining, Fluorescence, Two Tailed Test, Flow Cytometry, In Situ Hybridization

Journal: bioRxiv

Article Title: Hierarchical regulation of cerebellar neurogenesis by Sin3A-mediated gene repression

doi: 10.1101/2025.10.17.683101

Figure Lengend Snippet: (A) Venn diagram illustrating the identification of genomic loci directly regulated by the Sin3A/Hdac1 complex. (B) Heatmap and average intensity plots illustrating the H3K27ac ChIP-seq signal profiles surrounding 3439 genomic loci directly regulated by the Sin3A/Hdac1 complex in isolated GCP from both control and Sin3A CKO mice. (C) Selected motifs exhibit significant enrichment at genomic loci that are directly regulated by the Sin3A/Hdac1 complex. (D) Immunofluorescence staining for Atoh1-GFP and NeuroD1 at P6 in the cerebellum of control and Sin3A CKO mice. Scale bar indicates 250 μm. (E) Venn diagram illustrating the overlaps of Sin3A, Hdac1 and NeuroD1 binding sites in the genome of wild type cerebellum. (F) ChIP-seq tracks (fold changes of ChIP-seq signal relative to IgG) depicting the intensity of Sin3A, Hdac1, NeuroD1, and H3K27ac occupation at Atoh1 locus in the GCP of control, Sin3A CKO , and NeuroD1 LacZ/LacZ mice. The italicized text to the left of each track denotes the genotype of the mice used in ChIP-seq experiments, absence of genotype annotation indicates a wild-type animal. (G) Immunoblots for NeuroD1, Sin3A and Hdac1 binding in P7 cerebellum of wild-type mice. Lane 1-3: cell lysate of P7 cerebellum. Lane 4: Co-immunoprecipitation of rabbit IgG in cell lysate of P7 cerebellum. Lane 5: Co-immunoprecipitation of NeuroD1 in cell lysate of P7 cerebellum. (H) Immunofluorescence staining for Atoh1-GFP, NeuN, and Ki67 at P6 in the cerebellum of control and NeuroD1 LacZ/LacZ mice. Nuclei were counterstained with DAPI. Scale bar indicates 20 μm. (I) Boxplot showing the quantification of the relative fluorescence intensity of GFP in EGL cells of control and NeuroD1 lacZ/lacZ mice. Two-tailed t test: **** p ≤ 0.0001. n= 200 cells per group. (J) smFISH analysis showing the transcription levels of Atoh1 in the EGL of wild type and NeuroD1 lacZ/lacZ mice. Nuclei were counterstained with DAPI. Scale bar indicates 20 μm. (K) Quantification of the count of Atoh1 transcripts per 100 square micrometers in the EGL of wild type and NeuroD1 lacZ/lacZ mice. Shown are mean ± SEM. Two-tailed t test: ** p≤ 0.01. n= 3 mice per group. (L) Heatmap and average intensity plots of the H3K27ac ChIP-seq signals across 10 Kb from the center of Sin3A/Hdac1/NeuroD1 binding sites throughout the genome from isolated GCPs of control and NeuroD1 lacZ/lacZ mice.

Article Snippet: Primary antibodies used in this study are listed below: GFP (1:1000, Aves Labs, cat#: GFP-1020), Ki67 (1:1000, Thermo Fisher Scientific, cat#: 14-5698-82), Cdkn1b (1:500, Cell Signaling Technology, cat#: 3698), NeuroD1 (1:1000, Cell Signaling Technology, cat#: 62953), Sin3A (1:200, Novus Biologicals, cat#: NB600-1263), Sox2 (1:500, Thermo Fisher Scientific, cat#: 740013T), Ccnd1 (1:300, Cell Signaling Technology, cat#: 55506), Cleaved Caspase-3 Asp175 (1:200, Cell Signaling Technology, cat#: 9664), Cre Recombinase (1:500, Cell Signaling Technology, cat#: 15036), NeuN (1:500, Cell Signaling Technology, cat#: 24307).

Techniques: ChIP-sequencing, Isolation, Control, Immunofluorescence, Staining, Binding Assay, Western Blot, Immunoprecipitation, Fluorescence, Two Tailed Test