Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CHD8 dosage regulates transcription in pluripotency and early murine neural differentiation

doi: 10.1073/pnas.1921963117

Figure Lengend Snippet: Dosage-sensitive effect of CHD8 deletion on transcription in ESCs. (A) The gnomAD human gene constraint scores (missense and probability of intolerance to heterozygous pLoF variation [pLI] for all genes with CHD8 labeled). (B) Schematic showing the CHD8 CRISPR-mediated deletion approach and Western blot of CHD8 levels in Chd8+/− and Chd8−/− ESC lines. (C) Doubling time of WT, Chd8+/−, and Chd8−/− cells. Asterisks denote P < 0.05 (*). Values are expressed as the mean ± SE, n = 3. (D) Alkaline phosphatase staining of WT, Chd8+/−, and Chd8−/− ESCs in 2i media. Magnification: 200×. (E) Venn diagrams depicting overlap of DEGs in Chd8+/− and Chd8−/− ESCs. Genes with increased expression are on top, and genes with decreased expression are on the bottom (FDR < 0.05). (F) Heatmap showing dosage-sensitive transcriptional response for genes that are differentially expressed in Chd8−/−. (G) GO terms for DEGs (padj < 0.05).

Article Snippet: Antibodies used in this study are mouse a-FLAG M2 (1:1,000, Sigma-Aldrich), rabbit a-CHD8 (1:2,500, Novus Biologicals #NB100-60417; 1:2,000, Novus Biologicals #NB100-60418), mouse a-TBP (1:2,000, Abcam #ab818), rabbit a-DCX (1:200, CST #4604S), mouse a-Nestin (1:200, #MAB353), mouse a-Tuj1 (1:200, BioLegend #801201), rabbit a-Pax6 (1:200, BioLegend #901301), rabbit a-NeuroD1 (1:200, Proteintech #12081-1-AP), and mouse a-Map2a (1:200, M9942).

Techniques: Labeling, CRISPR, Western Blot, Staining, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CHD8 dosage regulates transcription in pluripotency and early murine neural differentiation

doi: 10.1073/pnas.1921963117

Figure Lengend Snippet: CHD8 deletion results in up-regulation of neuronal genes and Sox TFs during differentiation into NPCs. (A) Schematic showing the differentiation of WT, Chd8+/−, and Chd8−/− ESCs into NPCs. (B) Venn diagrams depicting overlap of DEGs in Chd8+/− and Chd8−/− NPCs. Genes with increased gene expression are on top, and genes with decreased expression are on bottom (FDR < 0.05). (C) Heatmap showing dosage sensitive transcriptional response for genes that are differentially expressed in Chd8−/−. (D) GO terms for DEGs. (E and F) Volcano plots of RNA-seq data for CHD8+/− and CHD8−/−. DEGs are highlighted in red (FDR < 0.05), and genes involved with mature neuronal development and Sox TFs are labeled. Genes labeled with yellow background were stained by IF in G. (G) Representative IF staining of day 8 NPCs, with antibodies listed on left side and respective genotypes on top (n = 2 biological replicates with similar results). Magnification: 200×.

Article Snippet: Antibodies used in this study are mouse a-FLAG M2 (1:1,000, Sigma-Aldrich), rabbit a-CHD8 (1:2,500, Novus Biologicals #NB100-60417; 1:2,000, Novus Biologicals #NB100-60418), mouse a-TBP (1:2,000, Abcam #ab818), rabbit a-DCX (1:200, CST #4604S), mouse a-Nestin (1:200, #MAB353), mouse a-Tuj1 (1:200, BioLegend #801201), rabbit a-Pax6 (1:200, BioLegend #901301), rabbit a-NeuroD1 (1:200, Proteintech #12081-1-AP), and mouse a-Map2a (1:200, M9942).

Techniques: Gene Expression, Expressing, RNA Sequencing, Labeling, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CHD8 dosage regulates transcription in pluripotency and early murine neural differentiation

doi: 10.1073/pnas.1921963117

Figure Lengend Snippet: CHD8 regulates accessibility at key regions in ESCs and during differentiation to NPCs. (A) Percentage of increased (blue) and decreased (orange) peaks for Chd8−/− ESCs. (B) Genomic annotation enrichment for increased and decreased sites for Chd8−/− ESC ATAC-seq datasets. (C) Percentage of increased (blue) and decreased (orange) peaks for Chd8−/− NPCs. (D) Genomic annotation enrichment for increased and decreased sites for Chd8+/−and Chd8−/− NPC ATAC-seq datasets. (E) Genome tracks showing gain of accessibility at Grb10 locus in ESCs. (F) Genome tracks showing loss of accessibility at Basp1 locus in NPCs.

Article Snippet: Antibodies used in this study are mouse a-FLAG M2 (1:1,000, Sigma-Aldrich), rabbit a-CHD8 (1:2,500, Novus Biologicals #NB100-60417; 1:2,000, Novus Biologicals #NB100-60418), mouse a-TBP (1:2,000, Abcam #ab818), rabbit a-DCX (1:200, CST #4604S), mouse a-Nestin (1:200, #MAB353), mouse a-Tuj1 (1:200, BioLegend #801201), rabbit a-Pax6 (1:200, BioLegend #901301), rabbit a-NeuroD1 (1:200, Proteintech #12081-1-AP), and mouse a-Map2a (1:200, M9942).

Techniques:

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: CHD8 dosage regulates transcription in pluripotency and early murine neural differentiation

doi: 10.1073/pnas.1921963117

Figure Lengend Snippet: CHD8 cooperates with Sox TFs to regulate accessibility and transcription. (A) Heatmap representation of ATAC-seq chromVAR bias-corrected deviations in the 50 most variable TF motifs across ESC WT and Chd8−/− ATAC-seq replicates. (B) Heatmap representation of ATAC-seq chromVAR bias-corrected deviations in the 50 most variable TF motifs across NPC WT and Chd8−/− ATAC-seq replicates. (C) Western blot showing co-IP of CHD8 with SOX2 in ESCs (antibodies used are the following: SOX2 A: sc-365823; SOX2 B: AB5603). (D) Heat map displaying CHD8 ChIP-seq at SOX2 peaks (41) (Left) and SOX2 ChIP-seq at CHD8 peaks (Right).

Article Snippet: Antibodies used in this study are mouse a-FLAG M2 (1:1,000, Sigma-Aldrich), rabbit a-CHD8 (1:2,500, Novus Biologicals #NB100-60417; 1:2,000, Novus Biologicals #NB100-60418), mouse a-TBP (1:2,000, Abcam #ab818), rabbit a-DCX (1:200, CST #4604S), mouse a-Nestin (1:200, #MAB353), mouse a-Tuj1 (1:200, BioLegend #801201), rabbit a-Pax6 (1:200, BioLegend #901301), rabbit a-NeuroD1 (1:200, Proteintech #12081-1-AP), and mouse a-Map2a (1:200, M9942).

Techniques: Western Blot, Co-Immunoprecipitation Assay, ChIP-sequencing

Journal: Cell reports

Article Title: Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes

doi: 10.1016/j.celrep.2024.114637

Figure Lengend Snippet: (A) Genetic strategy targeting exon 4 of the Chd8 gene. The exon was flanked with loxP sites to excise the loci, resulting in a frameshift mutation that disrupts the production of the CHD8 protein after Cre excision. (B and C) Representative images of CHD8 in the CA1 regions of adult mouse brains. CHD8 is expressed in neurons and astrocytes in controls (B). After tamoxifen administration, Chd8 fx/fx : CAGGS-CreER +/ − mouse brains showed non-detectable levels of CHD8 protein (C). In both (B) and (C), arrows point to astrocytes expressing CHD8 and its knockout thereafter. (D) Schematic diagram for tamoxifen-induced Chd8 cKO, the stab-wound injury model, and the analysis of reactive gliosis. (E and F) Response of GFAP + astrocytes and Iba1 + microglia after stab-wound injury. Control mice (E) exhibit astrocytic and microglial response as expected, while global Chd8 cKO mice (F) show reduced staining for both GFAP + and Iba1 + along the needle track. (G and H) High-magnification images from the injury site from the corresponding genotypes. Note the reduction in cell body size, process elongation, and polarity in astrocytes from global Chd8 cKO mice (H). (I and J) Decreased area occupied by GFAP + astrocytes (I) and Iba1 + microglia (J) in global Chd8 cKO mice compared to controls. In (B) and (C), scale bars indicate 20 μm; in (G) and (H), scale bars indicate 50 μm; in (E) and (F), scale bars indicate 500 μm. The dashed rectangle indicates the ROIs that were quantified. In (E)–(H), the dashed lines indicate the needle track of the injury. In (I) and (J), data points illustrate the quantified area from the six brain slices most proximal to the injury epicenter. Data are normalized to the means of the ipsilateral site in control mice. **** p < 0.0001, ns, not significant; statistical analysis was performed with two-way ANOVA; on the violin plots, dashed lines indicate the 25%, mean, and 75% percentile, respectively, from bottom to top; n = 4 mice per genotype.

Article Snippet: CHD8 Rabbit polyclonal 1 to 1000 , Novus Biologicals , NB 100-60418.

Techniques: Mutagenesis, Expressing, Knock-Out, Control, Staining

Journal: Cell reports

Article Title: Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes

doi: 10.1016/j.celrep.2024.114637

Figure Lengend Snippet: (A) Strategy for tamoxifen-induced, astrocyte-specific Chd8 cKO utilizing the Aldh1l1-CreERT2 line. Mice were crossed with the Ai14- tdTomato reporter line to visualize recombined cells. (B and C) Astrocyte-specific Chd8- cKO mice show non-detectable CHD8 protein expression in cortical slices. Controls are mice without the Chd8 floxed alleles but expressing Aldh1l1-CreERT2 to turn on the expression of tdTomato reporter. Arrows indicate CHD8 expression in tdTomato + astrocytes in control but not astrocyte Chd8 cKO. (D and E) GFAP and Iba1 staining of astrocytes and microglia, respectively, after stab-wound injury in control (D) and astrocyte cKO mice (E). (F and G) High-magnification images from the injury sites from the corresponding genotypes showing reduction in cell body size, process elongation, and polarity in astrocytes and reduced microglia numbers in astrocyte cKO mice. (H and I) Decreased area occupied by GFAP + astrocytes and their numbers in astrocyte cKO mice compared to controls. (J and K) Decreased area occupied by Iba1 + microglia and their numbers in astrocyte cKO mice compared to controls. In (H)–(K), data points represent the quantified area from the six brain slices most proximal to the injury epicenter. Data are normalized to the means of the ipsilateral site in control mice. In (B) and (C), scale bars indicate 20 μm; in (F) and (G), scale bars indicate 50 μm; in (D) and (E), scale bars indicate 500 μm. The dashed rectangle indicates the ROIs that were quantified. In (D)–(G), the dashed lines in merged images indicate the needle track of the injury. In (H)–(K), *** p < 0.001; **** p < 0.0001; ns, not significant; statistical comparisons were analyzed with two-way ANOVA; on the violin plots, dashed lines indicate the 25%, mean, and 75% percentile, respectively, from bottom to top; n = 6 mice per genotype.

Article Snippet: CHD8 Rabbit polyclonal 1 to 1000 , Novus Biologicals , NB 100-60418.

Techniques: Expressing, Control, Staining

Journal: Cell reports

Article Title: Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes

doi: 10.1016/j.celrep.2024.114637

Figure Lengend Snippet: (A) Heatmap of DEGs identified through RNA-seq between control and astrocyte Chd8 cKO mice after LPS treatment ( n = 5 mice per genotype). A total of 109 DEGs were identified, 76 of which were upregulated and 33 were downregulated (FDR < 0.05). (B) Volcano plot depicting the distribution of upregulated and downregulated genes, relative to their quantified fold change and their corresponding p values. The threshold was set at p (adjusted) < 0.05. (C) Venn diagram of the detected DEGs, depicting a subset of DEGs that correspond to genes whose expression is specific to astrocytes, neurons, and microglia. Of those, many DEGs (41) were determined to be astrocyte specific, while fewer were deemed to be specific in neurons (4) and microglia (1). (D) Bar plot showing the fold enrichment of the detected DEGs in our dataset, indicating significant enrichment of DEGs for astrocyte marker genes. Notably, no enrichment was detected when comparing neuronal or microglial genes to the cell-type markers from previous studies, as cited. *** p < 0.001; **** p < 0.0001. (E) GO terms analysis reveals changes associated with many cellular processes, including lipid and metabolic pathways in astrocyte cKO mice in response to LPS stimulation. (F) Heatmap of the top 20 DEGs identified through RNA-seq. Of these, 13 were upregulated and 7 were downregulated in astrocyte cKO mice treated with LPS vs. control mice treated with LPS. (G–O) qPCR analysis of Gstt3 (G), Acsl3 (H), Etnppl (I), Phykpl (J), Gjb6 (K), Slc9a8 (L), Agt (M), Tnfrsf25 (N), and Lcat (O) mRNA confirms the altered expression shown in (F) ( n = 4 mice per group). (P) Representative western blotting of TNFRSF25 from cortices of control and astrocyte cKO mice after LPS administration, showing reduced TNFRSF25 protein in the cortex of astrocyte cKO mice ( n = 7 mice per group). (Q) Representative western blotting of LCAT from cortices of control and astrocyte cKO mice after LPS administration showing reduced LCAT protein in astrocyte cKO mice ( n = 7). (R) Representative images of TNFRSF25 staining in the cortex of control and astrocyte cKO mice after LPS administration. Signal intensity quantification of TNFRSF25 staining ( n = 4 mice per genotype). * p < 0.05; scale bars indicate 200 μm. Data points are normalized to the mean of the control group. Error bars depict the SEM. Statistical comparisons were performed with the one-tailed Welch’s t test.

Article Snippet: CHD8 Rabbit polyclonal 1 to 1000 , Novus Biologicals , NB 100-60418.

Techniques: RNA Sequencing, Control, Expressing, Marker, Western Blot, Staining, One-tailed Test

Journal: Cell reports

Article Title: Autism-associated CHD8 controls reactive gliosis and neuroinflammation via remodeling chromatin in astrocytes

doi: 10.1016/j.celrep.2024.114637

Figure Lengend Snippet: (A) Schematic diagram illustrating the elements required for the designed AAV for astrocyte-specific Chd8 editing in vivo via CRISPR-SaCas9. (B) Diagram for the simultaneous AAV injection and stab-wound injury with analysis of reactive gliosis performed at 7 days post-injection. (C) Representative images near the needle track from control mice (Scramble-AAV injected). CHD8 is detectable in astrocytes (SOX9 + ) expressing SaCas9 (HA tag + ). (D) Representative images near the needle track from mice injected with the Chd8 -cKO AAVs. CHD8 is undetectable in the majority of HA + and SOX9 + astrocytes (white arrows), while fewer HA + and SOX9 + astrocytes still show CHD8 expression (yellow arrows). (E and F) GFAP and Iba1 staining of astrocytes and microglia, respectively, after stab-wound injury and AAV injection in the Scramble-AAV (E) and Chd8 -cKO-AAV groups (F). (G) Decreased area occupied by GFAP + astrocytes in the Chd8 -cKO-AAV mice. (H) Quantification of the area occupied by Iba1 + microglia between control and Chd8 -cKO-AAV mice. In (C) and (D), scale bars indicate 20 μm; in (E) and (F), scale bars indicate 500 μm. The dashed rectangle indicates the ROIs that were quantified in (G) and (H). The dashed lines in merged images indicate the needle track of the injury. In (G) and (H), statistical comparisons were performed with two-way ANOVA; ** p < 0.01. Data points indicate n = 3 mice per group. Data are normalized to the means of the ipsilateral site in the control group.

Article Snippet: CHD8 Rabbit polyclonal 1 to 1000 , Novus Biologicals , NB 100-60418.

Techniques: In Vivo, CRISPR, Injection, Control, Expressing, Staining

Journal: Nucleic acids research

Article Title: CHD8 suppression impacts on histone H3 lysine 36 trimethylation and alters RNA alternative splicing.

doi: 10.1093/nar/gkac1134

Figure Lengend Snippet: Figure 1. CHD8 suppression significantly impacts on histone H3K36me3 enrichment at transcriptional elongation sites. (A) Schematic representation of the study design and integrative approach used in this work. Human iPSC-derived NPCs (hiNPC) knocked down for CHD8 (Sh1-, Sh2- and Sh4- CHD8) and control hiNPCs (Sh-GFP and Sh-GFP2) (11), were analyzed via ChIP-seq for six histone marks representative of different chromatin regions: active promoters (H3K4me2 and H3K4me3), inactive promoters (H3K27me3), enhancers (H3K4me1 and H3K27ac) and actively transcribed regions (H3K36me3). ChIP-seq results were subsequently integrated with CHD8-binding sites and available transcriptomics (RNA-seq) datasets obtained from the same model system (11). (B) The heatmaps represent 10 different chromatin states (1, transcriptional initiation; 2, transcriptional elongation; 3, weakly

Article Snippet: For CHD8 immunoprecipitation, NB100-60417 and NB100-60418 (Novus Biotechnology), for hnRNPL D-5 D ow nloaded from https://academ ic.oup.com /nar/article/50/22/12809/6947080 by guest on 10 February 2024 (sc-48391, Santacruz) and for SETD2 (38633, SAB) were used, while rabbit IgG isotype control (10500C, Life) or mouse IgG (10400C, Invitrogen) were used as controls.

Techniques: Derivative Assay, Control, ChIP-sequencing, Binding Assay, RNA Sequencing

Journal: Nucleic acids research

Article Title: CHD8 suppression impacts on histone H3 lysine 36 trimethylation and alters RNA alternative splicing.

doi: 10.1093/nar/gkac1134

Figure Lengend Snippet: Figure 3. CHD8 suppression-elicited reduction in H3K36me3 correlates with significant alterations in RNA AS. (A, B) Venn diagrams represent the overlap between genes losing H3K36me3 peaks following CHD8 knockdown (losing H3K36me3) and genes presenting altered AS events as detected by SUPPA

Article Snippet: For CHD8 immunoprecipitation, NB100-60417 and NB100-60418 (Novus Biotechnology), for hnRNPL D-5 D ow nloaded from https://academ ic.oup.com /nar/article/50/22/12809/6947080 by guest on 10 February 2024 (sc-48391, Santacruz) and for SETD2 (38633, SAB) were used, while rabbit IgG isotype control (10500C, Life) or mouse IgG (10400C, Invitrogen) were used as controls.

Techniques: Knockdown

Journal: Nucleic acids research

Article Title: CHD8 suppression impacts on histone H3 lysine 36 trimethylation and alters RNA alternative splicing.

doi: 10.1093/nar/gkac1134

Figure Lengend Snippet: Figure 4. hnRNPL as a novel CHD8 interactor: bridging altered splicing to H3K36me3 enrichment. (A) Schematic representation of the MS/MS ex- perimental design and approach used in this work (figure created in BioRender.com). Nuclei from hiNPCs (11) were separated from the cytoplasmic fraction. The protein of interest was isolated from the nuclear lysate by specific primary antibodies followed by incubation with Sepharose beads. CHD8 immunoprecipitated proteins were then processed by in solution trypsin digestion prior to MS/MS analysis. (B) Representative western blot images depict immunoprecipitation by endogenous, full-length CHD8 in nuclear extracts by two different antibodies CHD8 NB100-60417 (CHD8 #17) and NB100- 60418 (CHD8 #18). A strong, reproducible enrichment compared with Input (Input, 15 g of nuclear lysate) and rabbit IgG control (IgG) is evident. High exp, 30 s; low exp = 4 s. (C) The volcano plots show CHD8-interacting proteins, significantly differentially enriched compared with IgG controls. Significantly enriched proteins are in blue, significantly depleted proteins in red and non-significant proteins in gray. The threshold for significance is set at a P-value of 0.05. Three independent experiments were averaged and analyzed together for each condition. CHD8, the more represented and enriched peptide with each of the two antibodies, was removed from the plots to optimize visualization of interactors. (D) Venn diagrams represent the overlap be- tween CHD8-interacting proteins identified by CHD8 #17 and CHD8 #18 antibodies. The analysis combines the statistically significant results from three independent biological replicates and two antibodies (Ab #17 A, B, C; Ab #18 A, B, C; see also Supplementary Figure S11). The number of proteins for each condition is indicated. The complete list of proteins is given in Supplementary Table S1. (E) Complete list of the 18 CHD8-interacting proteins identi- fied by CHD8 #17 and CHD8 #18 antibodies. (F) Representative western blot images from co-immunoprecipitation experiments demonstrate interaction between endogenous CHD8 and hnRNPL. Immunoprecipitations were conducted with the two antibodies (IP CHD8 #17 and IP CHD8 #18). A strong, reproducible CHD8 enrichment compared with Input (Input, 15 g of nuclear lysate, Input 5%, 0.75 g of nuclear lysate) and rabbit IgG control (IgG) is evident. Co-immunoprecipitation of hnRNPL is clearly visible at high exposure. CHD8 high exp, high exposure = 60 s; CHD8 low exp, low exposure = 20 s. HnRNPL high exp, high exposure = 240 s; CHD8 low exp, low exposure = 75 s. (G) Representative western blot images showing immunoprecipitation of endogenous CHD8 in the nuclear extract with different treatments: RNase A (RNaseA), EtBr or no treatment (NT). CHD8 high exp, high exposure = 30 s; CHD8 low exp, low exposure = 10 s. HnRNPL high exp, high exposure = 60 s; hnRNPL low exp, low exposure = 4 s. (H) Representative western blot images report co-immunoprecipitation between hnRNPL (anti-mouse) and SETD2 (anti-rabbit) antibodies. Endogenous hnRNPL interacts with SETD2, as demonstrated by enrichment over mouse IgG (IP IgG Mou) and INPUT (15 g of nuclear lysate). Reciprocal co-immunoprecipitation of endogenous SETD2 confirms the interaction, visible at high exposure, compared with IgG controls (IP IgG Rab). SETD2 high exp, high exposure = 20 s; SETD2 low exp, low exposure = 4 s. HNRNPL high exp, high exposure = 40 s; hNRNPL low exp, low exposure = 2 s.

Article Snippet: For CHD8 immunoprecipitation, NB100-60417 and NB100-60418 (Novus Biotechnology), for hnRNPL D-5 D ow nloaded from https://academ ic.oup.com /nar/article/50/22/12809/6947080 by guest on 10 February 2024 (sc-48391, Santacruz) and for SETD2 (38633, SAB) were used, while rabbit IgG isotype control (10500C, Life) or mouse IgG (10400C, Invitrogen) were used as controls.

Techniques: Tandem Mass Spectroscopy, Isolation, Incubation, Immunoprecipitation, Western Blot, Control