Journal: bioRxiv

Article Title: Molecular interactions of Chd8 in mouse brain highlights a role in chromatin-associated RNA processing

doi: 10.64898/2025.12.11.693549

Figure Lengend Snippet: A) Schematic of Chd8 protein domains with location of antibody epitopes indicated. The red line corresponds to the location of the 5bp deletion mutation to generate the Chd8 5bpdel/+ mouse line. B) Immunohistochemistry of Chd8 localization in PND2 mouse cortex, DIV14 primary neuron culture and HEK293T cells. Scale bar = 50µm. C) Schematic of endogenous immunoprecipitation approach. D) PCA plot showing peptide spectra variance of PC1 and PC2 in the four IP conditions, dimension reduction was performed on all proteins detected after IP. E) STRING Chd8 PPI network of all proteins considered significant for both Chd8 antibodies by SAINTexpress and DEP. Color of the outside ring indicates the annotated GO term. Inner circle color indicates the level of significance calculated via DEP. Network was made using default settings for a full STRING network indicating both functional and physical protein associations.

Article Snippet: CHD8 immunoprecipitation was performed with antibodies targeted to either the C- terminal (Bethyl, A301-225A) or N-terminal (Bethyl, A301-224A) end of CHD8.

Techniques: Mutagenesis, Immunohistochemistry, Immunoprecipitation, Functional Assay

Journal: bioRxiv

Article Title: Molecular interactions of Chd8 in mouse brain highlights a role in chromatin-associated RNA processing

doi: 10.64898/2025.12.11.693549

Figure Lengend Snippet: A) MCL clustering of STRING Chd8 protein-protein network with all proteins identified by either Chd8 antibody via either SAINTexpress or DEP at a P-value < 0.05 significance cutoff. MCL clustering was performed with the granularity parameter set to 6. Color of the outside ring indicates the annotated GO term. Inner circle color indicates if the protein was included in the more stringent 222 Chd8 interacting protein set. Network was made using default settings for a physical STRING network indicating the proteins are part of a physical complex. B) Western blotting of Co-IP validation of Chd8 protein interactions. Co-IP was performed using either the C-terminus or N- terminus Chd8 antibody, indicated by the top labels. The antibody used for western blotting is indicated to the right and the molecular weight (kDa) of the band to the left. C) Colocalization validation of protein interactions. D) Schematic of Proximity Ligation Assay (PLA) approach. PLA visualizes protein interactions via fluorescent signal amplification when targets are in close molecular proximity. E) PLA validation of Chd8 protein interactions. Nuclei were stained blue with Hoechst, with red dots indicating locations where the two target proteins are interacting. F-G) Western blotting of Chd8 Co-IP after RNase treatment. +/- indicates the presence or absence of RNase, respectively. The top labels indicate the antibody used for Co-IP. F) Co-IP of Rbm8a. G) Co-IP of Paf1.

Article Snippet: CHD8 immunoprecipitation was performed with antibodies targeted to either the C- terminal (Bethyl, A301-225A) or N-terminal (Bethyl, A301-224A) end of CHD8.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Biomarker Discovery, Molecular Weight, Proximity Ligation Assay, Amplification, Staining

Journal: bioRxiv

Article Title: Molecular interactions of Chd8 in mouse brain highlights a role in chromatin-associated RNA processing

doi: 10.64898/2025.12.11.693549

Figure Lengend Snippet: A. Schematic of the protein construct used for TurboID. B) Epifluorescence images validation of localization and biotinylation activity of CHD8-Turbo in HEK293T cells. Protein localization was stained by anti-HA antibodies and biotinylation activity was stained by fluorescently conjugated Neutravidin. C) STRING PPI network of the CHD8-Turbo fusion protein in HEK293T cells. Color of the outside ring indicates the annotated GO term. Network was made using default settings for a full STRING network indicating both functional and physical protein associations. D) Heatmap of GO term enrichment across multiple Chd8 pulldown conditions. The number of proteins included in each condition is indicated in parentheses.

Article Snippet: CHD8 immunoprecipitation was performed with antibodies targeted to either the C- terminal (Bethyl, A301-225A) or N-terminal (Bethyl, A301-224A) end of CHD8.

Techniques: Construct, Biomarker Discovery, Activity Assay, Staining, Functional Assay

Journal: bioRxiv

Article Title: Molecular interactions of Chd8 in mouse brain highlights a role in chromatin-associated RNA processing

doi: 10.64898/2025.12.11.693549

Figure Lengend Snippet: A) Schematic of PND2 forebrain RIP-seq method. B) Quantification of RNA that was isolated from Chd8 Co-IP from PND2 mouse forebrain. C) Quantification of RNA that was isolated from Chd8 Co-IP after DNase and RNase treatment. D) PCA plot showing transcriptional variance of PC1 and PC2 in the four IP conditions used for RIP-seq, dimension reduction was performed on the top 500 variable genes. Antibodies used for IP conditions are indicated by color. E) Volcano plot showing log fold change and significance of mRNA enriched after IP via the C-terminus antibody vs IgG control. Significant enrichment in C-terminus vs IgG is shown in red (P value < 0.05) with highly significant enrichment (FDR < 0.1) shown in dark red. 263 genes showed significant enrichment in the C-terminus RIP-seq vs IgG. F) Density plots showing the frequency of gene overlaps from permutation tests comparing the top 1000 C-terminus RIP-seq enriched genes and Chd8-bound genes from, the top 1000 Chd8 TaDa-seq (Wade 2021) genes (P = 0.0874), the top 1000 Chd8 CHIP- seq (Platt 2017) genes (P = 0.0232), and the 856 Chd8 CHIP-seq (Gompers 2016) genes (P = 0.0784). Observed intersections in shown in red. G) Gene ontology enrichment of genes significantly (P < 0.05) enriched in C-terminus RIP-seq. Strength and significance of enrichment, as well as number of DEGs present in gene set is indicated. Gene sets with DEGs highlighted in panel H are colored. H) Heatmap showing the z-scale normalized counts of DEGs present in indicated gene sets across C-terminus and IgG IP samples. Color to the left indicates the enriched gene set.

Article Snippet: CHD8 immunoprecipitation was performed with antibodies targeted to either the C- terminal (Bethyl, A301-225A) or N-terminal (Bethyl, A301-224A) end of CHD8.

Techniques: Isolation, Co-Immunoprecipitation Assay, Control, ChIP-sequencing

Journal: bioRxiv

Article Title: Molecular interactions of Chd8 in mouse brain highlights a role in chromatin-associated RNA processing

doi: 10.64898/2025.12.11.693549

Figure Lengend Snippet: A) Volcano plot of individual protein pulldown in Chd8 5bpdel/+ and wildtype mice including proteins identified by either Chd8 antibody via SAINTexpress or DEP at a P-value < 0.05 cutoff. Green and blue proteins are significantly differential for Chd8 5bpdel/+ or Chd8 +/+ forebrain at a significance of P- value < 0.05. B) GO terms enriched in proteins significantly increased in Chd8 5bpdel/+ or Chd8 +/+ forebrain. Set overlap indicates the number of significantly increased proteins and total detected indicates the total number from all proteins analyzed. C) Volcano plot of individual protein pulldown in Chd8 5bpdel/+ and wildtype mice. Color indicates the annotated GO term. D) The observed average fold change of multiple GO terms. SEM and p-values calculated by permutation test. Sample size for each permutation corresponds to the number of annotated proteins for each GO term. E) Heatmap comparing Odd Ratio values across Chd8 antibody and mouse genotype for all proteins associated with mRNA Splicing, via Spliceosome (GO:0000398).

Article Snippet: CHD8 immunoprecipitation was performed with antibodies targeted to either the C- terminal (Bethyl, A301-225A) or N-terminal (Bethyl, A301-224A) end of CHD8.

Techniques:

Journal: bioRxiv

Article Title: Molecular interactions of Chd8 in mouse brain highlights a role in chromatin-associated RNA processing

doi: 10.64898/2025.12.11.693549

Figure Lengend Snippet: A) Schematic of Chd8 protein domains with location of antibody epitopes indicated. The red line corresponds to the location of the 5bp deletion mutation to generate the Chd8 5bpdel/+ mouse line. B) Immunohistochemistry of Chd8 localization in PND2 mouse cortex, DIV14 primary neuron culture and HEK293T cells. Scale bar = 50µm. C) Schematic of endogenous immunoprecipitation approach. D) PCA plot showing peptide spectra variance of PC1 and PC2 in the four IP conditions, dimension reduction was performed on all proteins detected after IP. E) STRING Chd8 PPI network of all proteins considered significant for both Chd8 antibodies by SAINTexpress and DEP. Color of the outside ring indicates the annotated GO term. Inner circle color indicates the level of significance calculated via DEP. Network was made using default settings for a full STRING network indicating both functional and physical protein associations.

Article Snippet: CHD8 immunoprecipitation was performed with antibodies targeted to either the C- terminal (Bethyl, A301-225A) or N-terminal (Bethyl, A301-224A) end of CHD8.

Techniques: Mutagenesis, Immunohistochemistry, Immunoprecipitation, Functional Assay

Journal: bioRxiv

Article Title: Molecular interactions of Chd8 in mouse brain highlights a role in chromatin-associated RNA processing

doi: 10.64898/2025.12.11.693549

Figure Lengend Snippet: A) MCL clustering of STRING Chd8 protein-protein network with all proteins identified by either Chd8 antibody via either SAINTexpress or DEP at a P-value < 0.05 significance cutoff. MCL clustering was performed with the granularity parameter set to 6. Color of the outside ring indicates the annotated GO term. Inner circle color indicates if the protein was included in the more stringent 222 Chd8 interacting protein set. Network was made using default settings for a physical STRING network indicating the proteins are part of a physical complex. B) Western blotting of Co-IP validation of Chd8 protein interactions. Co-IP was performed using either the C-terminus or N- terminus Chd8 antibody, indicated by the top labels. The antibody used for western blotting is indicated to the right and the molecular weight (kDa) of the band to the left. C) Colocalization validation of protein interactions. D) Schematic of Proximity Ligation Assay (PLA) approach. PLA visualizes protein interactions via fluorescent signal amplification when targets are in close molecular proximity. E) PLA validation of Chd8 protein interactions. Nuclei were stained blue with Hoechst, with red dots indicating locations where the two target proteins are interacting. F-G) Western blotting of Chd8 Co-IP after RNase treatment. +/- indicates the presence or absence of RNase, respectively. The top labels indicate the antibody used for Co-IP. F) Co-IP of Rbm8a. G) Co-IP of Paf1.

Article Snippet: CHD8 immunoprecipitation was performed with antibodies targeted to either the C- terminal (Bethyl, A301-225A) or N-terminal (Bethyl, A301-224A) end of CHD8.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Biomarker Discovery, Molecular Weight, Proximity Ligation Assay, Amplification, Staining

Journal: bioRxiv

Article Title: Molecular interactions of Chd8 in mouse brain highlights a role in chromatin-associated RNA processing

doi: 10.64898/2025.12.11.693549

Figure Lengend Snippet: A. Schematic of the protein construct used for TurboID. B) Epifluorescence images validation of localization and biotinylation activity of CHD8-Turbo in HEK293T cells. Protein localization was stained by anti-HA antibodies and biotinylation activity was stained by fluorescently conjugated Neutravidin. C) STRING PPI network of the CHD8-Turbo fusion protein in HEK293T cells. Color of the outside ring indicates the annotated GO term. Network was made using default settings for a full STRING network indicating both functional and physical protein associations. D) Heatmap of GO term enrichment across multiple Chd8 pulldown conditions. The number of proteins included in each condition is indicated in parentheses.

Article Snippet: CHD8 immunoprecipitation was performed with antibodies targeted to either the C- terminal (Bethyl, A301-225A) or N-terminal (Bethyl, A301-224A) end of CHD8.

Techniques: Construct, Biomarker Discovery, Activity Assay, Staining, Functional Assay

Journal: bioRxiv

Article Title: Molecular interactions of Chd8 in mouse brain highlights a role in chromatin-associated RNA processing

doi: 10.64898/2025.12.11.693549

Figure Lengend Snippet: A) Schematic of PND2 forebrain RIP-seq method. B) Quantification of RNA that was isolated from Chd8 Co-IP from PND2 mouse forebrain. C) Quantification of RNA that was isolated from Chd8 Co-IP after DNase and RNase treatment. D) PCA plot showing transcriptional variance of PC1 and PC2 in the four IP conditions used for RIP-seq, dimension reduction was performed on the top 500 variable genes. Antibodies used for IP conditions are indicated by color. E) Volcano plot showing log fold change and significance of mRNA enriched after IP via the C-terminus antibody vs IgG control. Significant enrichment in C-terminus vs IgG is shown in red (P value < 0.05) with highly significant enrichment (FDR < 0.1) shown in dark red. 263 genes showed significant enrichment in the C-terminus RIP-seq vs IgG. F) Density plots showing the frequency of gene overlaps from permutation tests comparing the top 1000 C-terminus RIP-seq enriched genes and Chd8-bound genes from, the top 1000 Chd8 TaDa-seq (Wade 2021) genes (P = 0.0874), the top 1000 Chd8 CHIP- seq (Platt 2017) genes (P = 0.0232), and the 856 Chd8 CHIP-seq (Gompers 2016) genes (P = 0.0784). Observed intersections in shown in red. G) Gene ontology enrichment of genes significantly (P < 0.05) enriched in C-terminus RIP-seq. Strength and significance of enrichment, as well as number of DEGs present in gene set is indicated. Gene sets with DEGs highlighted in panel H are colored. H) Heatmap showing the z-scale normalized counts of DEGs present in indicated gene sets across C-terminus and IgG IP samples. Color to the left indicates the enriched gene set.

Article Snippet: CHD8 immunoprecipitation was performed with antibodies targeted to either the C- terminal (Bethyl, A301-225A) or N-terminal (Bethyl, A301-224A) end of CHD8.

Techniques: Isolation, Co-Immunoprecipitation Assay, Control, ChIP-sequencing

Journal: bioRxiv

Article Title: Molecular interactions of Chd8 in mouse brain highlights a role in chromatin-associated RNA processing

doi: 10.64898/2025.12.11.693549

Figure Lengend Snippet: A) Volcano plot of individual protein pulldown in Chd8 5bpdel/+ and wildtype mice including proteins identified by either Chd8 antibody via SAINTexpress or DEP at a P-value < 0.05 cutoff. Green and blue proteins are significantly differential for Chd8 5bpdel/+ or Chd8 +/+ forebrain at a significance of P- value < 0.05. B) GO terms enriched in proteins significantly increased in Chd8 5bpdel/+ or Chd8 +/+ forebrain. Set overlap indicates the number of significantly increased proteins and total detected indicates the total number from all proteins analyzed. C) Volcano plot of individual protein pulldown in Chd8 5bpdel/+ and wildtype mice. Color indicates the annotated GO term. D) The observed average fold change of multiple GO terms. SEM and p-values calculated by permutation test. Sample size for each permutation corresponds to the number of annotated proteins for each GO term. E) Heatmap comparing Odd Ratio values across Chd8 antibody and mouse genotype for all proteins associated with mRNA Splicing, via Spliceosome (GO:0000398).

Article Snippet: CHD8 immunoprecipitation was performed with antibodies targeted to either the C- terminal (Bethyl, A301-225A) or N-terminal (Bethyl, A301-224A) end of CHD8.

Techniques: