Journal: PLoS ONE

Article Title: Chromodomain Helicase Binding Protein 8 (Chd8) Is a Novel A-Kinase Anchoring Protein Expressed during Rat Cardiac Development

doi: 10.1371/journal.pone.0046316

Figure Lengend Snippet: Chd8-S arises from passage of transcription through the end of exon 9 into intron 10, where it terminates. , Chd8-S contains a single chromodomain (C1). Two longer isoforms contain the tandem chromodomains (C1 and C2) and helicase domain characteristic to Chd proteins , . Chd8-L1 and Chd8-L2 result from two different start sites for transcription , with the Chd8-L1 transcript encoding an amino terminus extension that encompasses a p53 binding domain . All three isoforms contain a series of five nuclear localization signals (NLS) and a β-catenin binding domain (β) , , that is also required for histone H1 binding and STAT3 binding. Chd8-L1 and Chd8-L2 also contain a pair of BRK domains, which mediate chromatin interaction via the histones and is required for interaction with CTCF . All three isoforms contain the AKAP domain (RII) characterized in this study.

Article Snippet: Taqman probes for Chd8 (Rn00576005_m1, designated Probe 1, and Rn01414467_m1, designated Probe 2), RIIα (Rn00709403_m1), and GAPDH (Rn 99999916_s1) were obtained from Applied Biosystems, and used in conjunction with Master Mix (Applied Biosystems, Carlsbad, California) as described for PCR amplification with a 7900HT Thermal Cycler (Applied Biosystems), as described.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Chromodomain Helicase Binding Protein 8 (Chd8) Is a Novel A-Kinase Anchoring Protein Expressed during Rat Cardiac Development

doi: 10.1371/journal.pone.0046316

Figure Lengend Snippet: Bioinformatics analyses of the Chd8 peptide identified in T7 phage display with JPRED2 (A) and Geneious (B) bioinformatics tools show predicted α-helices within the amino acid sequence. C) ClustalW alignment of Chd8 with known AKAP domains. Hydrophobicity plots for each peptide are listed below the amino acid sequence. D) A 2D helical wheel plot was generated for the predicted AKAP domain of Chd8. Hydrophobic amino acids are shaded in gray, and amino acids are numbered starting from the amino terminus. E) Alignment of the predicted AKAP domain (underlined) in Chd8 shows a high level of conservation between species. Sequences from H. sapiens (NP_001164100.1), M. musculus (NP_963999.2), R. norvegicus (NP_075222.2), B. taurus (NP_001179063.1), X. tropicalis (NP_001131089.2), and D. rerio (NP_001189381.1) were used. An asterisk (*) denotes a conserved amino acid, a colon (:) denotes strongly similar amino acids, and a period (.) denotes weakly similar amino acids.

Article Snippet: Taqman probes for Chd8 (Rn00576005_m1, designated Probe 1, and Rn01414467_m1, designated Probe 2), RIIα (Rn00709403_m1), and GAPDH (Rn 99999916_s1) were obtained from Applied Biosystems, and used in conjunction with Master Mix (Applied Biosystems, Carlsbad, California) as described for PCR amplification with a 7900HT Thermal Cycler (Applied Biosystems), as described.

Techniques: Sequencing, Generated

Journal: PLoS ONE

Article Title: Chromodomain Helicase Binding Protein 8 (Chd8) Is a Novel A-Kinase Anchoring Protein Expressed during Rat Cardiac Development

doi: 10.1371/journal.pone.0046316

Figure Lengend Snippet: A) PCR amplification of rat Chd8-S generated a construct with the predicted protein sequence of Chd8, as well as a c-terminal myc tag (not shown). The AKAP domain is underlined. Site-directed mutagenesis was used to mutate I464 (red) to a Pro for the Chd8 RII -P construct. B) Expression of constructs following induction with IPTG. Constructs were expressed in E. coli and grown to log phase before the addition of IPTG to induce expression. As a control for induction of protein, a myc-tagged LacZ construct was used (left lane, 140 kDa MW). Chd8 RII (second lane) and Chd8 RII -P (third lane) were expressed at the predicted molecular weight of approximately 22 kDa. C) RIIα overlay was conducted with Western blots of E. coli lysate from bacteria expressing inducible constructs. Top: In membranes incubated with RIIα, RIIα/β antibody detected bound RIIα to the lane expressing Chd8 RII , but not to the lane expressing Chd8 RII -P. Middle : Pre-incubation of RIIα with Ht31, an inhibitor of RII:AKAP interaction, resulted in loss of binding to Chd8 RII . Bottom : Pre-incubation of RIIα with Ht31P, a prolinated form of Ht31 unable to bind RII, did not prevent binding of RIIα to protein in the Chd8 RII lane. No corresponding bands were observed in the negative control (LacZ) lanes.

Article Snippet: Taqman probes for Chd8 (Rn00576005_m1, designated Probe 1, and Rn01414467_m1, designated Probe 2), RIIα (Rn00709403_m1), and GAPDH (Rn 99999916_s1) were obtained from Applied Biosystems, and used in conjunction with Master Mix (Applied Biosystems, Carlsbad, California) as described for PCR amplification with a 7900HT Thermal Cycler (Applied Biosystems), as described.

Techniques: Amplification, Generated, Construct, Sequencing, Mutagenesis, Expressing, Control, Molecular Weight, Western Blot, Bacteria, Incubation, Binding Assay, Negative Control

Journal: PLoS ONE

Article Title: Chromodomain Helicase Binding Protein 8 (Chd8) Is a Novel A-Kinase Anchoring Protein Expressed during Rat Cardiac Development

doi: 10.1371/journal.pone.0046316

Figure Lengend Snippet: A) Immunofluorescence of transfected cells shows nuclear localization of Chd8-S and Chd8-S-P constructs (red). Cells were imaged with inverted fluorescent microscopy at a magnification of 90X. Scale bar represents 25 µm. B) Western blot analysis of protein extracted from CHO cells transfected with Chd8-S, RIIα, or a combination of Chd8-S and RIIα or Chd8-S-P and RIIα. Chd8 constructs were detected by means of an antibody to a myc epitope tag. RIIα constructs were detected with a pan-RII antibody. GAPDH was used as a loading control. C) Cell lysate for single and co-transfections was subject to immunoprecipitation for myc-tagged constructs. In the single transfection of Chd8-S, immunoprecipitation with antibodies to the myc tag isolated Chd8-S. No product was observed in the single transfection with RIIα. For co-transfections, immunoblotting showed immunoprecipitation of RIIα with Chd8-S, but not Chd8-S-P. No target proteins were identified in immunoprecipitate from untransfected cells (NT). (n = 3, representative blots shown).

Article Snippet: Taqman probes for Chd8 (Rn00576005_m1, designated Probe 1, and Rn01414467_m1, designated Probe 2), RIIα (Rn00709403_m1), and GAPDH (Rn 99999916_s1) were obtained from Applied Biosystems, and used in conjunction with Master Mix (Applied Biosystems, Carlsbad, California) as described for PCR amplification with a 7900HT Thermal Cycler (Applied Biosystems), as described.

Techniques: Immunofluorescence, Transfection, Construct, Microscopy, Western Blot, Control, Immunoprecipitation, Isolation

Journal: PLoS ONE

Article Title: Chromodomain Helicase Binding Protein 8 (Chd8) Is a Novel A-Kinase Anchoring Protein Expressed during Rat Cardiac Development

doi: 10.1371/journal.pone.0046316

Figure Lengend Snippet: Protein extracted from HEK 293T cells was used for pulldown with cAMP-coupled agarose. Bound proteins were eluted and analyzed by Western blot. Top: Chd8 was detected in input lane (left lane) and with eluate from cAMP agarose. The cAMP analogue 8-Br-cAMP was used as a negative control, to compete binding to the cAMP-agarose. Chd8 was not detected in the negative control lane. Middle: Detection of RIIα with an RIIα monoclonal antibody showed RIIα in the input lane and the cAMP-agarose lane. Bottom: Detection of RIIβ with an RIIβ monoclonal antibody showed RIIβ in the input lane and cAMP-agarose lane. Addition of cAMP reduced, but did not entirely eliminate, pulldown of RIIα and RIIβ in this experiment. (n = 3, representative blots shown).

Article Snippet: Taqman probes for Chd8 (Rn00576005_m1, designated Probe 1, and Rn01414467_m1, designated Probe 2), RIIα (Rn00709403_m1), and GAPDH (Rn 99999916_s1) were obtained from Applied Biosystems, and used in conjunction with Master Mix (Applied Biosystems, Carlsbad, California) as described for PCR amplification with a 7900HT Thermal Cycler (Applied Biosystems), as described.

Techniques: Western Blot, Negative Control, Binding Assay

Journal: PLoS ONE

Article Title: Chromodomain Helicase Binding Protein 8 (Chd8) Is a Novel A-Kinase Anchoring Protein Expressed during Rat Cardiac Development

doi: 10.1371/journal.pone.0046316

Figure Lengend Snippet: A) RIIα, RIIα-SA, and RIIα-SD constructs were stably expressed in in CHO cells and visualized through a carboxyl CFP tag. Arrows point to a punctate distribution of the RIIα constructs observed in RIIα and RIIα-SD cell lines. Cells were imaged with inverted fluorescence microscopy and images taken at 90X magnification. Scale bar represents 25 µm. B) Western blot analysis of CHO cells stably expressing RIIα, RIIα-SA, or RIIα-SD alone, or transiently transfected with myc-tagged Chd8-S. Chd8 constructs were detected by means of an antibody to a myc epitope tag. RIIα constructs were detected with a pan-RII antibody. GAPDH was used as a loading control. C) CHO cell lysate was subject to immunoprecipitation with antibodies to the myc tag of Chd8-S. In co-transfected lanes, immunoblot of immunoprecipitate detected RIIα-SD and RIIα in immunoprecipitates of Chd8-S, but not RIIα-SA. No target proteins were detected in the CHO cell lysates expressing RIIα constructs alone. (n = 3, representative blots shown).

Article Snippet: Taqman probes for Chd8 (Rn00576005_m1, designated Probe 1, and Rn01414467_m1, designated Probe 2), RIIα (Rn00709403_m1), and GAPDH (Rn 99999916_s1) were obtained from Applied Biosystems, and used in conjunction with Master Mix (Applied Biosystems, Carlsbad, California) as described for PCR amplification with a 7900HT Thermal Cycler (Applied Biosystems), as described.

Techniques: Construct, Stable Transfection, Fluorescence, Microscopy, Western Blot, Expressing, Transfection, Control, Immunoprecipitation

Journal: PLoS ONE

Article Title: Chromodomain Helicase Binding Protein 8 (Chd8) Is a Novel A-Kinase Anchoring Protein Expressed during Rat Cardiac Development

doi: 10.1371/journal.pone.0046316

Figure Lengend Snippet: A) Immunofluorescence of endogenous Chd8 (green) identified nuclear Chd8, and also a discrete perinuclear staining (arrow, arrowhead). Immunofluorescence of the Golgi apparatus (red) with an antibody to human Golgi reveals distinct perinuclear localization. Merge shows that the perinuclear pool of Chd8 is in close proximity (arrowhead) to or overlapping with (arrow) the Golgi apparatus. Inset shows cell that was magnified 5.5× for the right panel. Cells were imaged with inverted fluorescence microscopy and images taken at 90× magnification. Scale bar represents 25 µm. B) Confocal microscopy of Chd8 (green) and Golgi (red) immunofluorescence in the same transverse slice. The graph represents the plot profile for signals across each channel in the same plane. Images taken at 63× magnification, the scale bar represents 10 µm. C) Costaining for RII (green) and Chd8 (red) in HeLa cells. The merge reveals overlapping signals between RII and Chd8 in the perinuclear staining (arrows). Inset shows cell that was magnified 4.5X for the right panel. Cells were imaged with inverted fluorescence microscopy and images taken at 90X magnification. Scale bar represents 25 µm.

Article Snippet: Taqman probes for Chd8 (Rn00576005_m1, designated Probe 1, and Rn01414467_m1, designated Probe 2), RIIα (Rn00709403_m1), and GAPDH (Rn 99999916_s1) were obtained from Applied Biosystems, and used in conjunction with Master Mix (Applied Biosystems, Carlsbad, California) as described for PCR amplification with a 7900HT Thermal Cycler (Applied Biosystems), as described.

Techniques: Immunofluorescence, Staining, Fluorescence, Microscopy, Confocal Microscopy

Journal: PLoS ONE

Article Title: Chromodomain Helicase Binding Protein 8 (Chd8) Is a Novel A-Kinase Anchoring Protein Expressed during Rat Cardiac Development

doi: 10.1371/journal.pone.0046316

Figure Lengend Snippet: A) A representation of the targets of the two sets of TaqMan probes used to measure Chd8 mRNA. Probe 1 (ABI IDRn00576005_m1) spans exon 2–3 and covers the RII binding domain. Probe 2 (ABI Rn01414467_m1) spans exons 12–13, which encode the helicase domain and detects only the two longest isoforms. B) Relative amounts of mRNA of Chd8 (Probe 1 and Probe 2) and RIIα (PKAR2A), normalized to GAPDH and calculated by the 2 −ΔΔCtt method. C) Western blot was used to detect Chd8-L1 and Chd8-L2 in NCMs. HeLa cell lysate was used as a positive control. D) NCMs were fixed at four days in culture and stained for Chd8 (green) and α-actinin (red). Arrows indicate myocytes, while arrowheads indicate fibroblasts. E) NCMs were fixed at four days in culture and stained for α-actinin (green) and RIIα/β (red). Arrows indicate myocytes, while arrowheads indicate fibroblasts. Cells were imaged with inverted fluorescence microscopy and images taken at 90X magnification. Scale bar represents 25 µm.

Article Snippet: Taqman probes for Chd8 (Rn00576005_m1, designated Probe 1, and Rn01414467_m1, designated Probe 2), RIIα (Rn00709403_m1), and GAPDH (Rn 99999916_s1) were obtained from Applied Biosystems, and used in conjunction with Master Mix (Applied Biosystems, Carlsbad, California) as described for PCR amplification with a 7900HT Thermal Cycler (Applied Biosystems), as described.

Techniques: Binding Assay, Western Blot, Positive Control, Staining, Fluorescence, Microscopy

Journal: Theranostics

Article Title: Growth hormone receptor disrupts glucose homeostasis via promoting and stabilizing retinol binding protein 4

doi: 10.7150/thno.61192

Figure Lengend Snippet: GHR promotes RBP4 protein homeostasis through the HIF1α/TTR axis. (A) Western blots analysis of GHR, p-SAT5 and STAT5 in the livers of AAV-infected mice as indicated. (B) Relative mRNA levels of RBP4 in the livers of AAV-infected mice as indicated (n=6). (C) The concentrations of serum RBP4 of AAV-infected mice as indicated (n=6). (D) Elution profile of chylomicrons in the serum of AAV-GFP (left) or AAV-GHR (right) mice. Purified proteins were detected in column eluents by monitoring absorbance at 280 nm. (E) Western blots of RBP4 and TTR in serum of AAV-GFP or AAV-GHR mice, which were separated by gel filtration chromatography and collected according to the ultraviolet absorption peak of fractions. (F) Western blots analysis of HIF1α in the livers of AAV-infected mice as indicated. (G) The schematic representation of the HIF1α binding site in the promoter region of TTR. (H) Western blots analysis of GHR, TTR and HIF1α in the livers of AAV-infected mice as indicated. (I) The HepG2 cells were transfected with pGL-3 or TTR promoter reporter plasmid or 5x HIF1α response elements (HRE) reporter. After transfection for 24 h, the cells were exposed to a hypoxic condition for 24 h. Then the luciferase activity was determined (n=6). (J and K) ChIP assay was performed by using anti-HIF1α antibody. The elutes were analyzed by using primers for VEGF HRE, TTR HRE, or non-HRE region. The quantitative results were obtained by real-time PCR (J, n=6) or electrophoretic assay (K). Data are expressed as the mean ± SD. ns, no significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student's t -test or one-way ANOVA).

Article Snippet: The primary antibodies for GHR (#sc-137185), PEPCK (#sc-32879), G6Pase (#sc-25840) (Santa Cruz Biotechnology, 1:1000), p-IR (#3024), IR (#3025), p-Akt (Ser473) (#9271), Akt(#9272), p-Foxo1 (#9464), Foxo1(#2880), p-GSK3β (#5558), GSK3β (#9315), HSL (#4107), ATGL(#2138), STAT5 (#94205), p-STAT5 (#9359) (Cell Signaling Technology, 1:1000), GAPDH (#10494-1-AP, 1:5000), PDH (#18068-1-AP), RBP4 (#11774-1-AP), TTR (#11891-1-AP), β-actin (#20536-1-AP) (ProteinTech Group, 1:1000), PDK4 (#ab38242), HIF1α (#ab228649) (Abcam, 1:1000), p-PDH (#ABS194, Merck Millipore, 1:1000) were used, and secondary antibodies including Peroxidase-AffiniPure Goat Anti-Rabbit IgG (#111-035-003) and Peroxidase-AffiniPure Goat Anti-Mouse IgG (#115-035-003) (Jackson ImmunoResearch, 1:2000-1:5000) were also used for Western blot analysis.

Techniques: Western Blot, Infection, Purification, Filtration, Chromatography, Binding Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction

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: 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 ) , 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 . ( B ) The heatmaps represent 10 different chromatin states (1, transcriptional initiation; 2, transcriptional elongation; 3, weakly transcribed; 4, strong enhancer; 5, weak/poised enhancer a; 6, weak/poised enhancer b; 7, active promoter; 8, inactive/poised promoter; 9, polycomb repressed; 10, heterochromatin/low signal), determined by the combination of different histone marks in control hiNPCs as defined by ChromHMM . The distribution of histone mark peaks across different chromatin states (see the Materials and Methods for details) is presented as a percentage of the total, and is color-coded in the heatmap (left). On the right, the heatmap describes the difference in number of peaks between two experimental conditions (controls versus CHD8 knockdown). Chromatin states enriched in the control are indicated in blue and chromatin states enriched in CHD8 knockdown in orange. H3K36me3 in transcriptional elongation is identified as the most affected chromatin state. ( C ) The bar plots represent the number of peaks for each histone mark identified at transcriptional initiation (left), elongation (center) and weakly transcribed (right) genomic regions. Gray bars indicate controls ( n = 2, Sh- GFP and Sh- GFP2 ) and white bars refer to CHD8 knockdown ( n = 2, Sh2- CHD8 and Sh4- CHD8 ). H3K36me3 peak loss upon CHD8 suppression was significant at the transcriptional elongation states (two biological replicates, t -test, P <0.05). ( D ) The volcano plot reports differentially enriched peaks for H3K36me3 as detected by DiffBind ( , ). Peaks significantly different (FDR <0.05) are shown in black. Peaks not significantly different (ns, FDR >0.05) are shown in gray. The dashed horizontal line represents FDR = 0.05. Peaks enriched in CHD8 knockdown compared with controls are represented on the right side of the plot, with positive log2(FC). Peaks depleted in CHD8 knockdown (enriched in controls) are represented on the left side of the plot, with negative log2(FC). ( E ) A representative image illustrating total histone levels (H3K36me3 and H3 total), comparing control (Sh- GFP ) and CHD8 knockdown clones (Sh1- CHD8 , Sh2- CHD8 and Sh4- CHD8 ) from western blotting experiments. Levels of H3K36me3 reduction are indicated as FC compared with control Sh- GFP . Comparable amounts of total protein were loaded. Total histone H3 was used as loading control. H3K36me3 exposure = 20 s; H3 total exposure = 20 s. ( F ) The bars in the chart represent normalized H3K36me3 (versus total histone H3) values relative to Sh- GFP controls. Mean values ± SE from independent biological replicates ( n = 4 for Sh4- CHD8 and n = 6 for the other samples) are plotted. A t -test for two mean populations was performed. * P ≤0.05. ( G ) The heatmap represents gene set enrichment P -values in –log10 scale for all genes losing H3K36me3 (as in D) following CHD8 knockdown. Gene lists related to ASD, neurodevelopment, co-expression modules in brain and intolerance to loss of function were tested for enrichment as described in the Materials and Methods. The full gene list description and enrichment results are available in .

Article Snippet: Membranes were blocked with 5% w/v non-fat dried milk and incubated with the following primary antibodies: anti-CHD8 (NB100-60417, Novus Biologicals) (1:1000), anti-HSP90 (4874S, Cell Signaling Tech.) (1:5000), anti-histone H3 (1:1.000) (4499, Cell Signaling Tech.) and anti-histone H3K36me3 (1:1.000) (Ab9050, Abcam).

Techniques: Derivative Assay, Control, ChIP-sequencing, Binding Assay, RNA Sequencing Assay, Knockdown, Clone Assay, Western Blot, Expressing

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: CHD8 suppression correlates with reduced H3K36me3 enrichment preferentially at CHD8-bound genes. ( A , B ) Metagene profiles display the average of histone H3K36me3 enrichment (scaled log2 ratio of normalized ChIP value over INPUT control; see also the Materials and Methods) in a region of ± 2 kbp upstream of the TSS and downstream of the TES, calculated for control (black line) and CHD8 knockdown (gray line) hiNPCs and for CHD8-bound (#988) (A) and CHD8-unbound genes (#4205) (B). The difference between H3K36me3 enrichment in control and CHD8 knockdown is significant for CHD8-bound genes, but not for CHD8-unbound genes (paired Cohen's d effect size statistics). ( C ) The violin plots represent the level of CHD8 binding enrichment [log2(ChIP/INPUT)] for three groups of genes as clustered by k-means. Cluster #1 composed of 1239 genes shows high CHD8 enrichment [mean log2(ChIP/INPUT) = 0.27], cluster #2 composed of 2429 genes shows medium to low CHD8 enrichment [mean log2(ChIP/INPUT) = –0.04] and cluster #3 composed of 1380 genes displays negligible CHD8 enrichment [mean log2(ChIP/INPUT) = –0.31]. ( D ) Metagene profiles show the average of CHD8 binding enrichment in a region of ± 2 kbp around the TSS, calculated for the three clusters #1, #2 and #3 as described in (C). ( E– G ) Metagene profiles display the average of histone H3K36me3 enrichment [log2(ChIP/INPUT)] in a region of ± 2 kbp upstream of the TSS and downstream of the TES, calculated for control (black line) and CHD8 knockdown (gray line) hiNPCs for each of the three clusters identified in (C). The difference between H3K36me3 enrichment in control and CHD8 knockdown is significant for cluster #1, but not for clusters #2 or #3 (paired Cohen's d effect size statistics, see ). ( H ) Bar plot presenting the top 20 biological process GO terms significantly enriched in genes belonging to clusters #1 and #2, with high/medium CHD8 binding enrichment in control hiNPCs and a lower H3K36me3 enrichment in CHD8 knockdown (in E and F). Bars are colored according to –log10 (adjusted P -values) and the x -axis represents the number of genes per term.

Article Snippet: Membranes were blocked with 5% w/v non-fat dried milk and incubated with the following primary antibodies: anti-CHD8 (NB100-60417, Novus Biologicals) (1:1000), anti-HSP90 (4874S, Cell Signaling Tech.) (1:5000), anti-histone H3 (1:1.000) (4499, Cell Signaling Tech.) and anti-histone H3K36me3 (1:1.000) (Ab9050, Abcam).

Techniques: Control, Knockdown, Binding Assay

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: 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 (AS SUPPA) (A), and the overlap between genes bound by CHD8 (CHD8-bound) and genes presenting altered AS events as detected by SUPPA (AS SUPPA) (B). The number of genes for each condition is indicated. The enrichment significance for each intersection is computed by Fisher's exact test and represented by colors. Color-coded key: –log10( P -value). The P -value and odds ratio are reported. ( C ) The stacked bar plot represents the 1862 differential AS events detected by SUPPA, distributed by event type. SE, skipped event; RI, retained intron; MX, mixed event; A3, alternative 3′; A5, alternative 5′; AF, alternative first exon; AL, alternative last exon. ( D ) The bar plot reports the overlap between sequences located ± 100 bp from all differentially spliced exons (all, light gray), differentially spliced and bound by CHD8 (AS-bound, gray) or differentially spliced and losing H3K36me3 (AS-K36, dark gray) and known RBP family motif matching. The six most representative RBP families are reported. The exact percentage of sequences matched is indicated within each bar. ( E ) The bar plot represents GO biological process and KEGG pathways terms significantly enriched in genes presenting altered AS events as detected by SUPPA and losing H3K36me3 peaks following CHD8 knockdown (H3K36me3 lost, top panel) or bound by CHD8 (CHD8 bound, bottom panel). The bars are ordered according to adjusted P -value in –log10 scale; the x -axis represents the number of genes enriched for each term. ( F ) The image displays sashimi plots (top), chromatin tracks of H3K36me3 enrichment (middle) and ENSEMBL transcript IDs (bottom) for the ITSN1 locus. Sh- CHD8 samples versus Sh- GFP controls are color-coded (in orange and blue, respectively). Numbers of junction reads are depicted on the top panel. The green box and green arrows highlight the skipped exon event. Green arrows on the bottom panel show the location of primers used to analyze the skipped exon event. ( G ) Gel images represent PCR products obtained using ITSN1 (top) and TBP reference primers (bottom). Using the ITSN1 primer set, two amplicons are obtained corresponding to target exon inclusion (351 bp) and exon skipped (138 bp). ( H ) The bar graph reports Image-J PCR band quantification of ITSN1 transcripts normalized using TBP as the reference gene. PSI (% spliced in) is obtained by calculating the ratio between ITSN1 spliced in transcripts and the sum of spliced in plus spliced out events: PSI [in/(in + out)]. Means ± SE are shown. One-tail t -test was performed with NS P >0.05, * P ≤0.05, ** P ≤0.01. ( I ) The bar plot shows CHD8 enrichment over the INPUT following CHD8-ChIP qPCR quantification. ITSN1 genomic primers (green arrows in F) and gene desert control regions ( HGD4 and HGD12 ) were amplified for Sh- CHD8 and Sh- GFP conditions.

Article Snippet: Membranes were blocked with 5% w/v non-fat dried milk and incubated with the following primary antibodies: anti-CHD8 (NB100-60417, Novus Biologicals) (1:1000), anti-HSP90 (4874S, Cell Signaling Tech.) (1:5000), anti-histone H3 (1:1.000) (4499, Cell Signaling Tech.) and anti-histone H3K36me3 (1:1.000) (Ab9050, Abcam).

Techniques: Knockdown, Control, Amplification

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: hnRNPL as a novel CHD8 interactor: bridging altered splicing to H3K36me3 enrichment. ( A ) Schematic representation of the MS/MS experimental design and approach used in this work (figure created in BioRender.com). Nuclei from hiNPCs 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 μg 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 between 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 ). The number of proteins for each condition is indicated. The complete list of proteins is given in . ( E ) Complete list of the 18 CHD8-interacting proteins identified 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 μg of nuclear lysate, Input 5%, 0.75 μg 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 μg 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: Membranes were blocked with 5% w/v non-fat dried milk and incubated with the following primary antibodies: anti-CHD8 (NB100-60417, Novus Biologicals) (1:1000), anti-HSP90 (4874S, Cell Signaling Tech.) (1:5000), anti-histone H3 (1:1.000) (4499, Cell Signaling Tech.) and anti-histone H3K36me3 (1:1.000) (Ab9050, Abcam).

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

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: siRNA-mediated hnRNPL reduction causes AS changes that partly mirror those elicited by CHD8 suppression. ( A ) The bar graph reports the normalized expression levels (2 −ΔΔCT ) of hnRNPL transcript following Si- C administration for 72 h in hiNPCs. Four different biological replicates per experimental condition are reported. Means ± SE are shown. One-tail t -test was performed, with *** P ≤0.001. ( B ) Representative western blot images illustrate total hnRNPL levels, comparing hiNPC exposed to siRNA against hnRNPL (Si- C ) and control (Si- Scr ). Two representative biological replicates per condition are presented. Comparable amounts of total protein were loaded as detected by the HSP90 loading control. ( C ) The bars in the chart represent normalized hnRNPL protein levels comparing hiNPCs exposed to siRNA against hnRNPL (Si- C ) and control (Si- Scr ). HnRNPL bands were normalized on HSP90 loading control. Mean values ± SE from four independent biological replicates are plotted. T -test for two mean populations was performed, * P ≤0.05. ( D ) Venn diagrams represent the overlap between genes presenting aberrant splicing following CHD8 suppression and genes presenting altered AS events after siRNA-mediated reduction of hnRNPL (72 h post-electroporation). The AS events are detected by SUPPA. The number of genes for each condition is indicated. The enrichment significance for the intersection is computed by Fisher's exact test and represented by colors. Color-coded key: –log10( P -value). The P -value and odds ratio are reported. ( E ) The stacked bar plot represents the 473 differential AS events detected by SUPPA in hiNPCs subjected to siRNA against hnRNPL distributed by event type. Event types: SE, skipped event; RI, retained intron; MX, mixed event; A3, alternative 3′; A5, alternative 5′; AF, alternative first exon; AL, alternative last exon. ( F ) The bar plot represents GO biological process and KEGG pathway terms significantly enriched in genes presenting altered AS events as detected by SUPPA following CHD8 and hnRNPL suppression (intersection in D). The bars are ordered according to adjusted P -values in –log10 scale; the x -axis represents the number of genes enriched for each term. ( G ) CHD8 functions at transcription initiation, elongation and regulation of AS. Schematic representation of the molecular mechanisms proposed to explain CHD8’s roles at promoters/enhancers and in the modulation of AS (figure created in BioRender.com). CHD8 interacts with hnRNPL, possibly through stabilizing RNA bridges (red hairpins). Thus, CHD8/hnRNPL association, possibly also recruiting the Mediator complex , might stabilize enhancer/promoter looping and transcriptional initiation. However, hnRNPL also solidly interacts with SETD2 at elongating RNAPII, thus implicating CHD8 in the regulation of RNA processing and AS. By comparing CHD8 and SETD2 MS experiments [our data and ], a number of other hnRNP interactors emerge, providing a functional link between SETD2, hnRNPs and CHD8 with elongating RNAPII, with functional consequences for the regulation of the splicing machinery. Differential alternative splicing events can be modulated by the CHD8/hnRNPL/SETD2 complex.

Article Snippet: Membranes were blocked with 5% w/v non-fat dried milk and incubated with the following primary antibodies: anti-CHD8 (NB100-60417, Novus Biologicals) (1:1000), anti-HSP90 (4874S, Cell Signaling Tech.) (1:5000), anti-histone H3 (1:1.000) (4499, Cell Signaling Tech.) and anti-histone H3K36me3 (1:1.000) (Ab9050, Abcam).

Techniques: Expressing, Western Blot, Control, Electroporation, Functional Assay, Alternative Splicing

Journal: bioRxiv

Article Title: Novel CHD8 genomic targets identified in fetal mouse brain by in vivo Targeted DamID

doi: 10.1101/2021.01.12.426468

Figure Lengend Snippet: ( A ) Plasmids used for TaDa. The top plasmid is a diagram for CHD8 TaDa experiments. The middle plasmid is a diagram for Dam-only experiments. The bottom plasmid is a diagram for the in utero electroporation control injected with the CHD8 TaDa or Dam-only plasmids. ( B ) Schematic and flowchart of TaDa-seq experiments. E13.5 mouse embryos were injected with CHD8 TaDa or Dam-only plasmid and the in utero electroporation control plasmid. Four CHD8 TaDa and three Dam-only brains from the same litter were dissected. Frozen brains were then processed for the pipeline indicated in the grey boxes. ( C ) Immunohistochemistry showing overlap between green fluorescence (in utero electroporation control), red fluorescence (mCherry expression upstream of the CHD8 TaDa open reading frame), and DAPI (nuclei) illustrates successful transfection of experimental plasmids. ( D ) TaDa-seq computational analysis pipeline used in this study. ( E ) Schematic showing example signal from CHD8 TaDa or Dam-only protein binding at genomic loci.

Article Snippet: To generate the experimental plasmid, pCAG-mCherry-intronDam-CHD8, encoding the Dam methylase fused to the human CHD8 open reading frame (hereafter CHD8 TaDa), a full-length CHD8 isoform (Origene, RG230753) was subcloned by Gibson assembly into pCAG-mCherry-intronDam, C-terminal to the Dam methylase and a myc-tag.

Techniques: Plasmid Preparation, In Utero, Electroporation, Injection, Immunohistochemistry, Fluorescence, Expressing, Transfection, Protein Binding

Journal: bioRxiv

Article Title: Novel CHD8 genomic targets identified in fetal mouse brain by in vivo Targeted DamID

doi: 10.1101/2021.01.12.426468

Figure Lengend Snippet: Data showing CHD8 binding at loci previously identified in CHD8 binding characterization studies, including RNA processing genes, Hnrnpll and Srsf7 in panel A, Srsf1 and Sf3b1 in panel B, and a chromatin remodeling gene, Top1 , in panel B. Grey boxes highlight CHD8 binding near identified promoters of interest. CHD8 TaDa, Dam-only, or Dam-only normalized CHD8 TaDa (TaDa Dam Norm.) experiment tracks are in blue (representative biological replicates shown), CHD8 ChIP-seq experiments are in grey, and datasets of histone and chromatin accessibility signatures from the ENCODE consortium are in black. Linear representations of genes from the mouse mm10 genome are shown below coverage tracks. Height of the y-axis is scaled to show the peak for each track separately.

Article Snippet: To generate the experimental plasmid, pCAG-mCherry-intronDam-CHD8, encoding the Dam methylase fused to the human CHD8 open reading frame (hereafter CHD8 TaDa), a full-length CHD8 isoform (Origene, RG230753) was subcloned by Gibson assembly into pCAG-mCherry-intronDam, C-terminal to the Dam methylase and a myc-tag.

Techniques: Binding Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: Novel CHD8 genomic targets identified in fetal mouse brain by in vivo Targeted DamID

doi: 10.1101/2021.01.12.426468

Figure Lengend Snippet: ( A ) Bar plots showing association of peaks with transcription start sites (TSS) using the GREAT online analysis tool. Bins along the x-axis represent 5, 50, 500, and greater than 500 kilobases away from the nearest TSS. ( B ) Genome-wide coverage heatmaps showing enrichment of signal at peaks for each dataset indicated on the left-hand side. Y axis of datasets were matched for visual comparison. Small line plots indicate the average normalized peak enrichment for each dataset with the color for each line next to each dataset name. Each peak is centered along the middle of each plot with a 3 kilobase pair window on each side. The legend indicates normalized enrichment. ( C ) Venn diagram showing the number of peaks annotated to genes overlapping with CHD8 TaDa, Embryonic CHD8 ChIP-seq, and Adult CHD8 ChIP-seq using stringent CHD8 TaDa-seq peak thresholding with peaks meeting an FDR < 0.00001 cutoff in at least 3 replicates (Top) or a looser threshold of peaks present in at least 3 replicates (Bottom). ( D ) Genome coverage correlation heatmap showing relationship between representative CHD8 TaDa-seq, Dam-only, CHD8 ChIP-seq, and ENCODE histone mark and chromatin accessibility datasets. Data are hierarchically clustered according to genome-wide similarity as indicated by a dendrogram. Legend indicates the correlation value between datasets. H3K27me3 is a histone mark associated with repressed DNA loci. H3K4me3 is a histone mark associated with actively transcribed promoters. ATAC-seq is sequencing data of open chromatin regions. H3K4me1 and H3K27ac are histone marks associated with putative enhancers. ( E ) Table showing functional annotations associated with CHD8 TaDa-seq called peaks. Region % refers to the percent of the total peak set annotated to each term.

Article Snippet: To generate the experimental plasmid, pCAG-mCherry-intronDam-CHD8, encoding the Dam methylase fused to the human CHD8 open reading frame (hereafter CHD8 TaDa), a full-length CHD8 isoform (Origene, RG230753) was subcloned by Gibson assembly into pCAG-mCherry-intronDam, C-terminal to the Dam methylase and a myc-tag.

Techniques: Genome Wide, ChIP-sequencing, Sequencing, Functional Assay

Journal: bioRxiv

Article Title: Novel CHD8 genomic targets identified in fetal mouse brain by in vivo Targeted DamID

doi: 10.1101/2021.01.12.426468

Figure Lengend Snippet: HOMER results of the top 30 motifs identified in CHD8 TaDa-seq peaks. Target sequences refer to sequences from CHD8 TaDa-seq peaks used as input. Background sequences refers to random sequence-content matched intervals in the genome.

Article Snippet: To generate the experimental plasmid, pCAG-mCherry-intronDam-CHD8, encoding the Dam methylase fused to the human CHD8 open reading frame (hereafter CHD8 TaDa), a full-length CHD8 isoform (Origene, RG230753) was subcloned by Gibson assembly into pCAG-mCherry-intronDam, C-terminal to the Dam methylase and a myc-tag.

Techniques: Sequencing

Journal: bioRxiv

Article Title: Novel CHD8 genomic targets identified in fetal mouse brain by in vivo Targeted DamID

doi: 10.1101/2021.01.12.426468

Figure Lengend Snippet: Genome coverage correlation heatmap showing relationship between CHD8 TaDa-seq replicates, Chd8 ChIP-seq replicates, and ENCODE histone mark and chromatin accessibility dataset replicates. Data are hierarchically clustered according to similarity as indicated by a dendrogram. Differences in signal intensity between CHD8 TaDa-seq replicates in the correlation heatmaps were due to differences in sequencing depth. The legend indicates the correlation value between datasets. H3K27me3 is a histone mark associated with repressed DNA loci. H3K4me3 is a histone mark associated with actively transcribed promoters. ATAC-seq identifies regions of open chromatin. H3K4me1 and H3K27ac are histone marks associated with putative enhancers. CHD8 TaDa Merge – Merged CHD8 TaDa-seq dataset. Dam-only Merge – Merged Dam-only dataset.

Article Snippet: To generate the experimental plasmid, pCAG-mCherry-intronDam-CHD8, encoding the Dam methylase fused to the human CHD8 open reading frame (hereafter CHD8 TaDa), a full-length CHD8 isoform (Origene, RG230753) was subcloned by Gibson assembly into pCAG-mCherry-intronDam, C-terminal to the Dam methylase and a myc-tag.

Techniques: ChIP-sequencing, Sequencing

Journal: bioRxiv

Article Title: Novel CHD8 genomic targets identified in fetal mouse brain by in vivo Targeted DamID

doi: 10.1101/2021.01.12.426468

Figure Lengend Snippet: ( A ) Box and whisker plots showing comparison between CHD8 TaDa-seq peak rank and an E17.5 Chd8 haploinsufficiency differential gene expression dataset. Change in log fold counts per million of genes according to CHD8 binding (Left). Change in log fold change of genes according to CHD8 binding (Right). Boxes were plotted according to CHD8 binding affinity bins: all genes meeting at least 0.1 count per million sequencing coverage (Expressed Genes), any genes having CHD8 binding (All Bound Genes), and the top 1000 genes near CHD8 peaks (Top 1000 Bound). Notches indicate values within the 95% confidence interval of the median. ( B ) Box and whisker plots showing comparison between Dam-only peak rank and an E17.5 Chd8 haploinsufficiency differential gene expression dataset. Change in log fold counts per million of genes according to Dam binding (Left). Change in log fold change of genes according to Dam binding (Right). Boxes were plotted according to CHD8 binding affinity bins: all genes meeting at least 0.1 count per million sequencing coverage (Expressed Genes), any genes having CHD8 binding (All Bound Genes), and top 1000 genes near CHD8 peaks (Top 1000 Bound). Notches indicate values within the 95% confidence interval of the median. ( C-D left ) Venn diagrams indicating the number of genes overlapping between the CHD8 TaDa-seq and E17.5 Chd8 haploinsufficiency significant (p < 0.05) downregulated and upregulated datasets. ( C-D right ) Tables showing functional annotations associated with genes having CHD8 binding in downregulated (C) and upregulated (D) genes from the E17.5 Chd8 haploinsufficiency dataset (p < 0.05) using goseq. Enrichment values indicate the percent of genes in the dataset that are differentially expressed and bound by CHD8 via TaDa-seq in relation to the total number of genes associated with each term.

Article Snippet: To generate the experimental plasmid, pCAG-mCherry-intronDam-CHD8, encoding the Dam methylase fused to the human CHD8 open reading frame (hereafter CHD8 TaDa), a full-length CHD8 isoform (Origene, RG230753) was subcloned by Gibson assembly into pCAG-mCherry-intronDam, C-terminal to the Dam methylase and a myc-tag.

Techniques: Whisker Assay, Expressing, Binding Assay, Sequencing, Functional Assay

Journal: bioRxiv

Article Title: Novel CHD8 genomic targets identified in fetal mouse brain by in vivo Targeted DamID

doi: 10.1101/2021.01.12.426468

Figure Lengend Snippet: ( A-C ) CHD8 binding near genes important for regulation of neuronal gene expression, Myt1l (A), and synaptic function, Ank3 (B) and Dlg4 (C). Grey boxes highlight CHD8 binding near select promoter and distal regions of interest overlapping with putative enhancer marks (H3K27ac and H3K4me1). CHD8 TaDa-seq experiment tracks are in blue, CHD8 ChIP-seq experiments are in grey, and datasets of histone and chromatin accessibility signatures from the ENCODE consortium are in black. Linear representations of genes from the mouse mm10 genome are shown below coverage tracks. Height of the y-axis is scaled to show the peak for each track separately. ( D ) Table showing functional annotations associated with promoter proximal (<1kb from TSS) (Top) and promoter distal (Bottom) regions. Rank refers to the rank within the dataset. A rank of 1 would mean the annotation with the smallest FDR value (aka the most significant). Region hits capture the number of peaks associated with each term. Region % captures the percent of regions captured compared to the total number of peaks.

Article Snippet: To generate the experimental plasmid, pCAG-mCherry-intronDam-CHD8, encoding the Dam methylase fused to the human CHD8 open reading frame (hereafter CHD8 TaDa), a full-length CHD8 isoform (Origene, RG230753) was subcloned by Gibson assembly into pCAG-mCherry-intronDam, C-terminal to the Dam methylase and a myc-tag.

Techniques: Binding Assay, Expressing, ChIP-sequencing, Functional Assay

Journal: bioRxiv

Article Title: Novel CHD8 genomic targets identified in fetal mouse brain by in vivo Targeted DamID

doi: 10.1101/2021.01.12.426468

Figure Lengend Snippet: Table showing functional annotations associated with peaks in CHD8 ChIP-seq datasets. Region hits capture the number of peaks associated with each term. Region % captures the percent of regions captured compared to the total number of peaks.

Article Snippet: To generate the experimental plasmid, pCAG-mCherry-intronDam-CHD8, encoding the Dam methylase fused to the human CHD8 open reading frame (hereafter CHD8 TaDa), a full-length CHD8 isoform (Origene, RG230753) was subcloned by Gibson assembly into pCAG-mCherry-intronDam, C-terminal to the Dam methylase and a myc-tag.

Techniques: Functional Assay, ChIP-sequencing