alexa fluor 594 conjugated rabbit anti chd7  (Novus Biologicals)

Journal: Human Genetics

Article Title: CHD7 variants associated with hearing loss and enlargement of the vestibular aqueduct

doi: 10.1007/s00439-023-02581-x

Figure Lengend Snippet: Seven variants were identified in CHD7 in seven families with bilateral sensorineural hearing loss and bilateral enlargement of the vestibular aqueduct (EVA)

Article Snippet: The preparations were then permeabilized for 30 min in PBS with 0.5% Triton X-100, blocked for 1 h at RT in PBS containing 1% BSA and 10% donkey serum, then incubated with the primary antibodies diluted in the same solution for 24 h at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\circ{\rm C}$$\end{document} ∘ C . Rabbit monoclonal anti-CHD7 (D3F5, #6505S, LOT: 1, Cell signaling Technology, Danvers, MA, USA, raised against a recombinant protein corresponding to the N-terminal region of human CHD7 protein), Alexa Fluor 594-conjugated rabbit anti-CHD7 (#NBP1-77393AF594, LOT: A-030520-AF594, Novus, Littleton, CO, USA, raised against the N-terminal region of the human CHD7 protein (within residues 25–200) [Swiss-Prot Q9P2D1]), and goat anti-FOXI1 (RRID:AB_732416, ab20454, Abcam, Cambridge, MA, USA) antibodies were used as primary antibodies at 1:50, 1:200 and 1:200 dilutions, respectively.

Techniques: Variant Assay, Mutagenesis

Journal: Human Genetics

Article Title: CHD7 variants associated with hearing loss and enlargement of the vestibular aqueduct

doi: 10.1007/s00439-023-02581-x

Figure Lengend Snippet: CHD7 variants and inner ear phenotypes. A Schematic representation of the coding exons of CHD7 and reported functional domains of the CHD7 protein. The variants NM_017780.4: c.3553A > G [p.(Met1185Val)] and c.5390G > C [p.(Gly1797Ala)] were identified in our EVA cohort in families 276 and 388, and are likely causal. The other variants presented in Table not thought to be pathogenic are not shown. B Segregation of EVA, SNHL, and CHD7 variants in families 276 and 388. Variants in bold are thought to be causal. One subject in family 388, who did not enroll in the study, was reported to have had bilateral hearing loss since 10 years of age which progressed to severe levels requiring bilateral hearing aids by the age of 39 years. This functional phenotype was not evaluated by radiologic imaging but was considered to be consistent with EVA (grey symbol). Other subjects had reported hearing loss whose onset and progression in adulthood was more consistent with aging as the primary etiology (presbycusis) rather than EVA (striped symbol). The genotype is indicated for all the gDNA available. “ + ” denotes a wild-type allele. C Chromatograms showing the presence of the c.3553A > G and c.5390G > C heterozygous variants of CHD7 detected in families 276 and 388. D Conservation of human CHD7 amino acid residues Met1185 and Gly1797 among vertebrate and invertebrate ( Drosophila melanogaster , Caenorhabditis elegans ) species. Blue highlighting reflects conservation among 11 (light blue) or 13–14 (dark blue) of the 14 orthologs shown. Sequences obtained through Uniprot website were analyzed in Jalview. Alignment was performed using ClustalW. Protein sequences identifiers for CHD7 orthologues are Q9P2D1 for Homo sapiens , F6PP91 for Macaca mulatta , F1PWD8 for Canis familiaris , G3UE09 for Loxodonta africana , F7G444 for Monodelphis domestica , A2AJK6 for Mus musculus , A0A452E916 for Capra hircus , A0A674K692 for Terrapene carolina triunguis , Q06A37 for Gallus gallus , U3JST3 for Ficedula albicollis , A0A1L8FT46 for Xenopus laevis , F1QGL1 for Danio rerio , M9NEL3 for Drosophila melanogaster , O61845 for Caenorhabditis elegans

Article Snippet: The preparations were then permeabilized for 30 min in PBS with 0.5% Triton X-100, blocked for 1 h at RT in PBS containing 1% BSA and 10% donkey serum, then incubated with the primary antibodies diluted in the same solution for 24 h at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\circ{\rm C}$$\end{document} ∘ C . Rabbit monoclonal anti-CHD7 (D3F5, #6505S, LOT: 1, Cell signaling Technology, Danvers, MA, USA, raised against a recombinant protein corresponding to the N-terminal region of human CHD7 protein), Alexa Fluor 594-conjugated rabbit anti-CHD7 (#NBP1-77393AF594, LOT: A-030520-AF594, Novus, Littleton, CO, USA, raised against the N-terminal region of the human CHD7 protein (within residues 25–200) [Swiss-Prot Q9P2D1]), and goat anti-FOXI1 (RRID:AB_732416, ab20454, Abcam, Cambridge, MA, USA) antibodies were used as primary antibodies at 1:50, 1:200 and 1:200 dilutions, respectively.

Techniques: Functional Assay, Imaging

Journal: Human Genetics

Article Title: CHD7 variants associated with hearing loss and enlargement of the vestibular aqueduct

doi: 10.1007/s00439-023-02581-x

Figure Lengend Snippet: Radiological images of temporal bones of affected individuals with p.(Met1185Val) and p.(Gly1797Val) variants of CHD7 . Montage of axial computed tomography (CT) (rows 1–4) and magnetic resonance (MR) (row 5) temporal bone imaging in a cohort subject with normal labyrinths and vestibular aqueducts (row 1), and from subjects with CHD7 variant p.(Met1185Val) (row 2) and CHD7 variant p.(Gly1797Ala) (rows 3–5). In row 1, normal anatomy is shown for reference, including the lateral semicircular canals (white arrows), which are often hypoplastic or absent in patients with CHARGE syndrome, and the normal vestibular aqueducts (white arrowheads) with a midpoint diameter less than 1 mm. For each patient (1810, 2106–2108), the lateral semicircular canals are well-formed, indistinguishable from normal (row 1, white arrow) and the vestibular aqueducts are enlarged (rows 2–5, white arrowheads) measuring 3 to 6 mm at the midpoint in each case. R right ear, L left ear

Article Snippet: The preparations were then permeabilized for 30 min in PBS with 0.5% Triton X-100, blocked for 1 h at RT in PBS containing 1% BSA and 10% donkey serum, then incubated with the primary antibodies diluted in the same solution for 24 h at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\circ{\rm C}$$\end{document} ∘ C . Rabbit monoclonal anti-CHD7 (D3F5, #6505S, LOT: 1, Cell signaling Technology, Danvers, MA, USA, raised against a recombinant protein corresponding to the N-terminal region of human CHD7 protein), Alexa Fluor 594-conjugated rabbit anti-CHD7 (#NBP1-77393AF594, LOT: A-030520-AF594, Novus, Littleton, CO, USA, raised against the N-terminal region of the human CHD7 protein (within residues 25–200) [Swiss-Prot Q9P2D1]), and goat anti-FOXI1 (RRID:AB_732416, ab20454, Abcam, Cambridge, MA, USA) antibodies were used as primary antibodies at 1:50, 1:200 and 1:200 dilutions, respectively.

Techniques: Computed Tomography, Imaging, Variant Assay

Journal: Human Genetics

Article Title: CHD7 variants associated with hearing loss and enlargement of the vestibular aqueduct

doi: 10.1007/s00439-023-02581-x

Figure Lengend Snippet: Structural models of human CHD7 in active and inactive states. A Structural models based upon the alignment shown in Fig. S3 of CHD7 containing two chromodomains (CRD 1 and 2, purple), an ATPase motor (lobe 1 in orange and lobe 2 in green) and a SANT–SLIDE domain (pink) that binds DNA. Domains are colored as in Farnung et al. (Farnung et al. ). The coordinates of the histone complex (shown in gray cylinders) and the double strand DNA (blue) in CHD7 active form were obtained for purpose of visual display, after structural superimposition of the active template structure onto the model obtained. For the inactive model, the coordinates of the histone complex and the double strand DNA were obtained after structural superimposition of lobe 1 of the ATPase motor and SANT–SLIDE domains of the active template onto the inactive model. C-alpha atoms of residues Met1185 and Gly1797 are shown as blue spheres, while the atoms forming the nucleotides are shown as yellow (C), red (O), orange (P), blue (N) spheres. BeF3 is shown as cyan spheres (right model). Structural changes in CHD7 between the active and inactive states are indicated with arrows: the 40° rotation of lobe 2 in the ATPase motor and the 15° rotation of the chromodomains with respect to the DNA. B , C Close-up views of the structural models of inactive and active states of wild-type CHD7, and with the substitutions p.(Met1185Val) ( B ) and p.(Gly1797Ala) ( C ). The residues at positions 1185 and 1797 as well as those within 6 Å of the variant site are shown as sticks. The interacting networks involving Met1185 and Gly1797, Val1185 and Ala1797 are indicated as dashed lines. The corresponding C-alpha atom of each of these residues is shown as a blue sphere

Article Snippet: The preparations were then permeabilized for 30 min in PBS with 0.5% Triton X-100, blocked for 1 h at RT in PBS containing 1% BSA and 10% donkey serum, then incubated with the primary antibodies diluted in the same solution for 24 h at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\circ{\rm C}$$\end{document} ∘ C . Rabbit monoclonal anti-CHD7 (D3F5, #6505S, LOT: 1, Cell signaling Technology, Danvers, MA, USA, raised against a recombinant protein corresponding to the N-terminal region of human CHD7 protein), Alexa Fluor 594-conjugated rabbit anti-CHD7 (#NBP1-77393AF594, LOT: A-030520-AF594, Novus, Littleton, CO, USA, raised against the N-terminal region of the human CHD7 protein (within residues 25–200) [Swiss-Prot Q9P2D1]), and goat anti-FOXI1 (RRID:AB_732416, ab20454, Abcam, Cambridge, MA, USA) antibodies were used as primary antibodies at 1:50, 1:200 and 1:200 dilutions, respectively.

Techniques: Variant Assay

Journal: Human Genetics

Article Title: CHD7 variants associated with hearing loss and enlargement of the vestibular aqueduct

doi: 10.1007/s00439-023-02581-x

Figure Lengend Snippet: Chd7 expression in the developing mouse endolymphatic sac and duct. A , B Single cell expression data (scRNA-seq (Honda et al. )) showing Chd7 expression in the different cell types of the developing mouse endolymphatic sac at E12.5, E16.5, P5 and P30 visualized in the gEAR portal ( https://umgear.org ) ( A ). Violin plots of Chd7 and Foxi1 expression in the cells of the endolymphatic sac and their precursor cells ( B ). While the transcription factor Foxi1 is expressed preferentially in MRCs and their precursor cells, Chd7 is expressed in all cell types of the endolymphatic sac. Each cell group is labelled based on expression patterns of canonical cell markers and results of gene ontology enrichment analysis. ProlC, proliferating cells (representative differentially expressed genes are Mki67, Birc5, Tpx2 ); ProgC, progenitor cells ( Lmx1a, Bmp3, Col11a1 ); MRC, mitochondria-rich cells ( Slc26a4, Foxi1, Atp6v0a4, Atp6v1b1 ); RRC, ribosome-rich cells ( Agt, Cav1, Clu, Dmkn ); Values: TPM transcript per million. C , D Developmental expression pattern of Chd7 in the endolymphatic sac and duct of Chd7 Gt /+ mice revealed by X-gal staining. Low magnification view of whole-mount X-gal staining of Chd7 Gt /+ endolymphatic sac and duct preparations show Chd7 expression at E14.5, E16.5 and P0 ( C ). Close-up views of the endolymphatic sac at higher magnification at E16.5, P5 and P30 ( D ). X-gal staining (blue dots) was detected in the different regions of the endolymphatic sac and along the endolymphatic duct at E14.5, E16.5, P0. Staining seemed to be more intense in the endolymphatic duct at E14.5, E16.5, as compared to the rest of the endolymphatic sac, whereas the staining seemed more homogenous at P0. At P5 and P30, staining was still detected in the endolymphatic sac. No staining was detected in endolymphatic sac and duct tissue from wild-type (WT) littermates processed in parallel. Three to four litters were studied for each datapoint. Whenever possible, animals of both sexes were studied and observed to have no obvious differences. Scale bars: 100 μm ( C ), 20 μm ( D )

Article Snippet: The preparations were then permeabilized for 30 min in PBS with 0.5% Triton X-100, blocked for 1 h at RT in PBS containing 1% BSA and 10% donkey serum, then incubated with the primary antibodies diluted in the same solution for 24 h at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\circ{\rm C}$$\end{document} ∘ C . Rabbit monoclonal anti-CHD7 (D3F5, #6505S, LOT: 1, Cell signaling Technology, Danvers, MA, USA, raised against a recombinant protein corresponding to the N-terminal region of human CHD7 protein), Alexa Fluor 594-conjugated rabbit anti-CHD7 (#NBP1-77393AF594, LOT: A-030520-AF594, Novus, Littleton, CO, USA, raised against the N-terminal region of the human CHD7 protein (within residues 25–200) [Swiss-Prot Q9P2D1]), and goat anti-FOXI1 (RRID:AB_732416, ab20454, Abcam, Cambridge, MA, USA) antibodies were used as primary antibodies at 1:50, 1:200 and 1:200 dilutions, respectively.

Techniques: Expressing, Staining

Journal: Human Genetics

Article Title: CHD7 variants associated with hearing loss and enlargement of the vestibular aqueduct

doi: 10.1007/s00439-023-02581-x

Figure Lengend Snippet: Immunolocalization of CHD7 in the developing mouse endolymphatic sac and duct. Confocal microscopy analysis of whole-mount endolymphatic sac and duct preparations from E16.5 mice labelled with antibodies directed against CHD7 (red) and FOXI1 (a mitochondria-rich cell marker, green). Hoechst 33342 (blue) was used to label cell nuclei. Experiments were conducted with two different antibodies directed against CHD7 N-terminal region, with similar results. Here are shown the results with Alexa Fluor 594 conjugated anti-CHD7 from Novus. Maximal intensity projection images at E16.5. A Low magnification images of the endolymphatic sac and duct. B Higher magnification images of the open endolymphatic sac. No labeling was detected in the absence of primary antibodies, or in skin tissue used as a negative control. CHD7 was detected in the nuclei of the cells all along the endolymphatic duct and in both mitochondria-rich cells and ribosome-rich cells in the endolymphatic sac. Scale bars: 100 μm ( A ), 10 μm ( B )

Article Snippet: The preparations were then permeabilized for 30 min in PBS with 0.5% Triton X-100, blocked for 1 h at RT in PBS containing 1% BSA and 10% donkey serum, then incubated with the primary antibodies diluted in the same solution for 24 h at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^\circ{\rm C}$$\end{document} ∘ C . Rabbit monoclonal anti-CHD7 (D3F5, #6505S, LOT: 1, Cell signaling Technology, Danvers, MA, USA, raised against a recombinant protein corresponding to the N-terminal region of human CHD7 protein), Alexa Fluor 594-conjugated rabbit anti-CHD7 (#NBP1-77393AF594, LOT: A-030520-AF594, Novus, Littleton, CO, USA, raised against the N-terminal region of the human CHD7 protein (within residues 25–200) [Swiss-Prot Q9P2D1]), and goat anti-FOXI1 (RRID:AB_732416, ab20454, Abcam, Cambridge, MA, USA) antibodies were used as primary antibodies at 1:50, 1:200 and 1:200 dilutions, respectively.

Techniques: Confocal Microscopy, Marker, Labeling, Negative Control

Journal: Cardiovascular Research

Article Title: CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression

doi: 10.1093/cvr/cvad059

Figure Lengend Snippet: Direct targets of CHD7 in the CPM, identified by CUT&RUN. ( A ) PCA of in vivo control ‘SHF’ and ‘HEART’ samples compared with in vitro CM differentiation time points from Wamstad et al. based on RNA-seq data. ( B ) Read density heatmaps (+/−2 kb relative to the centre of CHD7 peaks) and density profiles of CUT&RUN-seq in 3 biological replicates. Orange, CHD7 antibody; blue, IgG antibody; grey, input. ( C ) Distribution of distances from CHD7 peaks to the nearest TSS. ( D ) GO biological processes enriched in genes associated with CHD7 peaks. Terms are ranked by P -value from the Fisher exact test, by Enrichr. ( E ) Venn diagram of DEGs in Chd7 cKO ‘SHF’ with CHD7 binding, and GO terms enriched in these genes ( F ). ( G ) DEGs in Chd7 cKO ‘HEART’ with CHD7 binding, and GO terms enriched ( H ). P -values of Venn diagram overlaps from hypergeometric test and P -values of GO analysis from Fisher exact test.

Article Snippet: The immunoprecipitated samples were analysed by western blot, using antibodies against CHD7 (CST 6505) and ISL1 (DSHB 39.05D).

Techniques: In Vivo, Control, In Vitro, RNA Sequencing, Binding Assay

Journal: Cardiovascular Research

Article Title: CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression

doi: 10.1093/cvr/cvad059

Figure Lengend Snippet: Loss of Chd7 in CPM dysregulates the cardiogenic GRN. ( A ) Whole mount E9.5 embryo showing the regions micro-dissected for the ‘SHF’ and ‘HEART’ RNA-seq experiments. ( B–C ) MA plots showing global gene expression changes in ‘SHF’ ( B ) and ‘HEART’ ( C ) in Chd7 cKO embryos relative to controls. Significantly changed genes with FDR < 0.1 are highlighted in red (up-regulated) or blue (down-regulated). ( D ) GO enrichment and ( E ) Jensen disease analysis for the 222 genes down-regulated in Chd7 cKO compared to control ‘SHF’. ( F ) GO terms and ( G ) OMIM disease terms enriched in the 426 genes down-regulated in Chd7 cKO ‘HEART’. Terms are ranked by P -values from the Fisher exact test, by Enrichr. ( H–J ) Venn diagrams showing the overlap between DEGs in Chd7 cKO ‘SHF’ and ‘HEART’ samples ( H ), ‘HEART’ and E11.5 hearts ( I ), and between ‘HEART’ and E13.5 hearts ( J ). DEGs in E11.5 and E13.5 hearts were obtained from previously published data. P -values based on hypergeometric test.

Article Snippet: The immunoprecipitated samples were analysed by western blot, using antibodies against CHD7 (CST 6505) and ISL1 (DSHB 39.05D).

Techniques: RNA Sequencing, Gene Expression, Control

Journal: Cardiovascular Research

Article Title: CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression

doi: 10.1093/cvr/cvad059

Figure Lengend Snippet: Loss of CHD7 in the Meps1 lineage leads to reduced expression of anterior SHF markers. Whole mount in situ HCR staining of control and Chd7 cKO ( Mesp1-Cre; Chd7 fl/fl ) embryos at E9–9.5 for Fgf10 ( A–B’’’ ), Isl1 ( C–D’’’ ) and Mef2c ( E–F’’’ ). Boxed regions in ( A ), ( B ), ( C ), ( D ), ( E ), ( F ) are shown in neighbouring panels. Confocal maximum projection of the entire embryo and selected lateral or medial z-stacks are displayed. n = 4. See , for selected stacks highlighting domain differences. Arrowheads indicate cells in branchial arches; green bracket shows the SHF region. Scale bars represent 100 μm. ov, otic vesicle; SHF, second heart field; RV, right ventricle; FL, forelimb; DPW, dorsal pericardial wall; oft, outflow tract; mn, motor neurons.

Article Snippet: The immunoprecipitated samples were analysed by western blot, using antibodies against CHD7 (CST 6505) and ISL1 (DSHB 39.05D).

Techniques: Expressing, In Situ, Staining, Control

Journal: Cardiovascular Research

Article Title: CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression

doi: 10.1093/cvr/cvad059

Figure Lengend Snippet: Genes expressed in the venous pole progenitor cells (pSHF) are upregulated in Chd7 cKO embryos. Maximum intensity projections of whole mount in situ HCR of control and Chd7 cKO embryos for Tbx5 ( A–B’ ), Osr1 ( C–D’ ), Foxf1 ( E–F’ ) and Wnt4 ( G–H’ ). Boxed regions in ( A ), ( B ), ( C ), ( D ), ( E ), ( F ), ( G ), ( H ) are shown in the neighbouring panel. Arrows in ( A’ ) and ( B ’) indicate the expanded expression of Tbx5 in the cKO embryos. n = 4. Scale bars, 100 μm. FL, forelimb; pSHF, posterior second heart field.

Article Snippet: The immunoprecipitated samples were analysed by western blot, using antibodies against CHD7 (CST 6505) and ISL1 (DSHB 39.05D).

Techniques: In Situ, Control, Expressing

Journal: Cardiovascular Research

Article Title: CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression

doi: 10.1093/cvr/cvad059

Figure Lengend Snippet: CHD7 binds cardiac enhancers in CP cells. ( A ) Overlaps between CHD7 CUT&RUN peaks and active and poised cardiac enhancers. Odds ratio (OR) and P -value by Fisher’s test from each stage is presented, with the overlap defined by BEDTools. ( B ) Overlap of CHD7 peaks with enhancers during MES to CP transition, based on their activity. OR and P -value by Fisher’s test is presented. Genome browser snapshots at Isl1 ( C ) and Fgf10 ( D ) loci. Snapshots include tracks from CHD7 peaks (mid blue), ISL1 peaks (purple), CP enhancers from Wamstad et al. (dark blue), H3K4me1 from E10.5 hearts (light blue) and H3K27ac from E10.5 hearts (green). In ( C ), CP enhancers are expanded and their activity at each stage is indicated as absent (−1), poised (0) or active (1). Details on the tracks used can be found in the methods section.

Article Snippet: The immunoprecipitated samples were analysed by western blot, using antibodies against CHD7 (CST 6505) and ISL1 (DSHB 39.05D).

Techniques: Activity Assay

Journal: Cardiovascular Research

Article Title: CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression

doi: 10.1093/cvr/cvad059

Figure Lengend Snippet: CHD7 binding sites co-localize with a subset of ISL1-bound sites and are enriched for cardiac TFs. ( A–C ) TF motifs enriched in all CHD7 peaks ( A ) and in CHD7 peaks associated with ‘SHF’ DEGs ( B ) or ‘HEART’ DEGs ( C ), as discovered by HOMER. ( D ) GO:biological processes enriched in CHD7-ISL1 common peaks, ranked by P -values from the Fisher exact test, by Enrichr. ( E ) Distribution of distances from CHD7-ISL1 common peaks to the nearest TSS. ( F ) Top ten transcription factors bound at the promoters of genes associated with CHD7 peaks, based on the ChEA function of Enrichr ( P -value from Fisher exact test). HS, human; MM, mouse. ( G ) Co-immunoprecipitation with ISL1 antibody and western blot analysis for CHD7 (top) or ISL1 (bottom) in day 5 CP cells. 10% of each IP and 2.5% of input was run on 10% SDS-PAGE gel; arrow indicates CHD7 band at ∼340kD.

Article Snippet: The immunoprecipitated samples were analysed by western blot, using antibodies against CHD7 (CST 6505) and ISL1 (DSHB 39.05D).

Techniques: Binding Assay, Immunoprecipitation, Western Blot, SDS Page

Journal: Cardiovascular Research

Article Title: CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression

doi: 10.1093/cvr/cvad059

Figure Lengend Snippet: CHD7—proposed modes of action using Fgf10 enhancer as example. (An extended version of the model presented by Watanabe et al . ) As mentioned above, while CHD7 may participate in recruitment of histone modifiers and splicing the model focuses on its ATPase dependent nucleosome remodelling activity. Depending upon additional specific transcriptional regulators, opening of chromatin could be associated with either up or down regulation, and conversely closure could again be associated with increased or decreased target expression. In the context of the regulation of Fgf10 expression specifically via the intragenic enhancer bound by ISL1 (and TBX1), and its repression via NKX2.5, absence of CHD7 would be predicted to interfere with this balanced control as cells progress from a progenitor to differentiated state.

Article Snippet: The immunoprecipitated samples were analysed by western blot, using antibodies against CHD7 (CST 6505) and ISL1 (DSHB 39.05D).

Techniques: Activity Assay, Expressing, Control

Journal: Molecular Biology of the Cell

Article Title: ATM–dependent phosphorylation of CHD7 regulates morphogenesis-coupled DSB stress response in fetal radiation exposure

doi: 10.1091/mbc.e22-10-0450

Figure Lengend Snippet: FIGURE 2: Detection of radiation-induced unrepaired DSBs by anti-pCHD7 antibody staining. (A) Structure of the CHD7 protein and (B) potential site of ATM kinase–mediated phosphorylation. (C) pCHD7 and γH2AX antibody staining of 10 Gy-3 wk cultured cells. (D) Western blot analysis with anti-pCHD7, CHD7, and TBP antibody. TBP: TATA-binding protein as internal control. (E) Effect of ATM kinase inhibitor, KU55933, on the repair foci formation in cells at 1 Gy-1 h. pCHD7 staining appears red. (F) The effect of the polyubiquitination inhibitor MG132 on the repair foci formation in 1 Gy-1 h cells. Note that contrast was enhanced in some of the photo images, for example, in F, MG132 (+) 53BP1, to confirm the lack of positive signals in the very dark nuclear images. Scale bar, 5 μm.

Article Snippet: After the debris was removed, an anti-CHD7 antibody (CST; 6505S) was added and incubated overnight at 4°C.

Techniques: Staining, Phospho-proteomics, Cell Culture, Western Blot, Binding Assay, Control

Journal: Molecular Biology of the Cell

Article Title: ATM–dependent phosphorylation of CHD7 regulates morphogenesis-coupled DSB stress response in fetal radiation exposure

doi: 10.1091/mbc.e22-10-0450

Figure Lengend Snippet: FIGURE 3: Characterization of pCHD7 antibody by immunoprecipitation and cell staining. (A) Cell lysates from 5 Gy-1 h HCA2 were subjected to immunoprecipitation using the pCHD7 antibody. Then, the samples were analyzed by Western blotting using pCHD7, CHD7, or 53BP1 antibodies. Individual lanes indicate: T, total nuclear proteins; N, normal goat IgG used for the control of immunoprecipitation; and I.P, a pCHD7 antibody was used for immunoprecipitation. (B) Four-aa SSQP sequence including ATM kinase target SQ at the serine 2255 in CHD7 also exists at serine 831 in 53BP1. Furthermore, the serine position of CHD7 also matches at the serine 25 in 53BP1, with one aa shifted to make the four-aa sequence SQPE. (C) pCHD7 foci formation at DSB sites following irradiation in 53BP1-KO MEFs. Colocalization of pCHD7 and γH2AX was observed after irradiation (1 Gy in right panel) but no 53BP1 foci were observed (1 Gy in left panel). Scale bar, 5 μm.

Article Snippet: After the debris was removed, an anti-CHD7 antibody (CST; 6505S) was added and incubated overnight at 4°C.

Techniques: Immunoprecipitation, Staining, Western Blot, Control, Sequencing, Irradiation

Journal: Molecular Biology of the Cell

Article Title: ATM–dependent phosphorylation of CHD7 regulates morphogenesis-coupled DSB stress response in fetal radiation exposure

doi: 10.1091/mbc.e22-10-0450

Figure Lengend Snippet: FIGURE 5: Release of CHD7 proteins from the LCR HS2 sequence in the globin gene after radiation exposure. (A–C) ChIP assay. (A) Relative amount of CHD7 bound to LCR (HS2) sequence. (B) Relative amount of histone H3K4Me3 bound to LCR (HS2) sequence. (C) Relative amount of CHD7 or H3K4Me3 bound to LCR (HS2) 10 min after exposure at 5 Gy in cells with or without KU55933 pretreatment. The data are the average of four experiments with SD, t test, *p < 10–4. (D, E) Gel mobility shift assay of LCR sequence; in D the arrow in the left panel indicates the decrease of the LCR shifted band following irradiation. The arrow in the right panel indicates no change in the PE21 shifted band after irradiation in the control. Probe, radiolabeled LCR or PE21; NE, nuclear extract; competitor, excess amount of nonradiolabeled LCR or PE21. (E) Relative amount of nuclear protein binding to LCR or PE21. The data are the average of two experiments with SD; * p < 0.01.

Article Snippet: After the debris was removed, an anti-CHD7 antibody (CST; 6505S) was added and incubated overnight at 4°C.

Techniques: Sequencing, Mobility Shift, Irradiation, Control, Protein Binding

Journal: Molecular Biology of the Cell

Article Title: ATM–dependent phosphorylation of CHD7 regulates morphogenesis-coupled DSB stress response in fetal radiation exposure

doi: 10.1091/mbc.e22-10-0450

Figure Lengend Snippet: FIGURE 6: Targeted disruption of CHD7 and/or 53BP1 changes cell growth and radiation sensitivity. (A) Growth curve of each cell strain in comparison with WT HT1080. (B) Cell survival following radiation exposure. (C) Colony formation of each cell strain; 1600 cells of each strain were inoculated in duplicate dishes, and after 8 Gy x-ray irradiation, cells were cultured for 12 d to fix and stain. The data are shown as average with SD.

Article Snippet: After the debris was removed, an anti-CHD7 antibody (CST; 6505S) was added and incubated overnight at 4°C.

Techniques: Disruption, Comparison, Irradiation, Cell Culture, Staining

Journal: Molecular Biology of the Cell

Article Title: ATM–dependent phosphorylation of CHD7 regulates morphogenesis-coupled DSB stress response in fetal radiation exposure

doi: 10.1091/mbc.e22-10-0450

Figure Lengend Snippet: FIGURE 7: Loss of CHD7 induces error-prone NHEJ and mutations. (A) Structure of the NHEJ reporter plasmid pEJ. (B) Flow cytometric detection of I-SceI–induced GFP-positive cells. (C) Number of GFP-positive cells in 106 cells with or without pCBASceI transfection. Two independent clones, each from WT and KO cells, are shown. (D) Size distribution of deletions occurring at DSB sites. The data are shown as the average sizes of deletions in GFP-positive cells with SE: Parental #1, 0.04 ± 0.4; Parental #2, 2.5 ± 1.1; CHD7KO #1, 37.0 ± 5.9; CHD7KO #2, 20.3 ± 4.1; 53BP1KO #1, 1.2 ± 1.8; 53BP1KO #2, 5.0 ± 2.9; DKO #1, 5.9 ± 3.5; DKO #2, 4.2 ± 4.4. (E) Frequency of HPRT mutations in 106 viable cells after radiation. (F) Model depicting CHD7 involvement in DSB foci complex under the ATM control.

Article Snippet: After the debris was removed, an anti-CHD7 antibody (CST; 6505S) was added and incubated overnight at 4°C.

Techniques: Plasmid Preparation, Transfection, Clone Assay, Control

Journal: Stem Cells International

Article Title: CHD7 Regulates Osteogenic Differentiation of Human Dental Follicle Cells via PTH1R Signaling

doi: 10.1155/2020/8882857

Figure Lengend Snippet: The high expression of CHD7 in hDFCs after osteoinduction. (a) Immunohistochemical staining images unraveled that CHD7 is present in human dental follicle. Scale bar, 20 μ m. (b), (c) qRT-PCR and Western blot unraveled that the expression of CHD7 increased after 3-day and 7-day osteoinduction.

Article Snippet: After antigen blocking, the membranes were incubated in rabbit anti- α -tubulin antibody (Sigma, 1: 2500), rabbit anti-CHD7 antibody (Sigma, 1: 1000), and mouse anti-PTH1R antibody (Sigma, 1: 1000) at 4°C overnight.

Techniques: Expressing, Immunohistochemical staining, Staining, Quantitative RT-PCR, Western Blot

Journal: Stem Cells International

Article Title: CHD7 Regulates Osteogenic Differentiation of Human Dental Follicle Cells via PTH1R Signaling

doi: 10.1155/2020/8882857

Figure Lengend Snippet: Depletion of CHD7 decreases osteogenic differentiation of hDFCs. (a), (b) qRT-PCR and Western blot verified the knockdown efficiency of siCHD7. (c) Representative images and quantitative analyses of ALP and ARS staining of hDFCs in the siCHD7 and siCTRL group. (d) qRT-PCR analyses of the expression of RUNX2 , SP7 , BGLAP , DLX5 , BMP2 , and COL1A1 under osteogenic condition.

Article Snippet: After antigen blocking, the membranes were incubated in rabbit anti- α -tubulin antibody (Sigma, 1: 2500), rabbit anti-CHD7 antibody (Sigma, 1: 1000), and mouse anti-PTH1R antibody (Sigma, 1: 1000) at 4°C overnight.

Techniques: Quantitative RT-PCR, Western Blot, Staining, Expressing

Journal: Stem Cells International

Article Title: CHD7 Regulates Osteogenic Differentiation of Human Dental Follicle Cells via PTH1R Signaling

doi: 10.1155/2020/8882857

Figure Lengend Snippet: The overexpression of CHD7 promotes osteogenic differentiation of hDFCs. (a), (b) qRT-PCR and Western blot verified the overexpression efficiency of CHD7 . (c) Representative images and quantitative analyses of ALP and ARS staining of hDFCs in the LV-CHD7 and LV-GFP group. (d) qRT-PCR analyses of the expression of RUNX2 , SP7 , BGLAP , DLX5 , BMP2 , and COL1A1 under osteogenic condition.

Article Snippet: After antigen blocking, the membranes were incubated in rabbit anti- α -tubulin antibody (Sigma, 1: 2500), rabbit anti-CHD7 antibody (Sigma, 1: 1000), and mouse anti-PTH1R antibody (Sigma, 1: 1000) at 4°C overnight.

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Staining, Expressing

Journal: Stem Cells International

Article Title: CHD7 Regulates Osteogenic Differentiation of Human Dental Follicle Cells via PTH1R Signaling

doi: 10.1155/2020/8882857

Figure Lengend Snippet: RNA-seq revealed the downregulated enrichment of the PTH-related pathway after CHD7 depletion. (a) GO enrichment unraveled that skeletal system development and ossification were suppressed after CHD7 depletion. (b) Heatmap of representative osteogenesis associated genes. (c) KEGG enrichment unraveled that the PTH-related pathway was significantly suppressed after CHD7 depletion. (d) GSEA showed decreased enrichment of PTH-regulated genes in CHD7-deficient hDFCs.

Article Snippet: After antigen blocking, the membranes were incubated in rabbit anti- α -tubulin antibody (Sigma, 1: 2500), rabbit anti-CHD7 antibody (Sigma, 1: 1000), and mouse anti-PTH1R antibody (Sigma, 1: 1000) at 4°C overnight.

Techniques: RNA Sequencing Assay

Journal: Stem Cells International

Article Title: CHD7 Regulates Osteogenic Differentiation of Human Dental Follicle Cells via PTH1R Signaling

doi: 10.1155/2020/8882857

Figure Lengend Snippet: CHD7 regulates the expression of PTH1R. (a) qRT-PCR and Western blot of the PTH1R expression after CHD7 depletion. (b) qRT-PCR and Western blot of the PTH1R expression after the CHD7 overexpression. (c) Anti-CHD7 ChIP assay. CHD7 can bound to the promoter region of PTH1R, and the ChIP signaling was significantly suppressed after CHD7 depletion.

Article Snippet: After antigen blocking, the membranes were incubated in rabbit anti- α -tubulin antibody (Sigma, 1: 2500), rabbit anti-CHD7 antibody (Sigma, 1: 1000), and mouse anti-PTH1R antibody (Sigma, 1: 1000) at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Over Expression

Journal: Stem Cells International

Article Title: CHD7 Regulates Osteogenic Differentiation of Human Dental Follicle Cells via PTH1R Signaling

doi: 10.1155/2020/8882857

Figure Lengend Snippet: The overexpression of PTH1R partially rescues the osteogenic differentiation of CHD7-deficiency hDFCs. (a), (b) qRT-PCR and Western blot verified the overexpression efficiency of PTH1R . (c) Representative images and quantitative analyses of ALP and ARS staining of hDFCs in the siCTRL, siCHD7, and rescue group. (d) qRT-PCR analyses of the expression of RUNX2 , SP7 , BGLAP , DLX5 , BMP2 , and COL1A1 under osteogenic condition.

Article Snippet: After antigen blocking, the membranes were incubated in rabbit anti- α -tubulin antibody (Sigma, 1: 2500), rabbit anti-CHD7 antibody (Sigma, 1: 1000), and mouse anti-PTH1R antibody (Sigma, 1: 1000) at 4°C overnight.

Techniques: Over Expression, Quantitative RT-PCR, Western Blot, Staining, Expressing

Journal: Nature Communications

Article Title: CHARGE syndrome protein CHD7 regulates epigenomic activation of enhancers in granule cell precursors and gyrification of the cerebellum

doi: 10.1038/s41467-021-25846-3

Figure Lengend Snippet: a Heatmap of ChIP-seq signal for CHD7 (purple), H3K4me3 (orange), H3K27ac (blue), H3K4me1 (green) and ATAC-seq (black) centered on CHD7 genomic binding sites ( n = 22,515). Heatmaps are split into four groups (promoters, active enhancers, poised enhancers, and other). Anterior cerebellum from P4 mice were used ( n = 2 for CHD7 Ctrl, n = 1 for CHD7 cKO, n = 3 for histone modifications and ATAC-seq). b Pie chart depicting the proportion of CHD7-binding sites found to overlap ATAC-seq peaks (accessible regions) and not overlap (other). c WashU Epigenome Browser view of a promoter (left), active enhancer (middle), and poised enhancer (right) region (highlighted). Each site shows ChIP-seq coverage of CHD7, H3K4me3, H3K27ac, H3K4me1, and ATAC-seq. d Pie chart displaying regulatory element distribution of CHD7 peaks. Enh, enhancer. e Pie charts displaying the distribution of CHD7-bound P4-active (left) and P4-poised (right) enhancers at P4 ( n = 3) and P22 ( n = 3). f Heatmap of P4 and P22 H3K27ac ChIP-seq ( n = 3 for each timepoint) centered on CHD7 developmentally active enhancer binding sites ( n = 14,704). Heatmaps are split into three groups (P4-specific, P4/P22-active, and P4specific). g WashU Epigenome Browser view of a CHD7-bound P4 poised, P22-active enhancer (highlighted). ChIP-seq coverage for CHD7, H3K27ac, H3K4me1, as well as, ATAC-seq and RNA-seq is shown. h GREAT analysis showing enriched mouse phenotypes associated with P4-specific active enhancers (left) and P22-specific active enhancers (right).

Article Snippet: Antibodies to CHD7 (Immunofluorescence 1:2000, Immunoblot 1:1000, ChIP 1:100, Cell Signaling Technology, #6505 S), GAPDH (Immunoblot 1:500, Santa Cruz, #sc-32233), histone H3K27ac (ChIP 1:1000, Active Motif, #39133), H3K4me1 (ChIP 1:250, Active Motif, #39297), H3K4me3 (ChIP 1:125, Cell Signaling Technology, #9751 S), RNAP2 (ChIP 1:10, Santa Cruz, #sc-47701), CTCF (ChIP 1:200, Millipore, #07-729), RAD21 (ChIP 1:200, Abcam, #ab992), Calbindin (Immunofluorescence 1:5000, Swant, D-28K), GFP (Immunofluorescence 1:250, Abcam, #ab13970), GFAP (Immunofluorescence, Abcam, #ab7260), phospho-Histone H3 (Immunofluorescence 1:1000, Millipore, #06-570), S100β (Immunofluorescence 1:250, Abcam, #ab41548) were purchased.

Techniques: ChIP-sequencing, Binding Assay, RNA Sequencing

Journal: Nature Communications

Article Title: CHARGE syndrome protein CHD7 regulates epigenomic activation of enhancers in granule cell precursors and gyrification of the cerebellum

doi: 10.1038/s41467-021-25846-3

Figure Lengend Snippet: a Box-whisker plots showing the median, 1st quartile, 3rd quartile, and whiskers displaying the 5th and 95th percentiles of promoters ( n = 11,398, n = 5238), active enhancers ( n = 3535, n = 6531), and poised enhancers ( n = 25,231, n = 8039) representing median and distribution of the log2 fold-change of ATAC-seq signal in CHD7 cKO over CHD7 control. CHD7 unoccupied, (CHD7–) left, and occupied, (CHD7+) right, enhancers are shown. **** p < 10 −4 , unpaired two-sided t -test, Welch’s correction. b Pie chart representing proportion active enhancers that show significantly changed accessibility in the CHD7 cKO, which display either increased or reduced accessibility. c Box-whisker plots showing the median, 1st quartile, 3rd quartile, and whiskers displaying the 5th and 95th percentiles of active enhancers representing median and distribution of the log2 fold-change of H3K4me1 (left; n = 760, n = 2457), H3K27ac (middle; n = 1590, n = 2738), and RNApolII (right; n = 249, n = 1099) signal in CHD7 cKO over CHD7 control. *** p < 10 –3 , **** p < 10 − 4 , unpaired two-sided t -test, Welch’s correction. d Pie chart representing proportion of CHD7 unoccupied (CHD7−; left) and occupied (CHD7+; right) active enhancers displaying RNApolII binding. e WashU Epigenome Browser view of a CHD7-bound active enhancer (highlighted) near the Trhde gene. ChIP-seq coverage for CHD7, H3K27ac, H3K4me1, RNApolII, as well as ATAC-seq and RNA-seq is shown.

Article Snippet: Antibodies to CHD7 (Immunofluorescence 1:2000, Immunoblot 1:1000, ChIP 1:100, Cell Signaling Technology, #6505 S), GAPDH (Immunoblot 1:500, Santa Cruz, #sc-32233), histone H3K27ac (ChIP 1:1000, Active Motif, #39133), H3K4me1 (ChIP 1:250, Active Motif, #39297), H3K4me3 (ChIP 1:125, Cell Signaling Technology, #9751 S), RNAP2 (ChIP 1:10, Santa Cruz, #sc-47701), CTCF (ChIP 1:200, Millipore, #07-729), RAD21 (ChIP 1:200, Abcam, #ab992), Calbindin (Immunofluorescence 1:5000, Swant, D-28K), GFP (Immunofluorescence 1:250, Abcam, #ab13970), GFAP (Immunofluorescence, Abcam, #ab7260), phospho-Histone H3 (Immunofluorescence 1:1000, Millipore, #06-570), S100β (Immunofluorescence 1:250, Abcam, #ab41548) were purchased.

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

Journal: Nature Communications

Article Title: CHARGE syndrome protein CHD7 regulates epigenomic activation of enhancers in granule cell precursors and gyrification of the cerebellum

doi: 10.1038/s41467-021-25846-3

Figure Lengend Snippet: a Hi-C contact matrices of chromosome 10 in CHD7 control (top) and CHD7 cKO (bottom) cerebellum, visualized at chromosome-scale ( n = 3 for each condition). Pixel intensity represents the normalized number of contacts between a pair of loci. Contact frequencies calculated at 10 Kb. b Density plots comparing the log2 fold-change in ATAC-seq signal in each contact domain to the log2 fold-change in Hi-C contacts for that domain. Compartmental domains (top) and loop domains (bottom). c WashU Epigenome Browser view of a loop domain. ChIP-seq coverage for CHD7, H3K27ac, Rad21, CTCF, as well as ATAC-seq and Hi-C, in CHD7 control and CHD7 cKO are shown. d Density plot comparing the eigenvectors calculated for 150 Kb bins in CHD7 control and cKO cerebellum (left). WashU Epigenome Browser view of eigenvectors depicting A/B compartments and RNA levels in CHD7 control and cKO cerebellum (right). e Hierarchical clustering of gene expression genes detected as significantly dysregulated (false-discovery rate [FDR],<0.05) in analysis of RNA-seq from micro-dissected Ctrl and cKO P4 anterior cerebellum ( n = 5 biological replicates per genotype). Color represents Z -score of log2 cpm (counts per million) for a given gene. f Box-whisker plots showing the median, 1st quartile, 3rd quartile, and whiskers displaying the 5th and 95th percentiles of log2 fold-change of RNA signal for genes proximal to CHD7-bound sites with significant changes in accessibility in the CHD7 cKO. Active enhancers were divided into three groups based on significantly increasing or decreasing changes in ATAC-seq signal. We then quantified the change in gene expression of corresponding genes within groups defined by ATAC-seq changes. Genes must be found within 100 Kb and within the same TAD as the altered accessibility site to be linked to the site. Boxplots are divided into groups of decreasing (top; n = 255, n = 185, n = 129) and increasing (bottom; n = 39, n = 23, n = 22) ATAC-seq fold-change. **** p < 10 −4 , unpaired two-sided t -test, Tukey’s multiple comparison test. g Pie chart representing proportion of significantly changed enhancers in the CHD7 cKO for which transcript levels of the gene linked to that enhancer by an E–P loop change in the same direction as the enhancer. CHD7-occupied (CHD7+; right) enhancers are significantly different from unoccupied enhancers (CHD7–; left). p -value = 4.45E-32, one-sided Chi-squared test.

Article Snippet: Antibodies to CHD7 (Immunofluorescence 1:2000, Immunoblot 1:1000, ChIP 1:100, Cell Signaling Technology, #6505 S), GAPDH (Immunoblot 1:500, Santa Cruz, #sc-32233), histone H3K27ac (ChIP 1:1000, Active Motif, #39133), H3K4me1 (ChIP 1:250, Active Motif, #39297), H3K4me3 (ChIP 1:125, Cell Signaling Technology, #9751 S), RNAP2 (ChIP 1:10, Santa Cruz, #sc-47701), CTCF (ChIP 1:200, Millipore, #07-729), RAD21 (ChIP 1:200, Abcam, #ab992), Calbindin (Immunofluorescence 1:5000, Swant, D-28K), GFP (Immunofluorescence 1:250, Abcam, #ab13970), GFAP (Immunofluorescence, Abcam, #ab7260), phospho-Histone H3 (Immunofluorescence 1:1000, Millipore, #06-570), S100β (Immunofluorescence 1:250, Abcam, #ab41548) were purchased.

Techniques: Hi-C, Control, ChIP-sequencing, Gene Expression, RNA Sequencing, Whisker Assay, Comparison

Journal: Nature Communications

Article Title: CHARGE syndrome protein CHD7 regulates epigenomic activation of enhancers in granule cell precursors and gyrification of the cerebellum

doi: 10.1038/s41467-021-25846-3

Figure Lengend Snippet: a GREAT analysis showing enriched mouse phenotypes associated with CHD7-regulated genes. Cerebellar phenotypes shown in red text. b Hematoxylin and eosin staining of Ctrl (left) and CHD7 cKO (right) p56 sections of mouse cerebellum. Scale bar: 2 mm. c Zeiss Xradia Versa 520 XRM was used to perform a nano-computerized tomography (nano CT) scan of Ctrl (left) and cKO (right) p56 mouse cerebella. The cerebellar vermis is highlighted blue and corresponding hemisphere regions highlighted green and yellow. Scale bar: 2 mm. d Graph showing number of gyri in the cerebellar vermis of the anterior basal lobe of Ctrl and cKO mice ( n = 3 for each condition). e Bar graphs showing % change in total cerebellar (Cb) and anterior lobules (Ant Cb) lengths upon CHD7 cKO ( n = 3 for each condition). Length is shown as mean ± s.e.m for each measurement. p = 0.0188 (Cb), p = 0.0024 (Ant Cb) Paired two-sided t -test. f IHC (left) and H&E (right) of Ctrl and cKO p56 mouse cerebellar sections. Experiment was repeated three times for each condition. Scale bars: 100 µm. g Nano CT scans were taken of P3, P3.5, and P4 mice cerebella. Digital 2D sections were taken through anterior basal lobe lobules. Insets show entire cerebellar section. Yellow dashed squares define region of focus. Yellow arrowheads on P4 Ctrl section mark transient vermal indentations. Yellow asterisks on P4 cKO section mark aberrant sulci. Scale bars: 500 µm. h Dot plot showing relative distance of transient invaginations (TI) in P4 CHD7 ctrl mice ( n = 4) and sulci (S) formation in P4 CHD7 cKO mice ( n = 4).

Article Snippet: Antibodies to CHD7 (Immunofluorescence 1:2000, Immunoblot 1:1000, ChIP 1:100, Cell Signaling Technology, #6505 S), GAPDH (Immunoblot 1:500, Santa Cruz, #sc-32233), histone H3K27ac (ChIP 1:1000, Active Motif, #39133), H3K4me1 (ChIP 1:250, Active Motif, #39297), H3K4me3 (ChIP 1:125, Cell Signaling Technology, #9751 S), RNAP2 (ChIP 1:10, Santa Cruz, #sc-47701), CTCF (ChIP 1:200, Millipore, #07-729), RAD21 (ChIP 1:200, Abcam, #ab992), Calbindin (Immunofluorescence 1:5000, Swant, D-28K), GFP (Immunofluorescence 1:250, Abcam, #ab13970), GFAP (Immunofluorescence, Abcam, #ab7260), phospho-Histone H3 (Immunofluorescence 1:1000, Millipore, #06-570), S100β (Immunofluorescence 1:250, Abcam, #ab41548) were purchased.

Techniques: Staining, Tomography, Computed Tomography

Journal: Nature Communications

Article Title: CHARGE syndrome protein CHD7 regulates epigenomic activation of enhancers in granule cell precursors and gyrification of the cerebellum

doi: 10.1038/s41467-021-25846-3

Figure Lengend Snippet: a Immunohistochemistry analysis of Ctrl and cKO mice ( n = 3 for each condition) with antibodies recognizing phosphorylated histone H3 (pH3), a marker of dividing cells. Dividing cells were counted in the entirety of the region anterior to the primary fissure of the cerebellum (lobules I–V), referred to in shorthand as the anterior cerebellum. A representative image of the external granule layer is shown (left). Bar graphs (right) show mean pH3 positive cells per 100 µm 2 ± s.e.m. ns = non-significant. Paired two-sided t -test. Scale bar: 25 µm. b P6 mouse pups were electroporated ( n = 3 for each condition) with the GFP expression plasmid and killed 48 h later. Cerebella were removed, sectioned, and subjected to immunohistochemistry with antibodies recognizing GFP. Bar graphs show mean distance from EGL ± s.e.m. ns = non-significant. Paired two-sided t -test. Scale bar: 25 µm. c Image of P56 cerebellum obtained via nano CT scan (left) showing anterior–posterior axis (A–P) in green and medial–lateral axis (M–L) in yellow. d Immunohistochemistry analysis of axis of division of granule cell precursors, identified via pH3 staining in green. Dashed red line indicates the plane of division; dashed white line indicates the pial surface. Example of a granule cell precursor undergoing a vertical (top) and horizontal (bottom) division. Experiment was repeated three times for each condition. Scale bar: 5 µm. e Rose plot showing the distribution of axis of division angles (angle between pial surface and plane of division) for P3 Ctrl and cKO mice in A–P (top, blue) and M–L (bottom, green) axes. 180 cells (60 from each mouse, n = 3 for each condition) were analyzed for each rose plot. f Bar graph showing mean number of horizontal division per 100 µm 2 ± s.e.m. ( n = 3 for each condition). ns = non-significant. Paired two-sided t -test. g Bar graph showing mean ratio of vertical to horizontal division ± s.e.m. for Ctrl and cKO mice in both A–P and M–L axes ( n = 3 for each condition). * p = 0.0068 (AP), p = 0.0256 (ML) Paired two-sided t -test. h Cerebellar-dependent eye-blink conditioning learning paradigm was performed on CHD7 Ctrl and cKO ( n = 7 and n = 9, respectively) mice. Percent conditioned response (CR) is shown as mean ± SEM for each session day. ∗∗∗∗ p < 10 − 4 , ∗ p < 0.05 two-sided repeated-measures ANOVA, Sidak’s multiple comparison test. i Open-field assay was performed on CHD7 Ctrl and cKO mice ( n = 6 for each) to assess general locomotor activity. Two-sided t -test; ns, not significant.

Article Snippet: Antibodies to CHD7 (Immunofluorescence 1:2000, Immunoblot 1:1000, ChIP 1:100, Cell Signaling Technology, #6505 S), GAPDH (Immunoblot 1:500, Santa Cruz, #sc-32233), histone H3K27ac (ChIP 1:1000, Active Motif, #39133), H3K4me1 (ChIP 1:250, Active Motif, #39297), H3K4me3 (ChIP 1:125, Cell Signaling Technology, #9751 S), RNAP2 (ChIP 1:10, Santa Cruz, #sc-47701), CTCF (ChIP 1:200, Millipore, #07-729), RAD21 (ChIP 1:200, Abcam, #ab992), Calbindin (Immunofluorescence 1:5000, Swant, D-28K), GFP (Immunofluorescence 1:250, Abcam, #ab13970), GFAP (Immunofluorescence, Abcam, #ab7260), phospho-Histone H3 (Immunofluorescence 1:1000, Millipore, #06-570), S100β (Immunofluorescence 1:250, Abcam, #ab41548) were purchased.

Techniques: Immunohistochemistry, Marker, Expressing, Plasmid Preparation, Computed Tomography, Staining, Comparison, Activity Assay

Journal: Nature Communications

Article Title: CHARGE syndrome protein CHD7 regulates epigenomic activation of enhancers in granule cell precursors and gyrification of the cerebellum

doi: 10.1038/s41467-021-25846-3

Figure Lengend Snippet: a Immunohistochemistry analysis of 22 gestational week human cerebellum with antibodies recognizing hCHD7. Experiment was performed once on multiple sections. Scale bar: 100 µm. b Aggregate plot of ChIP-seq signal for hCHD7 (purple) and input (black) centered on hCHD7 genomic binding sites ( n = 17,100). 22 gestational week anterior human cerebellum ( n = 1 for CHD7 Ctrl, n = 1 for input). c Venn-diagram showing overlap of CHD7 mouse and human target genes with hypergeometric analysis. p -value = 4.48E-10, hypergeometric. See Methods section for more details on analysis. d Panther pathways analysis of mouse CHD7 direct target genes (left) and PMG-associated genes (right). Shared pathways are shown in red. e WashU Epigenome Browser view of two CHD7 direct target genes (TUBB2B, top; MAP1B, bottom) found in mouse and human cerebellum. Each site shows ChIP-seq coverage of CHD7 and H3K27ac. f H&E staining of 5-month-old clinically diagnosed CHARGE syndrome patient. Arrowheads point to sites or excessive ectopic cerebellar foliation. Zoom in of dashed rectangle shown below. EGL external granule layer; ML molecular layer; PCL Purkinje cell layer; IGL internal granule layer. Experiment was performed once on multiple sections for each region. Please refer to Methods for additional details regarding patient cerebellar sample.

Article Snippet: Antibodies to CHD7 (Immunofluorescence 1:2000, Immunoblot 1:1000, ChIP 1:100, Cell Signaling Technology, #6505 S), GAPDH (Immunoblot 1:500, Santa Cruz, #sc-32233), histone H3K27ac (ChIP 1:1000, Active Motif, #39133), H3K4me1 (ChIP 1:250, Active Motif, #39297), H3K4me3 (ChIP 1:125, Cell Signaling Technology, #9751 S), RNAP2 (ChIP 1:10, Santa Cruz, #sc-47701), CTCF (ChIP 1:200, Millipore, #07-729), RAD21 (ChIP 1:200, Abcam, #ab992), Calbindin (Immunofluorescence 1:5000, Swant, D-28K), GFP (Immunofluorescence 1:250, Abcam, #ab13970), GFAP (Immunofluorescence, Abcam, #ab7260), phospho-Histone H3 (Immunofluorescence 1:1000, Millipore, #06-570), S100β (Immunofluorescence 1:250, Abcam, #ab41548) were purchased.

Techniques: Immunohistochemistry, ChIP-sequencing, Binding Assay, Staining