Journal: iScience

Article Title: GRHL2 and AP2a coordinate early surface ectoderm lineage commitment during development

doi: 10.1016/j.isci.2023.106125

Figure Lengend Snippet: GRHL2 is required for AP2a activity at SE loci (A) Enrichment of various chromatin states in relation to GRHL2 ChIP-seq coordinates. Chromatin states were defined by ChromHMM using previously published histone mark ChIP-seq and ATAC-seq datasets in hESCs treated with RA/BMP4 for 7 days. (B) Expression of GRHL2-bound genes across integrated scRNA-seq identities. (C) Distribution of ATAC-seq signal (score based on number of reads per bin) relative to GRHL2 binding sites in WT and GRHL2 KO cells. (D) AP2a motif enrichment joint UMAP. (E) CRISPR strategy and immunoblot confirming AP2a KO hESCs. (F) Expression of GRHL2 and AP2a-dependent genes across integrated scRNA-seq identities. (G) Overlap of GRHL2 and AP2a-dependent and bound genes. Significance was calculated by Fisher exact test, ∗∗∗ indicates p value <0.0001. (H) Heatmap of read counts from WT, GRHL2 KO, and AP2a KO RNA-seq. (I) Expression of TFAP2A or GRHL2 in hESCs or WT, GRHL2 KO or AP2a KO hESCs treated with RA/BMP4. Error bars represent mean +/−SD, n= 2 biological replicates. (J) Immunoblot showing levels of AP2a protein in WT and GRHL2 KO cells treated with RA/BMP4. (K) Distribution of ATAC-seq signal (score based on number of reads per bin) relative to AP2a binding sites in WT and GRHL2 KO cells. Distribution of AP2a ChIP-seq signal (score based on number of reads per bin) relative to (L) all transcription star sites or (M) AP2a binding sites in WT and GRHL2 KO cells. (N) Representative bedgraphs of AP2a ChIP-seq in WT or GRHL2 KO cells.

Article Snippet: Anti-mouse AP2A , Santa Cruz , RRID: AB_667767.

Techniques: Activity Assay, ChIP-sequencing, Expressing, Binding Assay, CRISPR, Western Blot, RNA Sequencing

Journal: iScience

Article Title: GRHL2 and AP2a coordinate early surface ectoderm lineage commitment during development

doi: 10.1016/j.isci.2023.106125

Figure Lengend Snippet: AP2a restricts GRHL2 binding to appropriate target genes (A) Distribution of GRHL2 ChIP-seq signal (score based on number of reads per bin) relative to GRHL2 or AP2a binding sites in WT and AP2a KO cells. (B) Distribution of ATAC-seq signal (score based on number of reads per bin) relative to the new GRHL2 binding sites in WT or AP2a KO cells. (C) Enrichment of various chromatin states in relation to AP2a KO GRHL2 ChIP-seq coordinates. Chromatin states were defined by ChromHMM using histone mark ChIP-seq and ATAC-seq datasets in hESCs treated with RA/BMP4 for 7 days. (D) Percentage of GRHL2 peaks found at or connected to promoters or AP2a binding sites in both WT and AP2a KO cells. p values were calculated with two-tailed Fisher’s exact tests. ∗∗∗∗ indicates a p value <0.0001. (E) Number of chromatin contacts in WT and AP2a KO cells as measured by cohesin HiChIP. (F) Chromatin contact strength at ectopic GRHL2 binding sites in WT and AP2a KO cells. Boxes represent the median with interquartile range and error bars represent the minimum and maximum. (G) Empirical cumulative distribution function of the log 2 FoldChange in gene expression of promoters bound or looped to GRHL2 or AP2a binding sites (n = 6,564, purple) compared to all protein coding genes (n = 19,923, black) in WT vs. AP2a KO cells. p value <0.01 calculated by Student’s two-tailed test. (H) UMAP of integrated WT and AP2a KO cells colored by cell type. (I) Fold change in AP2a KO cell type proportions compared to WT, as measured by scRNA-seq. (J) Model of GRHL2 and AP2a regulation of gene expression. GRHL2 acts by blocking neural gene expression and promoting AP2a binding at surface ectoderm genes. In the absence of AP2a, GRHL2 binds unrestricted to inappropriate loci. (K) MA plot of differential expression analysis between WT and GRHL overexpression bulk RNA-seq. Transcript expression is either unchanged (gray), increased (blue, >2-fold), or decreased (red, <2-fold). p values for differentially expressed genes were calculated using DESEQ2 with a cutoff of 0.05. All differential genes and statistics can be found in Table S5 . (L) Overlap of GRHL2-regulated genes and genes altered by GRHL2 overexpression. p value was calculated with a Fisher exact test. (M) UMAP of integrated WT and GRHL2 over expression cells colored by cell type. (N) Fold change in GRHL2 overexpression cell type proportions compared to WT, as measured by scRNA-seq.

Article Snippet: Anti-mouse AP2A , Santa Cruz , RRID: AB_667767.

Techniques: Binding Assay, ChIP-sequencing, Two Tailed Test, HiChIP, Gene Expression, Blocking Assay, Quantitative Proteomics, Over Expression, RNA Sequencing, Expressing

Journal: iScience

Article Title: GRHL2 and AP2a coordinate early surface ectoderm lineage commitment during development

doi: 10.1016/j.isci.2023.106125

Figure Lengend Snippet: Cleft lip/palate genetic variants prioritized by integrating multiomic GRHL2/AP2a functional datasets (A) Pipeline overview, which connects single nucleotide polymorphisms (SNPs) in enhancers to distal genes via chromatin looping. (B) Flow chart illustrating how genes of interest were filtered using functional data. (C) Clinical significance of pipeline-identified output genetic variants. (D) Histogram of the minor allele frequency or (E) CADD scores of the genetic variants. Red dashed lines indicate standard cutoffs for minor allele frequency (MAF <0.02) or CADD score (CADD ≥15) for identifying disease-associated genetic variants. (F) Proportion of pipeline input or output genetic variants which were found directly from GWAS studies or are in linkage disequilibrium (LD). Significance was calculated with a Fisher’s exact test, ∗ indicates a p value <0.05. (G) The chromosomal location and (H) functional categories of pipeline-identified genetic variants. (I) Number of pipeline-identified genetic variants falling within GRHL2 or AP2a binding sites, or AP2a-dependent ATAC sites in WT (black) or AP2a KO cells (white). p values were calculated with two-tailed Fisher’s exact tests, ∗∗∗∗ indicates p value <0.0001. (J) Overlap of genetic variants identified in our day 7 RA/BMP4 cells compared to published neural crest studies. ,

Article Snippet: Anti-mouse AP2A , Santa Cruz , RRID: AB_667767.

Techniques: Functional Assay, Binding Assay, Two Tailed Test

Journal: iScience

Article Title: GRHL2 and AP2a coordinate early surface ectoderm lineage commitment during development

doi: 10.1016/j.isci.2023.106125

Figure Lengend Snippet: Cleft lip/palate genetic variants alter GRHL2 binding and downstream gene expression (A) Table of SNPs identified from previous GWAS studies showing whether they fall within GRHL2 or AP2a binding sites. (B) Feature linkage between the ABCA4 promoter and accessible chromatin from WT and GRHL2 KO single cell multiome datasets. Arc height corresponds to the absolute value of each linkage. Red = negative correlation and Blue = positive correlation. (C) Chromatin accessibility at the ABCA4 locus in each cell type identified by scATAC. (D) AP2a and GRHL2 ChIP-seq signal in WT and AP2a KO cells at the ABCA4 locus. (E) Raw cohesion HiChIP signal at the ABCA4 / ARHGAP29 locus in WT and AP2a KO cells. (F) Genetic variant preference at the SNP rs1211213 for AP2a or GRHL2 DNA binding as identified by ChIP-seq. Boxes represent median with interquartile ranges. (G) Expression of ABCA4 or (H) ARHGAP29 from bulk RNA-seq data. Error bars represent mean +/−SD, n= 2 biological replicates. (I) UMAP of ABCA4 or (J) ARHGAP29 expression from integrated scRNA-seq. (K) Raw sequencing data illustrating the single nucleotide change at rs1211213 made with CRISPR genome editing. (L) qPCR comparing homozygous vs heterozygous rs1211213 alleles. Bars indicated mean with SD. p values were calculated with two-tailed Student’s t test, n= 5 biological replicates run in triplicate. ∗∗∗∗ indicates p value <0.0001.

Article Snippet: Anti-mouse AP2A , Santa Cruz , RRID: AB_667767.

Techniques: Binding Assay, Gene Expression, ChIP-sequencing, HiChIP, Variant Assay, Expressing, RNA Sequencing, Sequencing, CRISPR, Two Tailed Test

Journal: iScience

Article Title: GRHL2 and AP2a coordinate early surface ectoderm lineage commitment during development

doi: 10.1016/j.isci.2023.106125

Figure Lengend Snippet:

Article Snippet: Anti-mouse AP2A , Santa Cruz , RRID: AB_667767.

Techniques: Virus, Recombinant, Blocking Assay, Plasmid Preparation, Magnetic Beads, Gene Expression, Purification, Cloning, dsDNA Assay, Sequencing, Software, Fluorescence

Journal: Biochemical and biophysical research communications

Article Title: Glycosaminoglycans promote osteogenesis from human induced pluripotent stem cells via neural crest induction.

doi: 10.1016/j.bbrc.2022.03.002

Figure Lengend Snippet: Fig. 1. Neural crest and mesenchymal stromal lineage differentiation from hiPSCs. (A) Schematic representation of stepwise differentiation into mature osteoblasts from hiPSCs via neural crest induction. (B) Induced NCLCs at day 10 expressed neural crest cell markers (AP2a, SOX10, NESTIN, and P75), but NCLCs did not express any pluripotent stem cell markers (NANOG, OCT3/4), as confirmed by immunofluorescence staining. (C) Induction efficiency of NCLCs was analyzed by counting the number of AP2a, SOX10 positive cells relative to the total number of nuclei in an average of five randomly selected images from three replicates. Data are represented as mean ± SD. (D) Mesenchymal cell markers CD29, CD44, CD73, and CD90 were detected in NCMCs at day 17 by flow cytometry analysis. Blue histogram; isotype control. (E) Expression of a cranial marker OTX2 in NCLCs and NCMCs, as observed by immunofluorescence staining. The cell nuclei were counterstained with Hoechst. Scale bars, 100 mm.

Article Snippet: Cells were then treated with 0.3% Triton X-100 and blocking buffer (Nacalai Tesque) at room temperature for 1 h, followed by overnight incubation in primary antibody solution at 4 C. The following primary antibodies were used: goat polyclonal anti-human SOX10 (1:200, R&D Systems), rabbit monoclonal anti-human AP2a (1:100, C83E10; Cell Signaling Technology), rabbit polyclonal anti-human NESTIN (1:200, Eurofins genomics), rabbit polyclonal anti-human P75 (1:200, Millipore), rabbit polyclonal anti-human NANOG (1:200, ReproCELL), mouse monoclonal anti-human OCT3/4 (1:200, C-10; Santa Cruz), goat polyclonal anti-human OTX2 (1:100, R&D Systems), rabbit polyclonal anti-human OPN (1:200, Abcam), and mouse polyclonal anti-human SOST (1:20, R&D Systems).

Techniques: Staining, Cytometry, Control, Expressing, Marker

Journal: Molecular Brain

Article Title: Stimulation-induced differential redistributions of clathrin and clathrin-coated vesicles in axons compared to soma/dendrites

doi: 10.1186/s13041-020-00683-5

Figure Lengend Snippet: Clathrin-coated vesicles in axon and soma/dendrite are of different sizes. Images were sampled from perfusion-fixed mouse brains (top row: a – c ), 3 wk-old (middle row: d – f ) and 4 DIV (bottom row: g – i ) dissociated rat hippocampal cultures. Synaptic vesicles (SV) were included as size references for CCV from the respective samples, and SVs from these three different groups of samples were of the same uniform size at ~ 40 nm in diameter ( a , d , and g ). CCVs in axon terminals from brains ( b1 – b4 ) were of the same size as SV. While the great majority of CCVs in axon terminals from dissociated cultures ( e1-3 ; h1-2 ) were also of the same size as SV, some CCVs ( e4 ; h3 ) were larger (~ 70 nm) than SV. In soma/dendrites ( c , f , i ), CCV were ~ 90 nm in diameter, much larger than those in the axon terminals. Immunogold labeling of 3 week-old cells illustrates that CCV in soma/dendrites labeled for clathrin ( f1 ), AP2 ( f2 ), and transferrin receptor (TfR, f3 ). j Histograms of size distribution of CCVs of soma/dendrites from brains (top panel), and dissociated neuronal cultures at 3 weeks (middle panel) and 4 days (bottom panel) in culture. The ranges of average diameter were similar (70–110 nm) among the three types of samples, and there was no statistical significance in mean values (ANOVA) or in median values (Wilcoxon test, median values at 85.8, 88.3, 88.3 nm, respectively). n = number of CCVs measured. Scale bar = 100 nm

Article Snippet: Mouse monoclonal antibody (mouse mAb) against clathrin (clone X22, 1:200–500) and AP2 (a clathrin adaptor protein-2, clone AP6, 1:100–200) were from Affinity Bioreagents (Golden, CO, USA); mouse mAb against transferrin receptor (TfR, clone OX-26) was from Chemicon (Temecula, CA, USA).

Techniques: Labeling

Journal: Molecular Brain

Article Title: Stimulation-induced differential redistributions of clathrin and clathrin-coated vesicles in axons compared to soma/dendrites

doi: 10.1186/s13041-020-00683-5

Figure Lengend Snippet: CCPs occasionally exist as multiples in close vicinity on somal/dendritic plasma membrane. The characteristic “coat” appearance was visible in samples treated with low concentrations of osmium tetroxide (0.2% in a, b), and labels for clathrin ( a ) and for AP2 ( b ) were specifically localized to the coat. The appearance of the coat was enhanced when tannic acid (1%) was added to fixative followed by regular osmium tetroxide (1% in c ), and less conspicuous with “reduced osmium” treatment (1% potassium ferrocyanide + 1% osmium tetroxide in d). All samples were 3 week-old dissociated hippocampal cultures. Scale bar = 100 nm

Article Snippet: Mouse monoclonal antibody (mouse mAb) against clathrin (clone X22, 1:200–500) and AP2 (a clathrin adaptor protein-2, clone AP6, 1:100–200) were from Affinity Bioreagents (Golden, CO, USA); mouse mAb against transferrin receptor (TfR, clone OX-26) was from Chemicon (Temecula, CA, USA).

Techniques:

Journal: Molecular Brain

Article Title: Stimulation-induced differential redistributions of clathrin and clathrin-coated vesicles in axons compared to soma/dendrites

doi: 10.1186/s13041-020-00683-5

Figure Lengend Snippet: Multivesicular bodies (MVB, top row) in neurons contain a dark patch (large arrows) that label for clathrin ( a ) but not for AP2 ( b ). In contrast, in the lower row, clathrin-coated vesicles in axons ( d , f ) and dendrites ( e , g ) label for both clathrin ( d , e ) and AP2 ( f , g ). In samples fixed with glutaraldehyde for better structural preservation (no label, right column), a two layered arrangement with a uniform periodicity is visible (small arrows in c ). The thickness of this patch is greater than those of the coated vesicles in axons ( h ) or in dendrites ( i ). MVBs in top row were sampled from neuronal soma ( a ) and dendrites ( b , c ). Scale bar = 100 nm

Article Snippet: Mouse monoclonal antibody (mouse mAb) against clathrin (clone X22, 1:200–500) and AP2 (a clathrin adaptor protein-2, clone AP6, 1:100–200) were from Affinity Bioreagents (Golden, CO, USA); mouse mAb against transferrin receptor (TfR, clone OX-26) was from Chemicon (Temecula, CA, USA).

Techniques: Preserving