Journal: Translational Psychiatry

Article Title: Enrichment of schizophrenia heritability in both neuronal and glia cell regulatory elements

doi: 10.1038/s41398-017-0053-y

Figure Lengend Snippet: . a Enrichment of schizophrenia heritability at H3K4me3 sites in post-mortem brain tissues. Y -axis is the enrichment value and x -axis is the different cell types. b −log10 P -values for the enrichment of schizophrenia heritability at H3K4me3 sites in post-mortem brain tissues. X -axis is the −log10 P -value derived from the z -score; y -axis is the different cell types. Red line is the significance threshold for a Bonferroni correction for multiple testing for total number of analyses undertaken (14 tests performed). c Enrichment of schizophrenia heritability at H3K4me3 sites identified in cultured cells. Y -axis is the enrichment value and x -axis is the different cell types. d −log10 P -values for enrichment of schizophrenia heritability at H3K4me3 sites identified in cultured cells. X -axis is the −log10 P -value derived from the z -score; y -axis is the different cell types Red line is the significance threshold for a Bonferroni correction for multiple testing for total number of analyses undertaken (14 tests performed)

Article Snippet: H3K4me3 chromatin immunoprecipitation (ChIP)-sequencing data from human embryonic stem cell-derived neurons were generated by the Epigenomics Roadmap .

Techniques: Derivative Assay, Cell Culture

Journal: Translational Psychiatry

Article Title: Enrichment of schizophrenia heritability in both neuronal and glia cell regulatory elements

doi: 10.1038/s41398-017-0053-y

Figure Lengend Snippet: Results from the partitioned heritability analysis for the cell types tested for enrichment of SNPs within H3K4me3 marks (a marker of active gene promoters)

Article Snippet: H3K4me3 chromatin immunoprecipitation (ChIP)-sequencing data from human embryonic stem cell-derived neurons were generated by the Epigenomics Roadmap .

Techniques: Marker

Journal: Science Advances

Article Title: Dedifferentiation and neuronal repression define familial Alzheimer’s disease

doi: 10.1126/sciadv.aba5933

Figure Lengend Snippet: ( A ) TSS heatmap coverage plots of H3K4Me3 and H3K27Me3 in NDC and PSEN1 M146L and PSEN1 A246E hiPSC-derived neurons as determined by ChIP-seq. ( B ) Annotation and directionality of differential ChIP-seq peaks in PSEN1 M146L and PSEN1 A246E hiPSC-derived neurons relative to NDC. ( C ) TF motif enrichment of regions with an increase in activating histone methylation status (increase in H3K4Me3 or decrease in H3K27Me3; top) or an increased in repressive histone methylation status (decrease in H3K4Me3 or increase in H3K27Me3; bottom) occurring within promoter or TSS-associated enhancer regions using GimmeMotifs and the HOCOMOCOv11 motif database. ( D and E ) chipenrich enrichment analysis of regions with an increase in activating histone methylation status (increase in H3K4Me3 or decrease in H3K27Me3; top) or an increased in repressive histone methylation status (decrease in H3K4Me3 or increase in H3K27Me3; bottom) occurring within promoter or TSS-associated enhancer regions using (D) ENCODE-ChEA consensus and ReMap TF-gene target databases or (E) GOBP and Hallmark ontology databases. ( F ) Venn-Euler diagram of overlap of DEGs with regions of differential H3K4Me3 or H3K27Me3 occurring within promoter or TSS-associated enhancer regions in PSEN1 M146L (top) and PSEN1 A246E (bottom) hiPSC-derived neurons. ( G ) Histone methylation density in PSEN1 M146L around DEGs with corresponding gain or loss of H3K4Me3 or H3K27Me3 status within promoter and enhancer regions. ( H and I ) Commonly enriched terms by hypergeometric (hg) enrichment analysis using the (H) ENCODE-ChEA Consensus, ReMap, Transfac/JASPAR (T/J), and TRRUST (T) TF-gene target databases or (I) GOBP and Hallmark databases for genes with directionally correlated increased gene expression and gain of activating H3KMe3 status (top) or decreased gene expression and gain of repressive H3KMe3 status (bottom).

Article Snippet: Five hundred nanograms of purified, sonicated chromatin was incubated with 5 μl of H3K4Me3 (Active Motif, catalog no. 39159) or H3K27Me3 (Active Motif, catalog no. 39155) rabbit polyclonal antibody overnight at 4°C.

Techniques: Derivative Assay, ChIP-sequencing, Methylation, Gene Expression

Journal: Science Advances

Article Title: Dedifferentiation and neuronal repression define familial Alzheimer’s disease

doi: 10.1126/sciadv.aba5933

Figure Lengend Snippet: ( A ) Venn diagram of overlap of DEGs, which have intersecting and directionally corresponding change in H3K4Me3 or H3K27Me3 status flanking regions of differential accessibility occurring within gene promoters or TSS-associated enhancers in PSEN1 M146L hiPSC-derived neurons. ( B and C ) Hypergeometric enrichment analysis using the (B) ENCODE-ChEA consensus and ReMap TF-gene target databases or (C) GOBP and Hallmark databases for genes with increased gene expression, chromatin accessibility, and gain of activating H3KMe3 status (top; yellow) or decreased gene expression, chromatin accessibility, and gain of repressive H3KMe3 status (bottom; blue). ( D to F ) ATAC-seq, H3K4Me3, and H3K27Me3 read profiles around the promoter regions of (D) nonectoderm lineage–controlling TFs with increased gene expression, (E) neuronal lineage– and function-controlling TFs with decreased gene expression, or (F) lineage-defining miRNAs with decreased expression.

Article Snippet: Five hundred nanograms of purified, sonicated chromatin was incubated with 5 μl of H3K4Me3 (Active Motif, catalog no. 39159) or H3K27Me3 (Active Motif, catalog no. 39155) rabbit polyclonal antibody overnight at 4°C.

Techniques: Derivative Assay, Gene Expression, Expressing

Journal: Nature Communications

Article Title: A biallelically active embryonic enhancer dictates GNAS imprinting through allele-specific conformations

doi: 10.1038/s41467-025-56608-0

Figure Lengend Snippet: A ENCODE ChIP-seq signal in hESCs showing histone modifications (H3K27ac, H3K4me3, and H3K27me3) and CTCF in STX16-ICR. The location of the CUT&RUN amplicon for panel ( B ) is shown at the bottom. B H3K27ac CUT&RUN-qPCR in STX16-ICR. Black, red, and blue bars represent WT, Δmat, and Δpat STX16-ICR hESCs, respectively. C ENCODE ChIP-seq signals in hESCs showing H3K27ac, H3K4me3, and CTCF in the NESP55 and XLαs regions. The locations of the CUT&RUN amplicons for panels ( D , E ) are shown at the bottom. D , E CUT&RUN-qPCR in hESCs. H3K4me3 in the NESP55 region ( D ) and in the putative XLαs promoter region ( E ) are shown. For B , D , and E , data are shown as mean ± SEM, and each dot represents an independent experiment (three biological replicates). Intergroup comparisons were performed by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: One μl of H3K27ac antibody (Cell Signaling Technology, 8173), 2 μl of H3K4me3 antibody (Cell Signaling Technology, 9751), 2 μl of H3K27me3 antibody (Cell Signaling Technology, 9733), 2 μl of CTCF antibody (Cell Signaling Technology, 3418), 2 μl of anti-OCT4 antibody (Abcam, ab200834), or 5 μl of normal rabbit IgG (Cell Signaling Technology, #66362) were added to 100 μl of cell suspension in antibody-binding buffer and incubated at 4 °C overnight.

Techniques: ChIP-sequencing, Amplification

Journal: Nature Communications

Article Title: A biallelically active embryonic enhancer dictates GNAS imprinting through allele-specific conformations

doi: 10.1038/s41467-025-56608-0

Figure Lengend Snippet: A (Top) Schematic locations of STX16-ICR with an OCT4 motif in intron 4 and the putative XLαs promoter. (Bottom) A UCSC browser screenshot magnifying the putative XLαs promoter region with FANTOM5 CAGE-seq and ENCODE H1ESCs H3K4me3 ChIP-seq signals. Each colored region corresponds to the region shown in the scheme for reporter constructs in ( B , C ). B , C Luciferase assays in WT hESCs. Forty-eight hours after transfection, firefly luciferase counts were measured and normalized by Renilla luciferase counts. B The effect of STX16-ICR on the activity of the full-length or truncated XLαs promoter (XLp) constructs was tested. From top to bottom, a negative control (promoterless), XLp-only, STX16-ICR with XLp, and STX16-ICR with three different truncated XLp constructs, as indicated by the colored regions. C The role of the OCT4 motif within STX16-ICR was tested. From top to bottom, a negative control (promoterless), XLp-only, WT STX16-ICR with XLp, and the OCT4 motif-deleted STX16-ICR with XLp constructs. For ( B and C ), experiments were repeated three times (biological replicates) with three technical replicates, and representative results are shown. Data are shown as mean ± SEM, and intergroup comparisons were performed by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: One μl of H3K27ac antibody (Cell Signaling Technology, 8173), 2 μl of H3K4me3 antibody (Cell Signaling Technology, 9751), 2 μl of H3K27me3 antibody (Cell Signaling Technology, 9733), 2 μl of CTCF antibody (Cell Signaling Technology, 3418), 2 μl of anti-OCT4 antibody (Abcam, ab200834), or 5 μl of normal rabbit IgG (Cell Signaling Technology, #66362) were added to 100 μl of cell suspension in antibody-binding buffer and incubated at 4 °C overnight.

Techniques: ChIP-sequencing, Construct, Luciferase, Transfection, Activity Assay, Negative Control

Journal: Nature Communications

Article Title: A biallelically active embryonic enhancer dictates GNAS imprinting through allele-specific conformations

doi: 10.1038/s41467-025-56608-0

Figure Lengend Snippet: A Schematic representation of XLαs exons and a heterozygous SNP in exon 5 (ex5), rs7121, in which T is maternal (Mat) and C is paternal (Pat). Arrows indicate RT-PCR primer locations. B – D Sanger sequencing of rs7121in XLαs RT-PCR products in hESCs. Results obtained from WT ( B ), STX16-ICR paternally deleted (Δpat STX16-ICR) ( C ), and STX16-ICR maternally deleted (Δmat STX16-ICR) ( D ) hESCs are shown. E – K Differentiation of hESCs into proximal renal tubule (PRT)-like cells. E The differentiation protocol. F , G Tetramethylrhodamine (TMR)-labeled parathyroid hormone (PTH) binding to hESCs ( F ) and differentiated PRT-like cells ( G ). Cells were incubated with TMR-PTH for 30 min, and fluorescence intensity was measured using a flow cytometer. H Quantitative PCR results of NESP55 and XLαs mRNAs in WT hESCs (ES, purple) and PRT-like cells (PRT-like, orange). Each dot represents the result of an independent experiment ( n = 4). Data are shown as mean ± SEM. The comparison was done by one-way ANOVA with Tukey’s post hoc test. I – K Sanger sequencing of rs7121 in XLαs RT-PCR products in differentiated PRT-like cells. Results obtained from WT ( I ), Δpat STX16-ICR ( J ), and Δmat STX16-ICR ( K ) cells are shown. L H3K27ac CUT&RUN qPCR in STX16-ICR using WT hESCs (purple) and WT PRT-like cells (orange). M , N 3C-qPCR detecting the interaction between STX16-ICR and the NESP55 region ( M ) and the XLαs region ( N ). Crosslinking frequencies were expressed as normalized values relative to the mean value of STX16-ICR-NESP55 interaction in hESCs. O – R CUT&RUN qPCR assays in WT hESCs (purple) and WT PRT-like cells (orange). O – Q In the NESP55 region, H3K4me3 ( O ), H3K27me3 ( P ), and CTCF ( Q ) were analyzed. R In the AS2 region, H3K4me3 was analyzed. L – R , each dot represents the result of an independent experiment ( n = 3). Data are shown as mean ± SEM. The comparison was done by two-tailed, unpaired t -tests.

Article Snippet: One μl of H3K27ac antibody (Cell Signaling Technology, 8173), 2 μl of H3K4me3 antibody (Cell Signaling Technology, 9751), 2 μl of H3K27me3 antibody (Cell Signaling Technology, 9733), 2 μl of CTCF antibody (Cell Signaling Technology, 3418), 2 μl of anti-OCT4 antibody (Abcam, ab200834), or 5 μl of normal rabbit IgG (Cell Signaling Technology, #66362) were added to 100 μl of cell suspension in antibody-binding buffer and incubated at 4 °C overnight.

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Labeling, Binding Assay, Incubation, Fluorescence, Flow Cytometry, Real-time Polymerase Chain Reaction, Comparison, Two Tailed Test

Journal: Nature communications

Article Title: Semiconductor-based DNA sequencing of histone modification states

doi: 10.1038/ncomms3672

Figure Lengend Snippet: ( a ) Density profiles of H3K4me3 (left) and polII ChIP-seq (right) from 2 million Ion Torrent reads (purple) and 15 million Illumina HiSeq reads (blue) over the Mx1 gene locus (top) and a larger region spanning the TNF locus (bottom). The scale of the density profile was set to the same level for each pair of samples, and is marked. All ChIP-seq experiments were performed with mDCs stimulated with LPS (0.1 ug/ml) for 2 hours. ( b ) A comparison of H3K4me3 (left) and polII (right) ChIP-seq peak enrichment scores (log 2 ) over a 500bp sliding window from 2 million Ion Torrent reads (X axis) and 15 million Illumina HiSeq reads (Y axis). The Pearson correlation coefficient is marked at the upper right corner. The grayscale represents a two-dimensional density plot, where the grid is divided into hexagonal bins and the color intensity reflects the density (counts) in the bin. An intensity bar of the counts is shown at the right bottom corner. ( c ) Saturation analysis to test for sufficient sequencing depth. Shown is the Pearson correlation coefficient (Y axis) calculated between decreasing sequencing reads randomly sub-sampled from the H3K4me3 ChIP-seq Ion Torrent reads (X axis) and either 15 million H3K4me3 ChIP-seq Illumina HiSeq reads (blue) or 2 million Ion Torrent reads (purple). ( d ) Density profiles of H3K4me3 ChIP-seq from 2 million Ion Torrent reads for libraries with 56ng, 4ng or 0.4ng of immunoprecipitated input DNA. ( e, f ) A comparison of H3K4me3 ChIP-seq peak enrichment scores (log 2 ) over a 500bp window from 2 million Ion Torrent reads with 56ng (Y axis) and 4ng (d, X axis) or 0.4ng (e, X axis) of immunoprecipitated input DNA. The Pearson correlation coefficient is marked at the upper right corner. The grayscale representation as in panel ( b ).

Article Snippet: We used 56ng, 4ng and 0.4ng of H3K4me3 ChIP DNA from a single ChIP experiment as input for our modified library construction protocol for Ion Torrent.

Techniques: ChIP-sequencing, Comparison, Sequencing, Immunoprecipitation

Journal: Nature communications

Article Title: Semiconductor-based DNA sequencing of histone modification states

doi: 10.1038/ncomms3672

Figure Lengend Snippet: ( a ) A comparison of accumulated H3K4me3 profiles in a pair of matching metastasis and primary tumor cell lines. Shown are the number of ChIP-seq reads over 1kb upstream and 1kb downstream of transcription start site of each of 20,000 human genes in a matched pair of primary melanoma tumor (WM115, black, X axis) and metastasis (WM266-4, blue, Y axis) cell lines derived from the same patient. The Pearson correlation coefficient is in the bottom right corner. ( b, c ) Density profiles of normalized reads from H3K4me3 ChIP-seq of primary melanoma tumor derived cell line (WM115) or metastasis derived cell line (WM266-4), at illustrative loci with either lower (b) or higher (c) levels of H3K4me3 in the metastasis derived cell line. ( d–g ) Gene sets enriched (by GSEA) among loci with different levels of histone modifications. In each case, shown are the names of the enriched gene sets (rows) along with the normalized enrichment score (NES), enrichment p value (P value) and False discovery rates (FDR). Each entry in the heat map indicates the percent overlap in ‘leading edge genes’ (those that contribute to the enrichment) between two enriched gene sets. (d) Genes with lower H3K4me3 in metastasis cell line (WM266-4) relative to control primary tumor cell line (WM115). (e) Genes with higher H3K4me3 in metastasis cell line (WM266-4) relative to control primary tumor (WM115). (f) Genes with lower H3K4me3 in metastatic melanoma tumors from patients (an average of seven tumors samples) relative to control normal skin melanocytes (an average of three biological samples). (g) Genes with higher H3K4me3 in metastatic melanoma tumors from patients relative to control normal skin melanocytes.

Article Snippet: We used 56ng, 4ng and 0.4ng of H3K4me3 ChIP DNA from a single ChIP experiment as input for our modified library construction protocol for Ion Torrent.

Techniques: Comparison, ChIP-sequencing, Derivative Assay, Control