Journal: Nucleic Acids Research

Article Title: Brain-specific deletion of histone variant H2A.z results in cortical neurogenesis defects and neurodevelopmental disorder

doi: 10.1093/nar/gkx1295

Figure Lengend Snippet: H2A.z is expressed in the embryonic cerebral cortex. ( A ) Western blot analysis of H2A.z in the development of the cerebral cortex. Brains in different developmental stages were isolated and lysed for western blot analysis with the antibodies indicated. ( B ) An overview of E13 brain sections labeled for H2A.z and DAPI. ( C ) Images of brain sections of the developing cerebral cortex labeled for H2A.z and DAPI. ( D ) H2A.z is abundantly expressed in Nestin positive neural progenitors and co labeled with Tuj1-positive neurons in the embryonic cortex. The E15 brain sections were immunostained with anti-H2A.z and anti-Nestin or anti-Tuj1 antibodies. ( E ) Western blot analysis reveals that the expression of H2A.z is effectively suppressed by H2A.z -shRNAs in primary neural progenitors. ( F ) Graph shows that the amount of H2A.z is obviously decreased in H2A.z -shRNA lentivirus-infected primary neural progenitors. n = 5 for all samples. ( G ) Electroporation of H2A.z -shRNAs results in abnormal cell distribution in the developing neocortex. The electroporation was performed at E13, and the mouse was sacrificed at E16. ( H ) Graph shows the percentage of GFP-positive cells distributed in the VZ/SVZ, IZ and CP. n = 5 for all samples. Representative images from at least three independent experiments. Error bars represent means ± S.E.M.; Two-tailed unpaired t -test, P < 0.05 (*), P < 0.01(**) or P < 0.001(***). n.s., not significant. CP, cortical plate; IZ, intermediate zone; VZ/SVZ, ventricular/subventricular zone. Scale bar represents 100 μm (B); 20 μm (C and D); 50 μm (G).

Article Snippet: The following antibodies were used: rat anti-BrdU (1:1000; Abcam; ab6326), rabbit anti-Ki67 (1:1000; Abcam; ab15580), rabbit anti-phospho-Histone H3 (pH3) (1:1000; Cell Signaling Technology; 3377S), mouse anti-Nestin (1: 200; Millipore; MAB353), rabbit anti-Pax6 (1:1000; Abcam; ab5790), mouse anti-Pax6 (1:100; DSHB), rabbit anti-Tbr2 (1:1000; Abcam; ab23345), mouse anti-Sox2 (1:1000; R&D; MAB2018), rabbit anti-Tuj1 (1:1000; Sigma; T2200), mouse anti-Tuj1 (1:2000; Millipore; MAB1637), rabbit anti-H2A.z (1:1000; Active Motif; 39943), rabbit anti-NeuN (1:1000; Abcam; ab177487), rabbit anti-Nkx2–4 (1:1000; Abcam; ab189202), mouse anti-β-actin (1:2000, Proteintech; 60008-1-Ig), rabbit anti-Flag (1:1000, Sigma; F1804), rabbit anti-IgG (1:1000, Bioss; bs-0295p), mouse anti-HA (1:1000; Abmart; M20003L), rabbit anti-H3K36me3 (1:1000; Cell Signaling Technology; 4909S), rabbit anti-H3K36me3 (Active Motif; 61101), rabbit anti-H3K27me3 (1:1000; Millipore; 07-449), rabbit anti-H3K9me3 (1:1000; Millipore; 07-442), rabbit anti-H3K4me3 (1:1000; Millipore; 07-473), and rabbit anti-H2A (1:500; Proteintech; 10445-1-AP).

Techniques: Western Blot, Isolation, Labeling, Expressing, shRNA, Infection, Electroporation, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Brain-specific deletion of histone variant H2A.z results in cortical neurogenesis defects and neurodevelopmental disorder

doi: 10.1093/nar/gkx1295

Figure Lengend Snippet: H2A.z regulates the proliferation of NPCs during brain development. ( A ) Brain sections of H2A.z fl/fl mice and H2A.z cKO mice at E16 were immunostained with mitotic marker pH3 and DAPI. ( B ) Statistics of pH3-positive cells in rostral, central, and caudal areas of the cortex. n = 5 for all samples. ( C ) Representative images of E16 coronal brain sections were immunostained for Tbr2 and BrdU. BrdU was injected intraperitoneally to pregnant mice at E16 for 2 h of pulse labeling. ( D ) Statistic of BrdU-positive cells per 100 μm 2 surface of VZ/SVZ. n = 7 for all samples. ( E ) Statistics of Tbr2-positive cells per 100 μm 2 surface of VZ/SVZ. n = 6 for all samples. ( F ) Percentage of Tbr2 + BrdU + cells among all Tbr2 + cells. n = 7 for all samples. ( G ) Neural progenitor cells isolated from H2A.z cKO mice form larger neurospheres. Primary neural progenitors were dissociated from E12 H2A.z cKO or H2A.z fl/fl mice and seeded at low density (50 cells/ml), then cultured in proliferated medium (suspension culture) for 3 days. ( H ) Statistic of the diameters of neurospheres. n = 8–9 for all samples. ( I ) Abnormal cell distribution is observed in H2A.z ablated neocortex. The GFP plasmid was electroporated into E13 mouse brains of H2A.z fl/fl mice and H2A.z cKO mice, and the mice were sacrificed at E16. ( J ) Graphs of the percentage of GFP-positive cells in the VZ/SVZ, IZ and CP. n = 8–10 for all samples. Representative images from at least three independent experiments. H2A.z fl/fl , WT; H2A.z cKO , cKO. CP, cortical plate; IZ, intermediate zone; VZ/SVZ, ventricular/subventricular zone. Error bars represent means ± S.E.M.; Two-tailed unpaired t-test, P < 0.05 (*), P < 0.01(**) or P < 0.001(***). n.s., not significant. Scale bar represents 15 μm (A); 20 μm (C); 80 μm (G); 50 μm (I).

Article Snippet: The following antibodies were used: rat anti-BrdU (1:1000; Abcam; ab6326), rabbit anti-Ki67 (1:1000; Abcam; ab15580), rabbit anti-phospho-Histone H3 (pH3) (1:1000; Cell Signaling Technology; 3377S), mouse anti-Nestin (1: 200; Millipore; MAB353), rabbit anti-Pax6 (1:1000; Abcam; ab5790), mouse anti-Pax6 (1:100; DSHB), rabbit anti-Tbr2 (1:1000; Abcam; ab23345), mouse anti-Sox2 (1:1000; R&D; MAB2018), rabbit anti-Tuj1 (1:1000; Sigma; T2200), mouse anti-Tuj1 (1:2000; Millipore; MAB1637), rabbit anti-H2A.z (1:1000; Active Motif; 39943), rabbit anti-NeuN (1:1000; Abcam; ab177487), rabbit anti-Nkx2–4 (1:1000; Abcam; ab189202), mouse anti-β-actin (1:2000, Proteintech; 60008-1-Ig), rabbit anti-Flag (1:1000, Sigma; F1804), rabbit anti-IgG (1:1000, Bioss; bs-0295p), mouse anti-HA (1:1000; Abmart; M20003L), rabbit anti-H3K36me3 (1:1000; Cell Signaling Technology; 4909S), rabbit anti-H3K36me3 (Active Motif; 61101), rabbit anti-H3K27me3 (1:1000; Millipore; 07-449), rabbit anti-H3K9me3 (1:1000; Millipore; 07-442), rabbit anti-H3K4me3 (1:1000; Millipore; 07-473), and rabbit anti-H2A (1:500; Proteintech; 10445-1-AP).

Techniques: Marker, Injection, Labeling, Isolation, Cell Culture, Suspension, Plasmid Preparation, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Brain-specific deletion of histone variant H2A.z results in cortical neurogenesis defects and neurodevelopmental disorder

doi: 10.1093/nar/gkx1295

Figure Lengend Snippet: H2A.z regulates the differentiation of NPCs. ( A ) The number of neurons is decreased in H2A.z -shRNA-electroporated brains. Neurons were labeled with anti-Tuj1 in E16 brain sections. ( B ) Percentage of Tuj1 + GFP + cells among GFP + cells. n = 4 for all samples. ( C ) Coronal brain slices of E16 H2A.z fl/fl and H2A.z cKO mice were immunostained using anti-Tuj1. ( D ) Relative Tuj1 + cells in the VZ of H2A.z fl/fl and H2A.z cKO mice. n = 8 for all samples. ( E ) The mRNA level of NeuroD1 and Tuj1 is decreased in H2A.z -deleted neurospheres. E12 primary neural progenitor cells (NPCs) were isolated from H2A.z cKO and H2A.z fl/fl mice, and cultured in proliferation medium (suspension culture) for 3 days, and then collected the neurospheres for further analysis (E and F). Actin mRNA served as an internal control. n = 4 for all samples. ( F ) Western blot analysis reveals that the expression levels of the neuron markers Tuj1 and NeuN are downregulated in H2A.z -loss neurospheres. ( G ) Statistics of the normalized density of H2A.z, Tuj1 and NeuN. n = 6 for all samples. ( H ) Cell cycle exit is decreased in H2A.z cKO mice. E16 brain sections were stained with anti-BrdU and anti-Ki67. H2A.z fl/fl mice and H2A.z cKO mice were administered BrdU (100 mg/kg) for 24 h and were euthanized at E16. ( I ) Percentage of cell cycle exit (BrdU + Ki67 − /Ki67 + ) in H2A.z fl/fl or H2A.z cKO mice. n = 6 for all samples. Representative images from at least three independent experiments. H2A.z fl/fl , WT; H2A.z cKO , cKO. VZ, ventricular zone. Error bars represent means ± S.E.M.; two-tailed unpaired t -test, P < 0.05 (*), P < 0.01(**) or P < 0.001(***). Scale bar represents 50 μm (A); 20 μm (C and H).

Article Snippet: The following antibodies were used: rat anti-BrdU (1:1000; Abcam; ab6326), rabbit anti-Ki67 (1:1000; Abcam; ab15580), rabbit anti-phospho-Histone H3 (pH3) (1:1000; Cell Signaling Technology; 3377S), mouse anti-Nestin (1: 200; Millipore; MAB353), rabbit anti-Pax6 (1:1000; Abcam; ab5790), mouse anti-Pax6 (1:100; DSHB), rabbit anti-Tbr2 (1:1000; Abcam; ab23345), mouse anti-Sox2 (1:1000; R&D; MAB2018), rabbit anti-Tuj1 (1:1000; Sigma; T2200), mouse anti-Tuj1 (1:2000; Millipore; MAB1637), rabbit anti-H2A.z (1:1000; Active Motif; 39943), rabbit anti-NeuN (1:1000; Abcam; ab177487), rabbit anti-Nkx2–4 (1:1000; Abcam; ab189202), mouse anti-β-actin (1:2000, Proteintech; 60008-1-Ig), rabbit anti-Flag (1:1000, Sigma; F1804), rabbit anti-IgG (1:1000, Bioss; bs-0295p), mouse anti-HA (1:1000; Abmart; M20003L), rabbit anti-H3K36me3 (1:1000; Cell Signaling Technology; 4909S), rabbit anti-H3K36me3 (Active Motif; 61101), rabbit anti-H3K27me3 (1:1000; Millipore; 07-449), rabbit anti-H3K9me3 (1:1000; Millipore; 07-442), rabbit anti-H3K4me3 (1:1000; Millipore; 07-473), and rabbit anti-H2A (1:500; Proteintech; 10445-1-AP).

Techniques: shRNA, Labeling, Isolation, Cell Culture, Suspension, Control, Western Blot, Expressing, Staining, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Brain-specific deletion of histone variant H2A.z results in cortical neurogenesis defects and neurodevelopmental disorder

doi: 10.1093/nar/gkx1295

Figure Lengend Snippet: The loss of H2A.z results in abnormal morphology of neurons. ( A ) Representative images of neurons after 4 days of culture in vitro . GFP-expressing plasmid was electroporated into the E13 cerebral cortices of H2A.z fl/fl or H2A.z cKO mice to mark neural progenitor cells. After 24 h, the GFP-positive cells were isolated and cultured in differentiation medium for 4 days. ( B ) Graph shows that the total dendritic length of neurons is decreased when H2A.z is deleted. n = 8 cells from three samples. ( C ) Statistics show that the number of primary dendrites is reduced upon H2A.z deletion. n = 8 cells from three samples. ( D ) Images of neurons cultured in differentiation medium for 6 days. ( E ) Sholl graphs of dendrites of neurons electroporated with GFP alone in H2A.z fl/fl or H2A.z cKO mice. The GFP-positive cells were isolated and cultured in differentiation medium for 6 days. H2A.z fl/fl , n = 6 cells; H2A.z cKO , n = 6 cells. ( F ) High-magnification confocal images of upper layer show that H2A.z knockout results in abnormally branched processes compared to the control. GFP plasmid was electroporated into the H2A.z fl/fl or H2A.z cKO embryonic brain at E13 and harvested at P0. ( G ) Quantification of dendritic numbers in H2A.z fl/fl and H2A.z cKO cortices. n = 6 for all samples. Representative images from at least three independent experiments. H2A.z fl/fl , WT; H2A.z cKO , cKO. Error bars represent means ± S.E.M., Kolmogorov–Smirnov test or two-tailed unpaired t -test, P < 0.05 (*), P < 0.01(**). Scale bar represents 15 μm (A and D); 10 μm (F).

Article Snippet: The following antibodies were used: rat anti-BrdU (1:1000; Abcam; ab6326), rabbit anti-Ki67 (1:1000; Abcam; ab15580), rabbit anti-phospho-Histone H3 (pH3) (1:1000; Cell Signaling Technology; 3377S), mouse anti-Nestin (1: 200; Millipore; MAB353), rabbit anti-Pax6 (1:1000; Abcam; ab5790), mouse anti-Pax6 (1:100; DSHB), rabbit anti-Tbr2 (1:1000; Abcam; ab23345), mouse anti-Sox2 (1:1000; R&D; MAB2018), rabbit anti-Tuj1 (1:1000; Sigma; T2200), mouse anti-Tuj1 (1:2000; Millipore; MAB1637), rabbit anti-H2A.z (1:1000; Active Motif; 39943), rabbit anti-NeuN (1:1000; Abcam; ab177487), rabbit anti-Nkx2–4 (1:1000; Abcam; ab189202), mouse anti-β-actin (1:2000, Proteintech; 60008-1-Ig), rabbit anti-Flag (1:1000, Sigma; F1804), rabbit anti-IgG (1:1000, Bioss; bs-0295p), mouse anti-HA (1:1000; Abmart; M20003L), rabbit anti-H3K36me3 (1:1000; Cell Signaling Technology; 4909S), rabbit anti-H3K36me3 (Active Motif; 61101), rabbit anti-H3K27me3 (1:1000; Millipore; 07-449), rabbit anti-H3K9me3 (1:1000; Millipore; 07-442), rabbit anti-H3K4me3 (1:1000; Millipore; 07-473), and rabbit anti-H2A (1:500; Proteintech; 10445-1-AP).

Techniques: In Vitro, Expressing, Plasmid Preparation, Isolation, Cell Culture, Knock-Out, Control, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Brain-specific deletion of histone variant H2A.z results in cortical neurogenesis defects and neurodevelopmental disorder

doi: 10.1093/nar/gkx1295

Figure Lengend Snippet: The loss of H2A.z results in behavioral deficits. ( A ) Representative tracing pathway of H2A.z fl/fl and H2A.z cKO mice in the open field test. ( B ) The total traveling distance was not different between H2A.z fl/fl and H2A.z cKO mice. ( C ) Time spent in the center is reduced in H2A.z cKO mice. ( D ) Representative tracing pathway of H2A.z fl/fl and H2A.z cKO mice in the elevated-plus maze test. ( E ) H2A.z cKO mice spent less time in the open arms. ( F ) H2A.z cKO mice spent more time in the closed arms. ( G ) Social interaction behavior is impaired in H2A.z- null mice. H2A.z cKO mice spent similar time in the chamber with the stranger 1 or stranger 2 mouse, while H2A.z fl/fl mice spent more time in the chamber with stranger 2 than with the stranger 1 mouse. ( H ) Close social interaction is affected by H2A.z deletion. H2A.z cKO mice did not spend more time in close interaction with stranger 2 than with stranger 1. ( I ) The immobile time of H2A.z cKO mice was more than that of H2A.z fl/fl mice during the forced swim test. ( J ) The percentage of freezing in H2A.z cKO mice was less than in H2A.z fl/fl mice, although they were explored in the same background. ( K ) Freezing response was also reduced in H2A.z cKO mice in the cued test. ( L ) Compared with H2A.z fl/fl mice, the entries of H2A.z cKO mice into new arms were decreased, while the entries into old arms were increased. ( M ) Compared with H2A.z fl/fl mice, H2A.z cKO mice spent less time in the new arms and more time in the old arms. All mice were male, 8–10 weeks old. H2A.z fl/fl , WT, n = 11–14; H2A.z cKO , cKO, n = 9–16. Error bars represent means ± S.E.M.; one-way ANOVA, P < 0.05 (*), P < 0.01(**) or P < 0.001(***). n.s., not significant. Analysis was performed by an experimenter blinded to experimental conditions.

Article Snippet: The following antibodies were used: rat anti-BrdU (1:1000; Abcam; ab6326), rabbit anti-Ki67 (1:1000; Abcam; ab15580), rabbit anti-phospho-Histone H3 (pH3) (1:1000; Cell Signaling Technology; 3377S), mouse anti-Nestin (1: 200; Millipore; MAB353), rabbit anti-Pax6 (1:1000; Abcam; ab5790), mouse anti-Pax6 (1:100; DSHB), rabbit anti-Tbr2 (1:1000; Abcam; ab23345), mouse anti-Sox2 (1:1000; R&D; MAB2018), rabbit anti-Tuj1 (1:1000; Sigma; T2200), mouse anti-Tuj1 (1:2000; Millipore; MAB1637), rabbit anti-H2A.z (1:1000; Active Motif; 39943), rabbit anti-NeuN (1:1000; Abcam; ab177487), rabbit anti-Nkx2–4 (1:1000; Abcam; ab189202), mouse anti-β-actin (1:2000, Proteintech; 60008-1-Ig), rabbit anti-Flag (1:1000, Sigma; F1804), rabbit anti-IgG (1:1000, Bioss; bs-0295p), mouse anti-HA (1:1000; Abmart; M20003L), rabbit anti-H3K36me3 (1:1000; Cell Signaling Technology; 4909S), rabbit anti-H3K36me3 (Active Motif; 61101), rabbit anti-H3K27me3 (1:1000; Millipore; 07-449), rabbit anti-H3K9me3 (1:1000; Millipore; 07-442), rabbit anti-H3K4me3 (1:1000; Millipore; 07-473), and rabbit anti-H2A (1:500; Proteintech; 10445-1-AP).

Techniques:

Journal: Nucleic Acids Research

Article Title: Brain-specific deletion of histone variant H2A.z results in cortical neurogenesis defects and neurodevelopmental disorder

doi: 10.1093/nar/gkx1295

Figure Lengend Snippet: H2A.z regulates neurogenesis by targeting Nkx2–4. ( A ) GO analysis for downregulated genes from E13 forebrain RNA-seq data sets in H2A.z -loss mice. ( B ) Analysis of gene ontology of biological functions for upregulated genes in H2A.z -depleted E13 forebrain. ( C ) Genome-wide distribution of H2A.z occupancies in E13 forebrain. ( D ) GO analyses of H2A.z binding genes. ( E ) Venn diagram showing the overlap between H2A.z bound genes by ChIP-seq with differentially expressed genes following H2A.z knockout (RNA-seq). ( F ) GO analyses for H2A.z-bound genes which are downregulated upon deletion of H2A.z . ( G ) GO analyses for H2A.z-bound genes which are upregulated upon deletion of H2A.z . ( H ) Volcano plots illustrate differentially expressed genes. Red dots are upregulated genes, and green dots are downregulated genes compared to the control. Nkx2–4 is one of the significantly differentially expressed genes. ( I ) H3K36me3 occupancy for all genes in E13 WT and H2A.z cKO forebrain. ( J ) RPKM values for all genes in E13 WT and H2A.z cKO forebrain.

Article Snippet: The following antibodies were used: rat anti-BrdU (1:1000; Abcam; ab6326), rabbit anti-Ki67 (1:1000; Abcam; ab15580), rabbit anti-phospho-Histone H3 (pH3) (1:1000; Cell Signaling Technology; 3377S), mouse anti-Nestin (1: 200; Millipore; MAB353), rabbit anti-Pax6 (1:1000; Abcam; ab5790), mouse anti-Pax6 (1:100; DSHB), rabbit anti-Tbr2 (1:1000; Abcam; ab23345), mouse anti-Sox2 (1:1000; R&D; MAB2018), rabbit anti-Tuj1 (1:1000; Sigma; T2200), mouse anti-Tuj1 (1:2000; Millipore; MAB1637), rabbit anti-H2A.z (1:1000; Active Motif; 39943), rabbit anti-NeuN (1:1000; Abcam; ab177487), rabbit anti-Nkx2–4 (1:1000; Abcam; ab189202), mouse anti-β-actin (1:2000, Proteintech; 60008-1-Ig), rabbit anti-Flag (1:1000, Sigma; F1804), rabbit anti-IgG (1:1000, Bioss; bs-0295p), mouse anti-HA (1:1000; Abmart; M20003L), rabbit anti-H3K36me3 (1:1000; Cell Signaling Technology; 4909S), rabbit anti-H3K36me3 (Active Motif; 61101), rabbit anti-H3K27me3 (1:1000; Millipore; 07-449), rabbit anti-H3K9me3 (1:1000; Millipore; 07-442), rabbit anti-H3K4me3 (1:1000; Millipore; 07-473), and rabbit anti-H2A (1:500; Proteintech; 10445-1-AP).

Techniques: RNA Sequencing, Genome Wide, Binding Assay, ChIP-sequencing, Knock-Out, Control

Journal: Nucleic Acids Research

Article Title: Brain-specific deletion of histone variant H2A.z results in cortical neurogenesis defects and neurodevelopmental disorder

doi: 10.1093/nar/gkx1295

Figure Lengend Snippet: H2A.z and Setd2-mediated H3K36me3 on Nkx2–4 promoter regulate the expression of Nkx2–4. ( A ) The interaction between H2A.z and Setd2 is verified by co-immunoprecipitation experiments. HA-Setd2-expressing plasmids were transfected into N2A cells. Three days later, the transfected cells were subjected to immunoprecipitation using HA beads. Bound proteins were detected by western blotting with the antibodies indicated. ( B ) H2A.z promotes Setd2-mediated H3K36me3 modification. HA-Setd2 plasmids were co-transfected with Flag-H2A.z or control into N2A cells. The cell lysates were immunoprecipitated with anti-HA, and bound proteins were detected by western blotting with the antibodies indicated. ( C ) Higher H3K36me3 levels were pulled down in both the H2A.z and Setd2 overexpression groups. Flag-H2A.z plasmids were co-transfected with HA-Setd2 or control into N2A cells. The cell lysates were immunoprecipitated with anti-Flag and blotted with the antibodies indicated. ( D ) The levels of H3K36me3 and Nkx2–4 are decreased when both H2A.z and Setd2 are silenced. Primary neural progenitors were infected with lentivirus indicated (D, E and F). Three days later, the cell lysates were analyzed by western blotting with the antibodies indicated. Graph below depicts quantification for Nkx2–4 and H3K36me3, normalized to actin. ( E ) Higher levels of Nkx2–4 and H3K36me3 are detected when H2A.z and Setd2 are overexpressed, while no obvious increase is investigated when H2A is overexpressed. Graph below depicts quantification for Nkx2–4 and H3K36me3, normalized to actin. ( F ) Overexpression of H2A.z, but not H2A, can rescue the downregulation of Nkx2–4 and H3K36me3 caused by H2A.z -sh1. Graph below depicts quantification for Nkx2–4 and H3K36me3, normalized to actin. ( G ) ChIP in primary neural progenitors overexpressing Setd2 with or without H2A.z -sh1, as indicated, performed with antibodies indicated. ( H ) ChIP in primary neural progenitors overexpressing H2A.z with or without Setd2-shRNA, as indicated, performed with antibodies indicated. ( I ) ChIP in primary neural progenitors overexpressing H2A.z with or without Setd2, as indicated, performed with antibodies indicated. ( J ) ChIP analysis of enrichment of H3K36me3 on promoters of Nkx2–4 in primary neural progenitors infected with control, Setd2 or Setd2 together with H2A.z -sh1 lentivirus. A total of 5% of input was loaded for western blotting. Representative images from at least three independent experiments. ChIP enrichment is expressed as percentage of input. n = 3–4 for all samples. Error bars represent means ± S.E.M.; two-tailed unpaired t -test, P < 0.05 (*), P < 0.01(**) or P < 0.001(***). n.s., not significant.

Article Snippet: The following antibodies were used: rat anti-BrdU (1:1000; Abcam; ab6326), rabbit anti-Ki67 (1:1000; Abcam; ab15580), rabbit anti-phospho-Histone H3 (pH3) (1:1000; Cell Signaling Technology; 3377S), mouse anti-Nestin (1: 200; Millipore; MAB353), rabbit anti-Pax6 (1:1000; Abcam; ab5790), mouse anti-Pax6 (1:100; DSHB), rabbit anti-Tbr2 (1:1000; Abcam; ab23345), mouse anti-Sox2 (1:1000; R&D; MAB2018), rabbit anti-Tuj1 (1:1000; Sigma; T2200), mouse anti-Tuj1 (1:2000; Millipore; MAB1637), rabbit anti-H2A.z (1:1000; Active Motif; 39943), rabbit anti-NeuN (1:1000; Abcam; ab177487), rabbit anti-Nkx2–4 (1:1000; Abcam; ab189202), mouse anti-β-actin (1:2000, Proteintech; 60008-1-Ig), rabbit anti-Flag (1:1000, Sigma; F1804), rabbit anti-IgG (1:1000, Bioss; bs-0295p), mouse anti-HA (1:1000; Abmart; M20003L), rabbit anti-H3K36me3 (1:1000; Cell Signaling Technology; 4909S), rabbit anti-H3K36me3 (Active Motif; 61101), rabbit anti-H3K27me3 (1:1000; Millipore; 07-449), rabbit anti-H3K9me3 (1:1000; Millipore; 07-442), rabbit anti-H3K4me3 (1:1000; Millipore; 07-473), and rabbit anti-H2A (1:500; Proteintech; 10445-1-AP).

Techniques: Expressing, Immunoprecipitation, Transfection, Western Blot, Modification, Control, Over Expression, Infection, shRNA, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Brain-specific deletion of histone variant H2A.z results in cortical neurogenesis defects and neurodevelopmental disorder

doi: 10.1093/nar/gkx1295

Figure Lengend Snippet:

Article Snippet: The following antibodies were used: rat anti-BrdU (1:1000; Abcam; ab6326), rabbit anti-Ki67 (1:1000; Abcam; ab15580), rabbit anti-phospho-Histone H3 (pH3) (1:1000; Cell Signaling Technology; 3377S), mouse anti-Nestin (1: 200; Millipore; MAB353), rabbit anti-Pax6 (1:1000; Abcam; ab5790), mouse anti-Pax6 (1:100; DSHB), rabbit anti-Tbr2 (1:1000; Abcam; ab23345), mouse anti-Sox2 (1:1000; R&D; MAB2018), rabbit anti-Tuj1 (1:1000; Sigma; T2200), mouse anti-Tuj1 (1:2000; Millipore; MAB1637), rabbit anti-H2A.z (1:1000; Active Motif; 39943), rabbit anti-NeuN (1:1000; Abcam; ab177487), rabbit anti-Nkx2–4 (1:1000; Abcam; ab189202), mouse anti-β-actin (1:2000, Proteintech; 60008-1-Ig), rabbit anti-Flag (1:1000, Sigma; F1804), rabbit anti-IgG (1:1000, Bioss; bs-0295p), mouse anti-HA (1:1000; Abmart; M20003L), rabbit anti-H3K36me3 (1:1000; Cell Signaling Technology; 4909S), rabbit anti-H3K36me3 (Active Motif; 61101), rabbit anti-H3K27me3 (1:1000; Millipore; 07-449), rabbit anti-H3K9me3 (1:1000; Millipore; 07-442), rabbit anti-H3K4me3 (1:1000; Millipore; 07-473), and rabbit anti-H2A (1:500; Proteintech; 10445-1-AP).

Techniques:

Journal: Cell Death & Disease

Article Title: Cortical microinfarcts potentiate recurrent ischemic injury through NLRP3-dependent trained immunity

doi: 10.1038/s41419-023-06414-7

Figure Lengend Snippet: Representative GFAP and Iba1 IHC staining ( A ) and quantitative analysis for Iba-1 ( B–D ) and GFAP- ( E ) positive astrocytes around the contralateral cortex following CMI, n = 12 mice per group. One-way ANOVA F 4, 55 = 2.808, p = 0.0342. Sham vs. 1w: p = 0.2748; Sham vs. 2w: p = 0.8075; Sham vs. 3w: p = 0.9621; Sham vs. 4w: p = 0.9515 ( B ); F 4, 55 = 34.01, p < 0.0001; Sham vs. 1w: p < 0.001; Sham vs. 2w: p < 0.001; Sham vs. 3w: p = 0.0142; Sham vs. 4w: p = 0.9215 ( C ); and F 4, 55 = 9.049, p < 0.0001; Sham vs. 1w: p = 0.0008; Sham vs. 2w: p = 0.0017; Sham vs. 3w: p = 0.0540; Sham vs 4w: p = 0.9999 ( E ) with Tukey’s correction. Scale bars, 25 μm. F Schematic diagram of experimental design. G Top 10 Gene Ontology (GO) enrichment terms from H3K4me3 ChIP-seq in contralateral microglia one week after CMI induction. H Top 10 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment components from H3K4me3 ChIP-seq in contralateral microglia one week after CMI induction. Quantitative analysis of H3K4me3 ChIP-qPCR on TNF-α ( I ), IL-6 ( J ), IL-1β ( K ), and IL-10 ( L ) promoters in contralateral microglia after CMI induction, n = 12 mice per group. One-way ANOVA. F 4, 55 = 7.884, p < 0.0001; Sham vs CMI1w: p = 0.0434; Sham vs. CMI 2w: p < 0.0001; Sham vs. CMI 3w: p = 0.0218; Sham vs. CMI 4w: p = 0.0256 ( I ); F 4, 55 = 5.031, p = 0.0016; Sham vs. CMI1w: p = 0.0091; Sham vs. CMI 2w: p = 0.9744; Sham vs. CMI 3w: p = 0.9477; Sham vs. CMI 4w: p = 0.9488 ( J ); F 4, 55 = 11.98, p < 0.0001; Sham vs. CMI1w: p < 0.0001; Sham vs. CMI 2w: p = 0.0037; Sham vs. CMI 3w: p = 0.7670; Sham vs. CMI 4w: p = 0.0183 ( K ); and F 4, 55 = 1.38, p = 0.253; Sham vs CMI1w: p = 0.6866; Sham vs. CMI 2w: p = 0.5709; Sham vs CMI 3w: p = 0.2671; Sham vs. CMI 4w: p = 0.2671 ( L ) with Tukey’s correction. Data are presented as mean ± standard deviation (SD), n.s., non-significant, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Chromatin precipitation was performed using a rabbit anti-mouse H3K4me3 IgG Ab (5326, Cell Signaling Technology, USA).

Techniques: Immunohistochemistry, ChIP-sequencing, ChIP-qPCR, Standard Deviation

Journal: Cell Death & Disease

Article Title: Cortical microinfarcts potentiate recurrent ischemic injury through NLRP3-dependent trained immunity

doi: 10.1038/s41419-023-06414-7

Figure Lengend Snippet: A Schematic diagram of experimental design. B – D Immunoblots and quantitative analysis of H3K4me1/3 showing microglial H3K4me1/3 expression in contralateral mouse cortex subjected to CMI and MLL1-shRNA treatment, n = 12 mice per group. Two-way ANOVA, for interaction: p < 0.0001. So, t-tests were used to detect the difference between groups. Sham vs. CMI: p < 0.0001; CMI vs. CMI+ MLL1-shRNA: p < 0.0001 ( C ) For interaction: p = 0.0774. For MLL1-shRNA factor: p < 0.0001. For CMI factor: p < 0.0001. Sham vs. CMI: p < 0.0001; CMI vs. CMI+ MLL1-shRNA: p < 0.0001 ( D ) with Turkey’s correction. Quantitative analysis of H3K4me3 ChIP-qPCR on TNF-α ( E ), IL-6 ( F ), IL-1β ( G ), and IL-10 ( H ) promoters in contralateral microglia 4 weeks after CMI and MLL1-shRNA treatment, n = 12 mice per group. Two-way ANOVA, for interaction p = 0.8532. For MLL1-shRNA factor, p < 0.0001. CMI vs. CMI+ MLL1-shRNA: p = 0.0129 ( E ); For interaction p = 0.3404. For MLL1-shRNA factor, p < 0.0001. CMI vs. CMI+MLL1-shRNA: p = 0.0238 ( F ); For interaction p = 0.7885. For MLL1-shRNA factor, p = 0.0002. CMI vs. CMI+ MLL1-shRNA: p = 0.0165 ( G ); For interaction p = 0.2121. For MLL1-shRNA factor, p = 0.0645. CMI vs. CMI+MLL1-shRNA: p = 0.9701 ( H ) with Tukey’s correction. I – M Immunoblots and quantitative analysis of TNF-α ( J ), IL-6 ( K ), IL-1β ( L ), and IL-10 ( M ) showing pro-inflammatory cytokine expression in peri-infarct region 12 h after PT stroke, n = 12 mice per group. Three-way ANOVA. For interaction, p < 0.0001. So, t-tests were used to detect the difference between groups. PT vs. CMI+PT: p < 0.0001; CMI+PT vs. CMI+PT+MLL1-shRNA: p < 0.0001 ( J ); For interaction, p = 0.0026. So, t-tests were used to detect the difference between groups. PT vs. CMI+PT: p = 0.0056; CMI+PT vs. CMI+PT+MLL1-shRNA: p = 0.8656 ( K ); For interaction, p < 0.0001. So, t-tests were used to detect the difference between groups. PT vs. CMI+PT: p < 0.0001; CMI+PT vs. CMI+PT+MLL1-shRNA: p < 0.0001 ( L) For interaction, p = 0.7475. PT vs. CMI+PT: p = 0.7733; CMI+PT vs. CMI+PT+MLL1-shRNA: p = 0.0188 ( M ) with Tukey’s correction. Representative Nissl and Iba1 IHC staining ( N ) and quantitative analysis of infarct size and microglial activation ( O – R ) showing PT stroke exacerbated by CMI was potentially mitigated by H3 methylation inhibition, n = 12 mice per group. Two-way ANOVA. For interaction, p = 0.0205. So, t-tests were used to detect the difference between groups. CMI+PT vs. CMI+PT+MLL1-shRNA: p = 0.0046 ( O ); For interaction, p = 0.2487. CMI+PT vs. CMI+PT+ MLL1-shRNA: p = 0.0228 ( Q ); For interaction, p < 0.0001. So, t-tests were used to detect the difference between groups. CMI+PT vs. CMI+PT++ MLL1-shRNA: p = 0.0001 ( R ) with Tukey’s correction. Scale bar, 200 μm for Nissl staining and 50 μm for Iba1 staining. Data are presented as mean ± standard deviation (SD), * p < 0.05, ** p < 0.01, *** p < 0.001, n.s., non-significant.

Article Snippet: Chromatin precipitation was performed using a rabbit anti-mouse H3K4me3 IgG Ab (5326, Cell Signaling Technology, USA).

Techniques: Western Blot, Expressing, shRNA, ChIP-qPCR, Immunohistochemistry, Activation Assay, Methylation, Inhibition, Staining, Standard Deviation

Journal: Cell Death & Disease

Article Title: Cortical microinfarcts potentiate recurrent ischemic injury through NLRP3-dependent trained immunity

doi: 10.1038/s41419-023-06414-7

Figure Lengend Snippet: A Immunoblots of NLRP3 expression in microglial whole-cell, cytoplasmic, and nuclear lysis after CMI, n = 12 mice per group. Quantitative ELISA analysis of TNF-α ( B ), IL-6 ( C ), IL-1β ( D ) and IL-10 ( E ) expression in the contralateral side of CMI showing that CMI did not influence the inflammatory cytokines expression in the chronic phase of CMI. ( n = 12 mice per group). One-way ANOVA F 4, 55 = 8.251, p < 0.0001. Sham vs. CMI1w: p = 0.0867; Sham vs. CMI 2w: p = 0.9064; Sham vs. CMI 3w: p < 0.0001; Sham vs. CMI 4w: p = 0.0331 ( B ); F 4, 55 = 3.113, p = 0.0222. Sham vs. CMI1w: p = 0.9992; Sham vs. CMI 2w: p = 0.9211; Sham vs. CMI 3w: p = 0.0390; Sham vs. CMI 4w: p = 0.9554 ( C ); F 4, 55 = 10.62, p < 0.0001. Sham vs. CMI1w: p = 0.2167; Sham vs. CMI 2w: p = 0.5763; Sham vs. CMI 3w: p = 0.9177; Sham vs. CMI 4w: p = 0.0027 ( D ); F 4, 55 = 4.098, p = 0.0056. Sham vs. CMI1w: p = 0.4233; Sham vs. CMI 2w: p > 0.9999; Sham vs. CMI 3w: p = 0.0266; Sham vs. CMI 4w: p = 0.9984 ( E ) with Tukey’s correction. F Representative immunofluorescence images of NLRP3 (red) expression in microglia (iba1, green) one week after CMI. Scale bars, 20 μm. G Immunoprecipitation and immunoblot analysis of MLL1 and NLRP3 in microglial lysates from mice subjected to sham and CMI. Lysates were immunoprecipitated by anti-MLL1 (IP) or immunoglobulin control (IgG) or directly subjected to immunoblot (Input), n = 3 mice per group. H Proximity ligation assay of microglial (iba1, green) NLRP3 and MLL1 (red dots) in sham and one-week post-CMI mice. Scale bars, 20 μm. I ChIP-seq analysis of NLRP3 and MLL1 binding to IL-1β in one-week post-CMI mice. J Representative images of sequence alignments showing interaction of NLRP3 (blue) with MLL1 (green). K Immunprecipitation and immunoblot analysis of MLL1 and Flag in microglial lysates from Cxc3r1 CreER mice transfected with 3xFlag-NLRP3 or truncated mutants (ΔPYD, ΔNACHT, ΔLRR) and subjected to CMI surgery. Lysates were immunoprecipitated by anti-MLL1 (IP) or immunoglobulin control (IgG) or directly subjected to immunoblot (Input), n = 3 mice per group. Quantitative analysis of H3K4me3 ChIP-qPCR on TNF-α ( L ), IL-6 ( M ), IL-1β ( N ), and IL-10 ( O ) promoters in contralateral microglia one week after CMI. Cxc3r1 CreER mice transfected with 3xFlag-NLRP3 or truncated mutants (ΔPYD, ΔNACHT, ΔLRR) and subjected to CMI surgery. One-way ANOVA, F 4, 55 = 42.25, p < 0.0001;Empty vs Full Length, p < 0.0001; Full Length vs ΔPYD: p = 0.7675; Full length vs ΔNACHT, p < 0.0001; Full Length vs ΔLRR: p = 0.1030 ( L ); F 4, 55 = 8.732, p < 0.0001; Empty vs. Full Length, p = 0.0077; Full Length vs ΔPYD: p = 0.9998; Full length vs ΔNACHT, p = 0.0001; Full Length vs. ΔLRR: p = 0.3921( M ); F 4, 55 = 122.5, p < 0.0001;Empty vs. Full Length, p < 0.0001; Full Length vs ΔPYD: p = 0.9868; Full length vs. ΔNACHT, p < 0.0001; Full Length vs. ΔLRR: p = 0.2638 ( N ); F 4, 55 = 2.246, p = 0.0758; Empty vs Full Length, p > 0.9999; Full Length vs ΔPYD: p = 0.9988; Full length vs ΔNACHT, p = 0.5413; Full Length vs. ΔLRR: p = 0.6035( O ). n = 12 mice per group. Data are presented as mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, n.s., non-significant.

Article Snippet: Chromatin precipitation was performed using a rabbit anti-mouse H3K4me3 IgG Ab (5326, Cell Signaling Technology, USA).

Techniques: Western Blot, Expressing, Lysis, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Immunoprecipitation, Control, Proximity Ligation Assay, ChIP-sequencing, Binding Assay, Sequencing, Transfection, ChIP-qPCR, Standard Deviation

Journal: Cell Death & Disease

Article Title: Cortical microinfarcts potentiate recurrent ischemic injury through NLRP3-dependent trained immunity

doi: 10.1038/s41419-023-06414-7

Figure Lengend Snippet: A Schematic diagram of experimental design. B Immunoblot of microglial NLRP3 expression in WT and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{NLRP}3}_{{cxc}3r1}^{-/-}$$\end{document} NLRP 3 cxc 3 r 1 − / − mice four weeks after CMI induction, n = 12 mice per group. C Immunoblot of microglial H3K4me1/3 expression in WT and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{NLRP}3}_{{cxc}3r1}^{-/-}$$\end{document} NLRP 3 cxc 3 r 1 − / − mice four weeks after CMI induction, n = 12 mice per group. Quantitative analysis of H3K4me3 ChIP-qPCR on TNF-α ( D ), IL-6 ( E ), IL-1β ( F ), and IL-10 ( G ) promoters among contralateral microglia four weeks after CMI induction in WT and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{NLRP}3}_{{cxc}3r1}^{-/-}$$\end{document} NLRP 3 cxc 3 r 1 − / − mice, n = 12 mice per group. Two-way ANOVA, for interaction: p = 0.0110. So, t-tests were used to detect the difference between groups. CMI vs. CMI+ \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{NLRP}3}_{{cxc}3r1}^{-/-}$$\end{document} NLRP 3 cxc 3 r 1 − / − : p = 0.0053 ( D ); For interaction: p = 0.7059. CMI vs. CMI+ \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{NLRP}3}_{{cxc}3r1}^{-/-}$$\end{document} NLRP 3 cxc 3 r 1 − / − : p = 0.9965 ( E ); For interaction: p = 0.0106. So, t-tests were used to detect the difference between groups. CMI vs. CMI+ \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{NLRP}3}_{{cxc}3r1}^{-/-}$$\end{document} NLRP 3 cxc 3 r 1 − / − : p = 0.0341 ( F ); For interaction: p = 0.0003. So, t-tests were used to detect the difference between groups. CMI vs. CMI+ \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{NLRP}3}_{{cxc}3r1}^{-/-}$$\end{document} NLRP 3 cxc 3 r 1 − / − : p = 0.6246 ( G ) with Tukey’s correction. Representative Nissl and Iba1 IHC staining ( H ) and quantitative analysis of infarct size and microglial activation ( I – L ) showing that NLRP3 blockade in microglia after CMI abolished the exacerbated infarct size and microglial activation by CMI, n = 12 mice per group. Two-way ANOVA, for interaction: p = 0.6293. CMI vs. CMI+ \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{NLRP}3}_{{cxc}3r1}^{-/-}$$\end{document} NLRP 3 cxc 3 r 1 − / − : p = 0.0231 ( I ), for interaction: p = 0.2277. CMI vs CMI+ \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{NLRP}3}_{{cxc}3r1}^{-/-}$$\end{document} NLRP 3 cxc 3 r 1 − / − : p = 0.0034 ( K ), for interaction: p = 0.0617. CMI vs. CMI+ \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{NLRP}3}_{{cxc}3r1}^{-/-}$$\end{document} NLRP 3 cxc 3 r 1 − / − : p = 0.0229 ( L ) with Tukey’s correction. Data are presented as mean ± standard deviation (SD), * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. non-significant.

Article Snippet: Chromatin precipitation was performed using a rabbit anti-mouse H3K4me3 IgG Ab (5326, Cell Signaling Technology, USA).

Techniques: Western Blot, Expressing, ChIP-qPCR, Immunohistochemistry, Activation Assay, Standard Deviation

Journal: Nature

Article Title: Re-engineering the zinc fingers of PRDM9 reverses hybrid sterility in mice

doi: 10.1038/nature16931

Figure Lengend Snippet: a , Enrichment of H3K4me3 marks at mouse motifs that are either within a B6 (left) or human (right) PRDM9 allele controlled DSB hotspot, or outside such a hotspot. The enrichment is relative to a control genomic track. Given the spread of the distributions, the interaction range between the histones and the DSB hotspot seems to be ~1.5 kb on each side of the motif. b , As a , for H3K36me3 marks. c , Mean coverage of H3K4me3 (left) or H3K36me3 (right) signal around the mouse motif nearest to each B6 DSB hotspot, split according to the strand on which the motif lies. d-h , As a , for H3K9ac ( d ), H3K27ac ( e ), H3K27me3 ( f ), H3K4me1 ( g ) and H3K79me2 ( h ) marks. All ChIP-seq data for histone modifications used in this analysis were obtained from the Mouse Encode Project.

Article Snippet: 5 μl rabbit polyclonal anti-H3K4me3 antibody (Abcam ab8580) was added to the remaining pre-cleared chromatin and incubated overnight at 4°C with rotation.

Techniques: ChIP-sequencing

Journal: Nature

Article Title: Re-engineering the zinc fingers of PRDM9 reverses hybrid sterility in mice

doi: 10.1038/nature16931

Figure Lengend Snippet: a , Distribution of the fraction of reads originating from the B6 chromosome in the infertile (PWD×B6)F1 PWD/B6 . PRDM9 control at each hotspot is attributed to B6 (blue), PWD (pink) or undetermined (grey). b , As a , but for non-shared hotspots, unique to either the rescue (PWD×B6)F1 PWD/H (top) or the infertile (PWD×B6)F1 PWD/B6 (bottom). c , Relative contributions of B6 and humanized PRDM9 to DMC1 signal in (B6/CAST)F2 B6/H . Bars represent the three possible genomic backgrounds. d , Individual chromosome effects (relative to Chromosome 1) when comparing DMC1 signals in (PWD×B6)F1 PWD/B6 relative to (PWD×B6)F1 PWD/H , for shared DSB hotspots. Bars: 1 SE. e , As d , but for H3K4me3. f , Comparison of DMC1 chromosome effects (as in d ) with the fitted chromosome effects, using a model including the symmetric hotspot measures for the three Prdm9 alleles. Bars: 3 SE (95% simultaneous confidence level for 19 chromosomes).

Article Snippet: 5 μl rabbit polyclonal anti-H3K4me3 antibody (Abcam ab8580) was added to the remaining pre-cleared chromatin and incubated overnight at 4°C with rotation.

Techniques:

Journal: Nature

Article Title: Re-engineering the zinc fingers of PRDM9 reverses hybrid sterility in mice

doi: 10.1038/nature16931

Figure Lengend Snippet: a-c , DSB hotspot asymmetry in (B6×PWD)F1 B6/PWD and in (B6×PWD)F1 H/PWD . a , Distribution of the fraction of (DMC1) informative reads originating from the B6 chromosome in the reciprocal (B6×PWD)F1 B6/PWD mouse. PRDM9 control at each DSB is attributed either to the B6 allele (blue) or the PWD allele (pink) or is undeterminable (grey). b-c , As a , but showing fractions only for non-shared hotspots, unique to either the reciprocal (B6×PWD)F1 B6/PWD ( b ) or the reciprocal rescue (B6×PWD)F1 H/PWD ( c ) mice. d-e , Comparison of the levels of asymmetric binding in the (PWD×B6)F1 PWD/B6 and (B6×CAST)F1 B6/CAST mice, using H3K4me3 signal. d , Distributions of the fraction of H3K4me3 reads from the B6 chromosome in the two mice. We used raw data from ref. for the (B6×CAST)F1 B6/CAST mouse, and processed both data sets in the same way. H3K4me3 heats were capped at the 95th percentile in each case, and only H3K4me3 binding peaks not inferred to be independent of PRDM9 binding , and overlapping with a DMC1 hotspot in the same mouse, were considered. e , Quantile-quantile plot for the distributions shown in d (blue). Dark grey: y = x line; light grey: 95% confidence band. f , Density plot comparing, for each hotspot in the (PWD×B6)F1 PWD/B6 mouse, its DMC1 and H3K4me3 asymmetries. The correlation between the two measures is 0.93. g , Mutations within 1kb regions around B6 and PWD PRDM9 motifs, on the B6 and PWD genomes. Main plot: For each combination of motif and lineage (PWD or B6), we plot the fraction of 30bp windows, along the 1kb regions surrounding motif occurrences within DSB hotspots, where at least one SNP or indel mutation occurred along the respective lineage. Inset plot: Distribution of motif score differences (derived-ancestral) for motif changes shown in the main plot. Motif score was defined as the logarithm of the probability that a motif was drawn from the motif’s position weight matrix, in the ancestral sequence and in the current-day mouse. A negative difference indicates the motif match worsened along the corresponding lineage. This panel is based on the (PWD×B6)F1 PWD/B6 DMC1 map. h , Mutations within 1kb regions around B6 PRDM9 motifs, on the B6 and CAST genomes, as in g , using the (B6×CAST)F1 B6/CAST DMC1 map. We see no evidence of erosion of B6 PRDM9 motifs on the CAST genome.

Article Snippet: 5 μl rabbit polyclonal anti-H3K4me3 antibody (Abcam ab8580) was added to the remaining pre-cleared chromatin and incubated overnight at 4°C with rotation.

Techniques: Binding Assay, Mutagenesis, Derivative Assay, Sequencing

Journal: Nature

Article Title: Re-engineering the zinc fingers of PRDM9 reverses hybrid sterility in mice

doi: 10.1038/nature16931

Figure Lengend Snippet: a , Individual chromosome effects (relative to chromosome 1) when comparing DMC1 signals in the B6 B6/H mouse relative to the B6 B6/B6 mouse, for the DSB hotspots that are shared between these two mice. b , Comparison of the observed chromosome effects for DMC1 signals with the fitted chromosome effects, using the 2-predictor model including the sum of symmetric H3K4me3 heats in B6 B6/B6 and in B6 H/H . Bars conservatively show 3 SEs in both plots.

Article Snippet: 5 μl rabbit polyclonal anti-H3K4me3 antibody (Abcam ab8580) was added to the remaining pre-cleared chromatin and incubated overnight at 4°C with rotation.

Techniques:

Journal: Nature

Article Title: Re-engineering the zinc fingers of PRDM9 reverses hybrid sterility in mice

doi: 10.1038/nature16931

Figure Lengend Snippet: a , Symmetry metric, as defined in the main text, for each sample (ALL), and for each autosome amongst those samples. Error bars represent bootstrap 95% CIs in all panels. b , Alternative symmetric metrics (to the ones reported in the main text), using only 10,000 hotspots per sample, or without weighting each chromosome specific metric, to compute the average metric genome-wide. Both metrics are computed using the DMC1 maps. c , Alternative symmetric metrics using H3K4me3 maps, similarly to b . The threshold of 12,540 hotspots per sample corresponds to the number of hotspots with ratio estimates in the (PWDxB6)F1 PWD/H mouse, which was the lowest amongst the three samples shown here.

Article Snippet: 5 μl rabbit polyclonal anti-H3K4me3 antibody (Abcam ab8580) was added to the remaining pre-cleared chromatin and incubated overnight at 4°C with rotation.

Techniques: Genome Wide

Journal: Nature

Article Title: Re-engineering the zinc fingers of PRDM9 reverses hybrid sterility in mice

doi: 10.1038/nature16931

Figure Lengend Snippet: a , For the PWD allele in the humanized rescue (PWD×B6)F1 PWD/H mouse, mean DMC1 signal is plotted in decile bins of H3K4me3 enrichment on the B6 chromosome (or the PWD X-chromosome), with error bars showing 95% CIs and lines of best fit (as in ). The slope of the line for asymmetric hotspots is 2.5-fold greater than that of the symmetric hotspots, and the slope for hotspots on the X-chromosome is 5.2-fold greater, illustrating that the DMC1 signal at asymmetric sites is elevated in a similar fashion to hotspots on the X-chromosome, which do not repair until late in meiosis. We found similar results in all cases tested. b , Comparison of DMC1 heats on B6 chromosome for hotspots shared between the humanized B6 H/H and the humanized rescue (PWD×B6)F1 PWD/H mice, under humanized PRDM9 control. We show symmetric hotspots (fraction of DMC1 informative reads between 0.4 and 0.6, green), and hotspots opposite a deletion on the PWD chromosome (deletion of at least 200bp, encompassing a human PRDM9 binding motif, black). The black line represents the median DMC1 heat for symmetric hotspots. c , As b , but showing the asymmetric hotspots (fraction of DMC1 informative reads above 0.9, red), with the corresponding median line. Hotspots opposite PWD deletion show a significant elevation in DMC1 heat relative to symmetric hotspots (14/16 hotspots above the symmetric median line, p=0.004). This elevation is similar to the one showed by asymmetric hotspots (9/16 hotspots above the asymmetric median line, p=0.80). d , Barplot showing the genome-wide ratio of mean DMC1 heat to mean H3K4me3 enrichment for asymmetric hotspots relative to symmetric hotspots in 9 scenarios studied, each for a different combination of mouse, Prdm9 allele, and haplotype, with error bars representing 95% bootstrap CIs for the ratio of means. In all cases, asymmetric hotspots show an elevation in DMC1 signal for a given H3K4me3 signal. e , Ratio of mean DMC1 and H3K4me3 signals on the B6 chromosome for the humanized allele in the humanized rescue mouse. Hotspots are clustered according to the fractions of their H3K4me3 signal that is on the B6 chromosome (r), and the ratio of the mean DMC1 and H3K4me3 signals in each class is shown here. The whiskers show 95% CIs for the mean, estimated using bootstrapping. When r>0.5, the B6 chromosome has greater H3K4me3 than the PWD chromosome, and vice versa. The ratio could not be estimated for r<=0.01 due to H3K4me3 levels being zero or nearly zero in those cases. f , (Left) Ratio of mean DMC1 and H3K4me3 signals on the B6 chromosome compared with the H3K4me3 signal on the PWD chromosome (log scale) in the infertile mouse. Asymmetric hotspots were defined as those with H3K4me3 fraction on the B6 chromosome > 0.9, and symmetric hotspots were those with the fraction between 0.1 and 0.9. Hotspots that we estimated to be completely asymmetric (H3K4me3 fraction=0 on either chromosome) or those with H3K4me3 enrichment on either chromosome close to zero (enrichment < 0.05) were excluded to avoid singularities on either axis. Asymmetric hotspots were binned into 4 bins of equal size and symmetric hotspots were binned into 10 bins of equal size. Different numbers of bins were used for asymmetric and symmetric hotspots to get approximately similar confidence intervals (error bars represent 95% CIs) to enable comparison. We did not observe many weak symmetric hotspots as we have limited power to detect such hotspots, which is why there are no symmetric bins with very low H3K4me3 levels on the homologue (Right). As (Left), but with the ratio determined for the PWD chromosome relative to H3K4me3 on the B6 chromosome. Accordingly, asymmetric hotspots are defined as those with H3K4me3 fraction on the PWD chromosome > 0.9.

Article Snippet: 5 μl rabbit polyclonal anti-H3K4me3 antibody (Abcam ab8580) was added to the remaining pre-cleared chromatin and incubated overnight at 4°C with rotation.

Techniques: Binding Assay, Genome Wide

Journal: Nature

Article Title: Re-engineering the zinc fingers of PRDM9 reverses hybrid sterility in mice

doi: 10.1038/nature16931

Figure Lengend Snippet: a , Comparison of B6 H/H and (PWD×B6)F1 PWD/H DMC1 signals (medians shown). Signals are compared for symmetric and asymmetric hotspots in (PWD×B6)F1 PWD/H , on the shared B6 chromosomes. Bars: 95% CIs. b , As a , but for H3K4me3. c , Comparison of DMC1 signals in (PWD×B6)F1 PWD/B6 , at symmetric and asymmetric hotspots, binned by H3K4me3 enrichment (medians shown). H3K4me3 and DMC1 signals are estimated on the PWD chromosome only, for hotspots associated with B6 PRDM9.

Article Snippet: 5 μl rabbit polyclonal anti-H3K4me3 antibody (Abcam ab8580) was added to the remaining pre-cleared chromatin and incubated overnight at 4°C with rotation.

Techniques:

Journal: Nature

Article Title: Re-engineering the zinc fingers of PRDM9 reverses hybrid sterility in mice

doi: 10.1038/nature16931

Figure Lengend Snippet: a-f , Comparison of DMC1 signals in the infertile (PWD×B6)F1 PWD/B6 mouse, at symmetric and asymmetric hotspots respectively, binned by H3K4me3 enrichment, after matching symmetric and asymmetric hotspots on various features: a , DMC1 heat in B6 B6/B6 ; b , local heterozygosity outside the PRDM9 binding motif, in a 500bp window; c , as b , but for a 1kb window; d , number of SNPs in binding motif; e , number of indels in binding motif; f , local GC content, computed in a 200bp window around hotspot centre. g , Distributions of the ratios of H3K4me3 heats on the B6 chromosome, in the rescue (PWD×B6)F1 PWD/H vs humanized B6 H/H mice, for the symmetric (fraction of informative DMC1 reads in the range 0.4-0.6, green) and asymmetric (fraction 0.9-1, orange) hotspots under humanized PRDM9 control shared between the two mice. The distributions are very close, suggesting similar trimethylation by PRDM9 on the B6 chromosome in both mice. h , As g , but for the DMC1 heats. Despite similar trimethylation marking by PRDM9 in both mice, we observed striking changes in the distribution of DMC1 ratios. This could be due to either more breaks occurring at the asymmetric sites, or a longer time taken to repair them. i , Quantile-quantile plots of DMC1 heats for hotspots under the control of the human allele on the B6 chromosome in the rescue (PWD×B6)F1 PWD/H (y-axis, left) vs the humanized B6 H/H (x-axis) mice, for symmetric (green) and asymmetric (orange) hotspots. Dotted line represents the ratios of asymmetric to symmetric quantiles (excluding distribution tails; y-axis, right). Dashed line represents expected ratio if there were no differences between symmetric and asymmetric hotspots. The observed ratio of DMC1 quantiles is constant across DMC1 heats, emphasizing that the increase in DMC1 heat at asymmetric sites is very similar across the whole range of DMC1 heats, and does not simply result from a few outlying hotspots.

Article Snippet: 5 μl rabbit polyclonal anti-H3K4me3 antibody (Abcam ab8580) was added to the remaining pre-cleared chromatin and incubated overnight at 4°C with rotation.

Techniques: Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: SMYD3 synergises with RACK1 to promote colorectal cancer lung metastasis by recruiting SMAD3

doi: 10.1186/s12964-026-02687-5

Figure Lengend Snippet: RACK1 recruits SMAD3 to SMYD3 and promotes the transcriptional activation of the genes downstream of SMYD3-SMAD3. A Effect of RACK1 knockdown on TSKU, H3K4me3, p-SMAD3 protein expression in RKO cells. B Effect of RACK1 overexpression on TSKU, H3K4me3, p-SMAD3 protein expression in HCT8 cells. C Effect of RACK1 knockdown on TSKU mRNA expression in RKO cells. D Effect of RACK1 overexpression on TSKU mRNA expression in HCT8 cells. E ChIP-qPCR with anti-Flag antibody showed binding of SMYD3 to TSKU. F ChIP-qPCR with anti-His antibody showed binding of SMAD3 to TSKU Data were shown as mean ± SD. The data were analyzed by Two-way ANOVA. *** p < 0.001

Article Snippet: The sheared chromatin was pre-conjugated to Protein G magnetic beads, and after 2 h, each mixture of chromatin and Protein G magnetic beads was incubated with 4 μg of anti-Flag antibody (CST, #14793S), anti-H3K4me3 antibody (CST, #9751S), anti-His antibody (Proteintech, #66,005–1-Ig), or normal rabbit IgG (Active Motif) overnight at 4 °C.

Techniques: Activation Assay, Knockdown, Expressing, Over Expression, ChIP-qPCR, Binding Assay

Journal: bioRxiv

Article Title: Yersinia remodels epigenetic histone modifications in human macrophages

doi: 10.1101/2021.09.07.459276

Figure Lengend Snippet: A, Left: Immunofluorescence staining of primary human macrophages mock infected or infected with WA314-YopE-ALFA for 6 h with MOI 10, 25, 50 or 100. Cells were stained with anti-ALFA nanobody-635 (red) to visualize YopE, Alexa568 phalloidin (green) to visualize actin and DAPI (blue) to visualize nucleus. Right: Quantification of YopE-ALFA positive cells. Bars show mean and error bars represent standard deviation when analyzing two different coverslips. B, Line plot showing time course H3K4me3 (IL1B, SOCS3, IDO1 genes) and H3K27ac (IL6, SOCS3 genes) ChIP-qPCR analysis of WAC infected primary human macrophages with MOI of 100. Lines represent means and error bars represent standard deviation from two independent macrophage donors/ biological replicates. C, Bar plot showing membrane permeability due to cell death in primary human macrophages mock infected, infected with WA314 (MOI of 100) or treated with positive (pos.) control (1 % TRITON-X-100) for 6 h. Increased membrane permeability is expressed as higher SYTOX Green intensity, where the dye binds to DNA and emits fluorescence when plasma membrane integrity is compromised. Bars represent means of two biological replicates/ macrophage donors (dots with different shapes). D, Western blot analysis of primary human macrophages showing caspase-3 cleavage in mock or WA314 (MOI 100) infected cells or treated with positive control Staurosporine (5 μM) for 6 h. * indicates full length caspase-3 (35 kDa) and cleavage fragments (17 and 19 kDa). GAPDH was used as a loading control. Data are representative of four biological replicates/ different macrophage donors. E, Bar plot showing colony forming unit (CFU) analysis of Y. enterocolitica WA314 growth during infection of primary human macrophages for 0 h, 1.5 h or 6 h at 37 °C. Bars represent means of two different biological replicates (dots with different shapes). F, Flow cytometry analysis of monocyte-derived primary human macrophages after 6 days of differentiation. Live cells (L/D negative) were selected (blue areas; first top panel) and doublets excluded (second top panel). CD14 positive macrophages were selected (third top panel) and analyzed for expression of macrophage markers CD163, CD38, CD86, HLA-DR,CD206 and CD16 (bottom panels).

Article Snippet: Antibodies used for ChIP were anti-H3K4me3 (Merck Millipore, 04–745, 4 μl per ChIP), anti-H3K27me3 (Merck Millipore, 07-449, 4 μl per ChIP), anti-H3K27ac (abcam, ab4729, 4 μg per ChIP) and anti-H3K4me1 (Cell Signaling, 5326S, 5 μl per ChIP).

Techniques: Immunofluorescence, Staining, Infection, Standard Deviation, Permeability, Fluorescence, Western Blot, Positive Control, Flow Cytometry, Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Yersinia remodels epigenetic histone modifications in human macrophages

doi: 10.1101/2021.09.07.459276

Figure Lengend Snippet: A, Experimental design. CD14 + monocytes were isolated and differentiated into macrophages by cultivation with 20 % human serum for 6 days. Macrophages from > two independent donors were mock infected or infected with avirulent Y. enterocolitica strain WAC, wild type strain WA314 or the single Yop-mutant strains WA314ΔYopM and WA314ΔYopP with multiplicity-of-infection (MOI) of 100 for 6 h. Samples were subjected to ChIP-seq for histone modifications H3K27ac, H3K4me3 and RNA-seq. H3K4me1 and H3K27me3 ChIP-seq was performed only for mock, WAC and WA314 infected macrophages. For all replicates used in the analysis see Table S17. B, Bar plot showing proportions of dynamic and constant regions of ChIP-seq data obtained from mock-, WAC- and WA314 infected macrophages as described in (A) . Dynamic regions were defined as MACS (H3K4me3, H3K27ac) or SICER (H3K4me1, H3K27me3) peaks overlapping significantly (≥ 2-fold change, adjusted P-value ≤ 0.05) up- or down-regulated differentially enriched regions from diffReps in the three pairwise comparisons WAC vs mock, WA314 vs mock and WAC vs WA314. C, Bar plot showing distribution of dynamic regions from (B) at gene promoters and enhancers. D, Heatmap showing Spearman correlation (cor.) of H3K27ac tag density at all H3K27ac peaks from mock-, WAC- and WA314-infected samples and publicly available H3K27ac ChIP-seq data of naive* and LPS* treated macrophages. Low to high correlation is indicated by blue-white-red colour scale. E, Number of differentially enriched regions (diffReps ≥ 2-fold change, adjusted P-value ≤ 0.05) for the indicated histone marks. Total (unique) refers to all unique regions in each row or column after merging overlapping regions. F, G, Venn diagrams showing overlaps of differentially enriched regions from diffReps depicting suppression of WAC induced upregulation of H3K4me3 marks by WA314 (F) and suppression of WAC induced up-regulation of H3K27ac marks by WA314 or prevention of WAC induced down-regulation of H3K27ac marks by WA314 (G) . H, Bar plot showing the number of genes associated with promoters, enhancers or both for the indicated histone marks associated with dynamic regions in (E) or when taking all regions together (total).

Article Snippet: Antibodies used for ChIP were anti-H3K4me3 (Merck Millipore, 04–745, 4 μl per ChIP), anti-H3K27me3 (Merck Millipore, 07-449, 4 μl per ChIP), anti-H3K27ac (abcam, ab4729, 4 μg per ChIP) and anti-H3K4me1 (Cell Signaling, 5326S, 5 μl per ChIP).

Techniques: Isolation, Infection, Mutagenesis, ChIP-sequencing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Yersinia remodels epigenetic histone modifications in human macrophages

doi: 10.1101/2021.09.07.459276

Figure Lengend Snippet: A, Bar plot showing H3K27me3 (left) and H4K4me3 (right) ChIP-qPCR from primary human macrophages mock infected or infected with WAC or WA314 for 6 h with MOI of 100. HOXC12 and NXPH4 are positive controls for H3K27me3 and ADH5 and TUBA1C are positive controls for H3K4me3. Data are representative of at least two experiments with different biological replicates/ macrophage donors. B, Peak tracks showing tag density of H3K4me3 (red) or H3K27ac (blue) ChIP-seq from primary human macrophages infected for 6 h with indicated strains (MOI of 100) (left). qPCR target refers to the primer binding site used for fragment amplification in ChIP-qPCR analysis shown as a dot plot (right). The ChIP-qPCR signal was expressed as relative (rel.) enrichment (enr.) vs mock. Lines represent means from at least two different biological replicates/ macrophage donors (dots with different shapes). C, Dot plots showing gene expression analysis of PPARGC1B, ABI1, TNF and IL1B genes with RT-qPCR from primary human macrophages infected for 6 h with indicated strains (MOI 100). The RT-qPCR signal was expressed as fold vs mock. Lines represent means from at least two different biological replicates/ macrophage donors (dots with different shapes).

Article Snippet: Antibodies used for ChIP were anti-H3K4me3 (Merck Millipore, 04–745, 4 μl per ChIP), anti-H3K27me3 (Merck Millipore, 07-449, 4 μl per ChIP), anti-H3K27ac (abcam, ab4729, 4 μg per ChIP) and anti-H3K4me1 (Cell Signaling, 5326S, 5 μl per ChIP).

Techniques: Infection, ChIP-sequencing, Binding Assay, Amplification, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Yersinia remodels epigenetic histone modifications in human macrophages

doi: 10.1101/2021.09.07.459276

Figure Lengend Snippet: A, Heatmap showing clustering of all H3K4me3 (module P1) and H3K27ac (module P2) differentially enriched regions at promoters in mock-, WAC- and WA314 infected human macrophages. P1 contains dynamic H3K4me3 regions and P2 contains dynamic H3K27ac regions at largely constant H3K4me3 regions. H3K4me3 and H3K27ac tag densities are shown in both modules. The identified clusters (colour coded bars on the left side) were grouped in classes P1a-P2d. B, Boxplots of H3K4me3 and H3K27ac tag counts for the classes in (A) . Profiles describing relation of histone mark levels between mock, WAC and WA314 are indicated on top. Data are representative of at least two independent experiments. C Peak tracks of H3K4me3 (red) and H3K27ac (blue) ChIP-seq tag densities at promoter regions of the genes OASL from Suppression profile classes P1a and ACOT7 from Prevention profile class P1b (left) and ZNF513 from Down profile class P2b and SLC30A3 from Up profile class P2c (right). D, Heatmap showing clustering of H3K27ac differentially enriched regions at enhancers of mock-, WAC- and WA314 infected macrophages. H3K4me1 tag counts are shown for the associated regions. Clustering analysis yielded 6 clusters (color coded, left side) which were assembled into classes E1-4. E, Boxplot of H3K27ac tag counts of classes E1-E4 in (D) . Profiles describing relation of histone mark levels between mock, WAC and WA314 are indicated on top. Data are representative of two independent experiments. F, Heatmap showing H3K4me1 and H3K27ac tag counts at latent enhancers induced by WAC vs mock. For (A) , (D) and (F) rows are genomic regions from −10 to +10 kb around the centre of the analyzed regions. “n” indicates number of regions.

Article Snippet: Antibodies used for ChIP were anti-H3K4me3 (Merck Millipore, 04–745, 4 μl per ChIP), anti-H3K27me3 (Merck Millipore, 07-449, 4 μl per ChIP), anti-H3K27ac (abcam, ab4729, 4 μg per ChIP) and anti-H3K4me1 (Cell Signaling, 5326S, 5 μl per ChIP).

Techniques: Infection, ChIP-sequencing

Journal: bioRxiv

Article Title: Yersinia remodels epigenetic histone modifications in human macrophages

doi: 10.1101/2021.09.07.459276

Figure Lengend Snippet: A-D, Bar plots showing fraction (%) of genes associated with H3K4me3 at promoter (A) , H3K27ac at promoter (B) , H3K4me3 and H3K27ac at promoter (C) and H3K27ac at enhancer (D) changes for comparisons between mock, WAC and WA314 showing significant (≥ 2-fold change, adjusted P-value ≤ 0.05) associated change in gene expression (overlap RNA).

Article Snippet: Antibodies used for ChIP were anti-H3K4me3 (Merck Millipore, 04–745, 4 μl per ChIP), anti-H3K27me3 (Merck Millipore, 07-449, 4 μl per ChIP), anti-H3K27ac (abcam, ab4729, 4 μg per ChIP) and anti-H3K4me1 (Cell Signaling, 5326S, 5 μl per ChIP).

Techniques: Expressing

Journal: bioRxiv

Article Title: Yersinia remodels epigenetic histone modifications in human macrophages

doi: 10.1101/2021.09.07.459276

Figure Lengend Snippet: A, Principal component analysis of H3K27ac tag counts in classes P1a-P2d and E1-4 in two replicates of macrophages infected with the indicated strains. B, Number of statistically significant (≥ 2-fold change, adjusted P-value ≤ 0.05) DEGs and differentially enriched H3K4me3 and H3K27ac regions for WA314ΔYopM vs WA314 and WA314ΔYopP vs WA314. C, D, Percentage of WA314 effect on expression of Rho GTPase pathway (C) or Inflammatory response (D) genes that is caused by YopP. Genes with associated promoter or enhancer changes from the same profile and significant change for RNA-seq (DESeq2) and ChIP-seq (diffReps) between WAC vs WA314 are shown. The percentage value was calculated from the ratio of fold change (FC) between WA314ΔYopP vs WA314 and WA314 vs WAC for Suppression (C, D) and Prevention (C) profiles. E, Peak tracks of ChIP-seq tag densities showing H3K4me3 and H3K27ac changes associated with IL2RA gene, whose suppression of gene expression by WA314 is produced in a YopP- dependent manner (D) . H3K4me3 and H3K27ac densities at the IL2RA promoter (green shade) and H3K27ac densities at the enhancers (purple shade) are not affected by YopP (compare effects of WA314 and WA314ΔYopP). F, Dot plots of H3K4me3 and H3K27ac ChIP-qPCR signals at indicated genes in macrophages not infected (mock) or infected with indicated strains. WA314ΔYopP infections occurred in the absence and presence of MAPK inhibitors (inh). The ChIP-qPCR signal was expressed as relative (rel.) enrichment (enr.) vs mock. Lines represent mean of 3 biological replicates (dots with different shapes). SOCS3: Suppressor of cytokine signaling 3, IDO1: Indoleamine 2,3-Dioxygenase 1, PTGS2: Prostaglandin-Endoperoxide Synthase 2. G, Western blot analysis (top) and quantification (bottom) of ABI1 levels in primary human macrophages infected with WA314, WA314ΔYopP and WA314ΔYopP+inh (MAPK inhibitors) at an MOI of 100 for 6 h. GAPDH was used as a loading control and for normalization of ABI1 signal. Western blot is representative of three independent experiments shown in quantification. H, Immunofluorescence staining of primary human macrophages infected with WA314, WA314ΔYopP+inh (MAPK inhibitors) and WA314ΔYopP with MOI of 100 for 6 h. Cells were stained with Alexa568 phalloidin (red) to visualize actin and antibodies for ABI1 (cyan), vinculin (blue) and ZO-1 (green). Fourth column shows an overlay of actin and ABI1 (top) and actin, vinculin and ZO-1 (bottom) for WA314ΔYopP infection. Data are representative of two independent experiments. I, Quantification of actin (left) and ABI1 (right) signal intensity at cell junctions from immunofluorescence of primary human macrophages infected with WA314, WA314ΔYopP+inh (MAPK inhibitors) and WA314ΔYopP with MOI of 100 for 6 h. Data are representative of two independent experiments. J, Immunofluorescence staining of primary human macrophages infected with WA314, WA314ΔYopP+C646 (p300 inhibitor) and WA314ΔYopP with MOI of 100 for 6 h (left). Cells were stained with Alexa568 phalloidin (red) to visualize actin and antibody for ABI1 (cyan). Quantification of ABI1 signal at cell cell contacts is shown on the right. Data are representative of two independent experiments. In (I) and (J) bars show mean and error bars represent standard deviation. ****: P-adjusted < 0.0001 by unpaired Wilcoxon test.

Article Snippet: Antibodies used for ChIP were anti-H3K4me3 (Merck Millipore, 04–745, 4 μl per ChIP), anti-H3K27me3 (Merck Millipore, 07-449, 4 μl per ChIP), anti-H3K27ac (abcam, ab4729, 4 μg per ChIP) and anti-H3K4me1 (Cell Signaling, 5326S, 5 μl per ChIP).

Techniques: Infection, Expressing, RNA Sequencing Assay, ChIP-sequencing, Produced, Western Blot, Immunofluorescence, Staining, Standard Deviation

Journal: bioRxiv

Article Title: Yersinia remodels epigenetic histone modifications in human macrophages

doi: 10.1101/2021.09.07.459276

Figure Lengend Snippet: A, Principal component analysis of H3K4me3 tag counts in classes P1a-b of macrophages infected with the indicated strains for all biological replicates used in the analysis. B, Percentage YopP effect (median value) for H3K4me3 (P1a-b classes) and H3K27ac (P2a-d, E1-4 classes) when compared to WA314 vs mock (Up and Down profiles) or WA314 vs WAC (Suppression and Prevention profiles). C, Percentage YopP effect (median value) for DEGs associated with respective histone modification changes at promoters or enhancers. “Median”: median value when taking % YopP effect from all classes together. D, Scatter plot of % YopP effect for RNA-seq DEGs and ChIP-seq differential regions for genes with associated gene expression and histone modification change from Rho GTPase and Inflammatory response pathways from . Purple dots associated with ABI1 gene are indicated. E, Top: Dot plots showing ChIP-qPCR of H3K27ac at enhancer within ABI1 gene and H3K4me3 at ABI1 promoter from primary human macrophages infected for 6 h with indicated strains (MOI of 100). Lines represent means from three different biological replicates/ macrophage donors (dots with different shapes). Bottom: Peak tracks showing tag density of H3K27ac at enhancer (blue) and H3K4me3 at promoter (red) ChIP-seq and primer binding site used for fragment amplification in ChIP-qPCR analysis (qPCR target) shown in dot plots. The ChIP-qPCR signal was expressed as relative (rel.) enrichment (enr.) vs mock. inh: MAPK inhibitors. F, Dot plots showing RT-qPCR gene expression analysis of ABI1 from primary human macrophages infected for 6 h with indicated strains (MOI of 100). The RT-qPCR signal was expressed as fold vs mock. Lines represent means from three different biological replicates/ macrophage donors (dots with different shapes). inh: MAPK inhibitors. G, Immunofluorescence staining of primary human macrophages infected with WA314ΔYopP+C646 (p300 inhibitor) and WA314ΔYopP with MOI of 100 for 6 h. Cells were stained with antibody for H3K27ac. Images are representative of two independent experiments.

Article Snippet: Antibodies used for ChIP were anti-H3K4me3 (Merck Millipore, 04–745, 4 μl per ChIP), anti-H3K27me3 (Merck Millipore, 07-449, 4 μl per ChIP), anti-H3K27ac (abcam, ab4729, 4 μg per ChIP) and anti-H3K4me1 (Cell Signaling, 5326S, 5 μl per ChIP).

Techniques: Infection, Modification, RNA Sequencing Assay, ChIP-sequencing, Expressing, Binding Assay, Amplification, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Yersinia remodels epigenetic histone modifications in human macrophages

doi: 10.1101/2021.09.07.459276

Figure Lengend Snippet: Plasmid-encoded virulence factors suppress upregulation (Suppression profile) and prevent downregulation (Prevention profile) of PAMP induced H3K27ac and H3K4me3 changes at gene promoters and/ or enhancers and consequently the expression of associated genes. Suppression profile is associated with genes from Inflammatory response and Type I IFN signaling, whereas Prevention profile is associated with genes from Metabolic pathways and Rho GTPase signaling. Representative genes belonging to each pathway are shown. Enhancer activity is indicated with arched arrows. Increased promoter activity is indicated with more mRNAs.

Article Snippet: Antibodies used for ChIP were anti-H3K4me3 (Merck Millipore, 04–745, 4 μl per ChIP), anti-H3K27me3 (Merck Millipore, 07-449, 4 μl per ChIP), anti-H3K27ac (abcam, ab4729, 4 μg per ChIP) and anti-H3K4me1 (Cell Signaling, 5326S, 5 μl per ChIP).

Techniques: Plasmid Preparation, Expressing, Activity Assay