Journal: Translational Psychiatry

Article Title: Schizophrenia-associated differential DNA methylation in brain is distributed across the genome and annotated to MAD1L1 , a locus at which DNA methylation and transcription phenotypes share genetic variation with schizophrenia risk

doi: 10.1038/s41398-022-02071-0

Figure Lengend Snippet: Differentially methylated sites in SZ.

Article Snippet: cg04594439 , −0.025 , 0.061 , PASK.

Techniques: Methylation

Journal: Nutrients

Article Title: Roseburia intestinalis Supplementation Could Reverse the Learning and Memory Impairment and m6A Methylation Modification Decrease Caused by 27-Hydroxycholesterol in Mice

doi: 10.3390/nu16091288

Figure Lengend Snippet: Primers used in this study.

Article Snippet: The antibodies used were as below: β-actin (ABclonal, AC026, 1:50,000, Wuhan, China), METTL4 (bioss, bs-18851R, 1:1000, Beijing, China), PSD-95 (Abcam, ab18258, 1:1000, Cambridge, UK), occludin (Abcam, ab216327, 1:1000, Cambridge, UK), claudin-1 (Abcam, ab180158, 1:1000, Cambridge, UK).

Techniques: Sequencing

Journal: Nutrients

Article Title: Roseburia intestinalis Supplementation Could Reverse the Learning and Memory Impairment and m6A Methylation Modification Decrease Caused by 27-Hydroxycholesterol in Mice

doi: 10.3390/nu16091288

Figure Lengend Snippet: Levels of the m6A modification and its related enzyme expression. ( A ) m6A methylation in the brain cortex (ng/200ng RNA). ( B ) Western blot result of METTL4. ( C ) METTL4 mRNA relative expression level in brain cortex. ( D ) METTL4 protein expression level in the brain cortex. ( E ) METTL14 mRNA relative expression level in the brain cortex, ( F ) YTHDF-1 mRNA relative expression level in the brain cortex. ( G ) FTO mRNA relative expression level in the brain cortex. ( H ) WTAP mRNA relative expression level in the brain cortex. n = 4–5 mice/group. All the data are presented as means ± SEM. * p < 0.05. ** p < 0.01. *** p < 0.001. ns p > 0.05.

Article Snippet: The antibodies used were as below: β-actin (ABclonal, AC026, 1:50,000, Wuhan, China), METTL4 (bioss, bs-18851R, 1:1000, Beijing, China), PSD-95 (Abcam, ab18258, 1:1000, Cambridge, UK), occludin (Abcam, ab216327, 1:1000, Cambridge, UK), claudin-1 (Abcam, ab180158, 1:1000, Cambridge, UK).

Techniques: Modification, Expressing, Methylation, Western Blot

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet:

Article Snippet: Plasmid: p3xFlag-Mettl4 , This study , Addgene plasmid 86665.

Techniques: Luciferase, Recombinant, SYBR Green Assay, Sequencing, Methylated DNA Immunoprecipitation, RNA Sequencing Assay, Plasmid Preparation, CRISPR, Software

Journal: Scientific Reports

Article Title: METTL3/IGF2BP2 axis affects the progression of colorectal cancer by regulating m6A modification of STAG3

doi: 10.1038/s41598-023-44379-x

Figure Lengend Snippet: Antibodies used in the study are shown.

Article Snippet: METTL14 , 1: 1000 , Rabbit , 55 , orb28269, Biorbyt.

Techniques:

Journal: Scientific Reports

Article Title: METTL3/IGF2BP2 axis affects the progression of colorectal cancer by regulating m6A modification of STAG3

doi: 10.1038/s41598-023-44379-x

Figure Lengend Snippet: METTL3 mediated m6A methylation of STAG3. ( A ) In CRC and adjacent normal tissues, the protein expression levels of METTL3, METTL14, ALKBH5, and FTO were determined by WB. ( B,C ) The mRNA and protein expression levels of METTL3 in HCT116 cells were measured using qRT-PCR and WB. ( D ) The STAG3 m6A modification level in HCT116 cells was evaluated by Me-RIP. *P < 0.05 vs. normal, & P < 0.05 vs. sh-NC, # P < 0.05 vs. oe-NC. Superscript a: P < 0.05, superscript b: P < 0.01, superscript d: P < 0.0001.

Article Snippet: METTL14 , 1: 1000 , Rabbit , 55 , orb28269, Biorbyt.

Techniques: Methylation, Expressing, Quantitative RT-PCR, Modification

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: Mettl4 and Alkbh4 Catalyze Deposition and Erasure, Respectively, of 6mA (A) Schematic presentation of Mettl4 protein structure and linear arrangement of conserved motifs in methyltransferase domain (amino acids 257–471), showing detailed alignment to motif IV sequence logo in catalytic site of MT-A70 adenine N6-methyltransferases. (B) Clustering of proteins within the MT-A70 family. RNA-specific methyltransferases form a tight cluster of closely related sequences, while Mettl4 and the DNA adenine N6-methyltransferases DAMT-1 ( C. elegans ) and M. MunI (Mycoplasma) (red circles) are positioned apart. (C) HEK293T cells transfected with empty vector (EV) or expressing Flag-Dam or the wild-type or DPPW catalytic-site mutant (PPmut) variants of Flag-Mettl4 were harvested and proteins in whole-cell extracts resolved by SDS-PAGE and immunoblotting with Flag antisera. Actin, loading control. 6mA and 5mC in the same samples were detected by dot blot of genomic DNA (n = 3 experiments), and mean 6mA levels relative to dA were quantified by LC-MS/MS analysis of genomic DNA hydrolysates (n = 2 experiments). (D) Levels of 6mA relative to total dA in genomic DNA (left panel) and m6A relative to total adenosine (A) in mRNA (right panel), purified from WT and Mettl4 KO ESCs, as determined by quantitative LC-MS/MS. Dashed line represents limit of detection. n.d., not detected. Data are plotted as mean with SD (n = 2 experiments). (E) Overlaid extracted LC-MS chromatograms of dA and 6mA in genomic DNA hydrolysates prepared from WT and Mettl4 KO spleens. (F) In vitro 6mA demethylation assay. Recombinant His-Alkbh4 was purified from bacterial cell extracts (left panel). Irrelevant lanes were omitted from the gel. Human genomic DNA containing 6mA was incubated in buffer supplemented with Fe 2+ and 2-oxoglutarate cofactors in the absence or presence of His-Alkbh4 or vitamin C (VitC) for the indicated times. 6mA in each sample was detected by dot blot analysis (n = 3 experiments). (G) Alkbh4 demethylates 6mA in double-stranded (ds) DNA. Single-stranded (ss) or double-stranded DNA oligonucleotide containing unmodified adenine or 6mA was incubated in the absence or presence of His-Alkbh4 for the indicated times. Following the reactions, 6mA in 10 pmol of each DNA sample was detected by dot blot analysis (n = 3 experiments). See also and .

Article Snippet: Plasmid: Mettl4 HDR , Santa Cruz Biotechnology , Cat#sc-429415-HDR.

Techniques: Sequencing, Transfection, Plasmid Preparation, Expressing, Mutagenesis, SDS Page, Western Blot, Control, Dot Blot, Liquid Chromatography with Mass Spectroscopy, Purification, In Vitro, Demethylation Assay, Recombinant, Incubation

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: Embryonic Sublethality and Craniofacial Dysmorphism in Mettl4 KO Incross Progeny (A) Genotypic analysis of progeny from intercrossed Mettl4 ± heterozygous (Het) mice, showing expected and observed frequencies for each genotype. n.s., not significant, Chi-square test. (B) Mating scores of strain-matched wild-type (WT) controls, intercrossed Mettl4 ± heterozygotes, and incrossed Mettl4 KO mice. Black dots indicate the numbers of pups in each litter at the day of birth or recovered by Caesarian section at E18.5. Red lines indicate the mean litter size. p value was calculated by two-tailed t test. ∗∗ p < 0.01. The percentages of progeny that displayed craniofacial or limb dysmorphism are shown below each plot. (C) Neonatal Mettl4 KO pups from a single litter of incrossed Mettl4 KO parents. White arrow indicates mandibular malformation. Scale bar, 10 mm. (D) Representative image of spleens recovered from individual adult (10–12 weeks old) WT or Mettl4 KO mice. Rulers show scale in cm. Separate images for WT and KO spleens were juxtaposed at the dashed line. (E) Bar graph representation of spleen mass divided by total body mass for adult WT and Mettl4 KO mice (n = 6 mice per group). Error bars indicate SD ∗∗ p < 0.01, two-tailed t test. (F) (Left panel) Peripheral blood count of white cells (WBC) and lymphocytes (left-hand scale) and red blood cells (RBC, right-hand scale) in adult WT and Mettl4 KO mice. (Right panel) Hematocrit (HCT) analysis. Each point corresponds to values from an individual mouse. Brackets indicate normal range and mean values in WT mice. p values were calculated using unpaired, two-tailed t test. ∗ p < 0.05. See also and .

Article Snippet: Plasmid: Mettl4 HDR , Santa Cruz Biotechnology , Cat#sc-429415-HDR.

Techniques: Two Tailed Test

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: 6mA Deposition Triggers Proteolysis of the Sensor Proteins ASXL1 and MPND (A) Domain structures of ASXL1, showing the positions of the HARE-HTH and PHD domains and the central proline-rich region (PRR), and the MYSM1 and MPND deubiquitinases, with the percentage amino acid identity and similarity between RAMA domains. JAMM/DUB denotes the deubiquitinase catalytic domain. (B) In vitro DNA pull-down assays. Purified recombinant HARE-HTH and RAMA domains were incubated in the presence of untreated resin or resin coated with duplex DNA containing unmodified adenine (A) or 6mA. After extensive washing, bound protein was resolved by SDS-PAGE and detected by immunoblotting using the indicated antisera (n = 3 experiments). (C) HEK293T cells expressing Flag-ASXL1.591 and either empty vector, Flag-Mettl4, or Flag-Dam were harvested and proteins in whole-cell extracts resolved by SDS-PAGE and immunoblotting with the indicated antisera (upper panels). Dot blot analysis shows relative 6mA levels in genomic DNA recovered from the same samples (n = 3 experiments). (D) Cells expressing Flag-RAMA, Flag-Mettl4, or Flag-Dam were harvested and proteins in whole-cell extracts resolved by SDS-PAGE and immunoblotting (upper panels). Dot blot analysis shows relative 6mA levels in genomic DNA recovered from the same samples (n = 2 experiments). (E) Accumulation of Asxl1 and reduction of H2A-K119Ub and H3K27me3 in Mettl4 KO cells. Whole-cell extracts (upper panels) and purified histones (lower panels) were prepared from spleens isolated from individual WT or Mettl4 KO mice and proteins resolved by SDS-PAGE and immunoblotting (n = 2 experiments). (F) Accumulation of Mpnd in Mettl4 KO cells. Protein extracts were prepared from spleens isolated from individual WT or Mettl4 KO mice and resolved by SDS-PAGE and immunoblotting (n = 2 experiments). See also and .

Article Snippet: Plasmid: Mettl4 HDR , Santa Cruz Biotechnology , Cat#sc-429415-HDR.

Techniques: In Vitro, Purification, Recombinant, Incubation, SDS Page, Western Blot, Expressing, Plasmid Preparation, Dot Blot, Isolation

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: The E3 Ubiquitin Ligase TRIP12 Mediates Proteolysis of ASXL1 (A) Lysates prepared from HEK293T cells expressing the indicated combinations of Flag-ASXL1.591, Flag-Mettl4, or GFP-TRIP12 were immunoprecipitated using TRIP12 antisera or isotype-matched control IgG. Inputs and immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotting with Flag or TRIP12 antisera (n = 2 experiments). (B) HEK293T cells expressing Flag-ASXL1.591 and either a control shRNA targeting GFP or independent shRNAs (sh1 or sh2) targeting TRIP12 were lysed and protein extracts resolved by SDS-PAGE and immunoblotting. Tubulin, loading control (n = 3 experiments). (C) Protein extracts prepared from cells expressing Flag-ASXL1.591, in the absence or presence of Flag-Mettl4 or sh1- TRIP12 , were resolved by SDS-PAGE and immunoblotting with the indicated antisera (n = 3 experiments). (D) Model depicting 6mA deposition by Mettl4, recruitment of ASXL1/PR-DUB to 6mA, and engagement of PR-DUB with the E3 ubiquitin ligase TRIP12. These steps are proposed to stimulate ASXL1 proteolysis, inactivating PR-DUB and thereby preserving the Polycomb repressive mark H2A-K119Ub in chromatin.

Article Snippet: Plasmid: Mettl4 HDR , Santa Cruz Biotechnology , Cat#sc-429415-HDR.

Techniques: Ubiquitin Proteomics, Expressing, Immunoprecipitation, Control, SDS Page, Western Blot, shRNA, Preserving

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: Ectopic Asxl1 and Mpnd Correspond with Loss of Polycomb Silencing in Mettl4 KO ESCs (A) Pie chart illustrating the distribution of called 6mA peaks (n = 4,922) across promoter (−2 kb to TSS), TSS downstream (0–2 kb downstream of TSS), 5′ and 3′ UTR, coding exon, intron, and intergenic regions. Red numbers indicate the fold enrichment or depletion of 6mA at each feature relative to a random distribution. (B) Venn diagram showing overlap of 6mA peaks identified by MeDIP and called 6mA bases identified by PacBio SMRT sequencing analysis ( Wu et al., 2016 ). p value, Fisher’s exact test. (C) Normalized 6mA tag density plotted 1 kb upstream of the TSS, across the first 3 kb of a metagene, and 1 kb downstream of the TES for all genes (black), the top 10% of highly expressed genes (blue), and the bottom 10% of least expressed genes in WT ESCs (red). (D) Venn diagrams showing the number and relative distribution of Asxl1, Bap1, O-GlcNAc, Mpnd, and H2A-K119Ub (H2A-Ub) peaks in WT and Mettl4 KO ESCs. (E) Normalized tag density of ectopic Asxl1 peaks induced in Mettl4 KO ESCs (left) with heatmap representation of peaks ranked-ordered by the mean signal (right), each plotted across a window centered on the TSS. (F) Genome browser view showing profiles of 6mA or isotype-matched control IgG in WT ESCs (top) and Asxl1, Mpnd, H2A-Ub, Bap1, and O-GlcNAc in WT and Mettl4 KO ESCs at two representative loci. Shaded vertical bars highlight regions containing ectopic Asxl1 or Mpnd and depletion of H2A-Ub in Mettl4 KO ESCs. RefSeq exon structures (blue) for each annotated gene are shown at the bottom. (G) ChIP-qPCR analysis of the Rpl13 and Dvl3 genes in WT and Mettl4 KO ESCs. Antisera specific for Asxl1, H2A-K119Ub002C or Bap1 were used for chromatin immunoprecipitation. The mean fold enrichments normalized to isotype-matched IgG control are shown for each condition. Error bars indicate SEM (n = 2 experiments). (H) RT-qPCR analysis of Rpl13 and Dvl3 transcript levels in WT and Mettl4 KO ESCs. The mean value of WT control samples is set as 1. Error bars indicate SEM (n = 2 experiments). (I) Volcano plot presentation of transcript levels for genes expressed in WT and Mettl4 KO ESCs as determined by RNA-seq. Genes strongly up- or downregulated (FC > 2.0, FDR-adjusted p < 0.05) in Mettl4 KO cells are identified and indicated in red or green, respectively. (J) Gene ontology analysis of differentially expressed genes induced in Mettl4 KO cells showing involvement in embryonic development and tissue patterning. The yellow vertical line indicates the threshold for significance. See also and .

Article Snippet: Plasmid: Mettl4 HDR , Santa Cruz Biotechnology , Cat#sc-429415-HDR.

Techniques: Methylated DNA Immunoprecipitation, Sequencing, Control, ChIP-qPCR, Chromatin Immunoprecipitation, Quantitative RT-PCR, RNA Sequencing

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet:

Article Snippet: Plasmid: Mettl4 HDR , Santa Cruz Biotechnology , Cat#sc-429415-HDR.

Techniques: Luciferase, Virus, Recombinant, SYBR Green Assay, cDNA Synthesis, Sequencing, Methylated DNA Immunoprecipitation, RNA Sequencing, Control, Plasmid Preparation, CRISPR, Software

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: Mettl4 and Alkbh4 Catalyze Deposition and Erasure, Respectively, of 6mA (A) Schematic presentation of Mettl4 protein structure and linear arrangement of conserved motifs in methyltransferase domain (amino acids 257–471), showing detailed alignment to motif IV sequence logo in catalytic site of MT-A70 adenine N6-methyltransferases. (B) Clustering of proteins within the MT-A70 family. RNA-specific methyltransferases form a tight cluster of closely related sequences, while Mettl4 and the DNA adenine N6-methyltransferases DAMT-1 ( C. elegans ) and M. MunI (Mycoplasma) (red circles) are positioned apart. (C) HEK293T cells transfected with empty vector (EV) or expressing Flag-Dam or the wild-type or DPPW catalytic-site mutant (PPmut) variants of Flag-Mettl4 were harvested and proteins in whole-cell extracts resolved by SDS-PAGE and immunoblotting with Flag antisera. Actin, loading control. 6mA and 5mC in the same samples were detected by dot blot of genomic DNA (n = 3 experiments), and mean 6mA levels relative to dA were quantified by LC-MS/MS analysis of genomic DNA hydrolysates (n = 2 experiments). (D) Levels of 6mA relative to total dA in genomic DNA (left panel) and m6A relative to total adenosine (A) in mRNA (right panel), purified from WT and Mettl4 KO ESCs, as determined by quantitative LC-MS/MS. Dashed line represents limit of detection. n.d., not detected. Data are plotted as mean with SD (n = 2 experiments). (E) Overlaid extracted LC-MS chromatograms of dA and 6mA in genomic DNA hydrolysates prepared from WT and Mettl4 KO spleens. (F) In vitro 6mA demethylation assay. Recombinant His-Alkbh4 was purified from bacterial cell extracts (left panel). Irrelevant lanes were omitted from the gel. Human genomic DNA containing 6mA was incubated in buffer supplemented with Fe 2+ and 2-oxoglutarate cofactors in the absence or presence of His-Alkbh4 or vitamin C (VitC) for the indicated times. 6mA in each sample was detected by dot blot analysis (n = 3 experiments). (G) Alkbh4 demethylates 6mA in double-stranded (ds) DNA. Single-stranded (ss) or double-stranded DNA oligonucleotide containing unmodified adenine or 6mA was incubated in the absence or presence of His-Alkbh4 for the indicated times. Following the reactions, 6mA in 10 pmol of each DNA sample was detected by dot blot analysis (n = 3 experiments). See also and .

Article Snippet: Additional KO lines were generated by transfecting Mettl4 heterozygous ESC with Mettl4 CRISPR/Cas9 KO Plasmid and HDR vector (Santa Cruz Biotechnology) containing a puromycin resistance cassette.

Techniques: Sequencing, Transfection, Plasmid Preparation, Expressing, Mutagenesis, SDS Page, Western Blot, Control, Dot Blot, Liquid Chromatography with Mass Spectroscopy, Purification, In Vitro, Demethylation Assay, Recombinant, Incubation

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: Embryonic Sublethality and Craniofacial Dysmorphism in Mettl4 KO Incross Progeny (A) Genotypic analysis of progeny from intercrossed Mettl4 ± heterozygous (Het) mice, showing expected and observed frequencies for each genotype. n.s., not significant, Chi-square test. (B) Mating scores of strain-matched wild-type (WT) controls, intercrossed Mettl4 ± heterozygotes, and incrossed Mettl4 KO mice. Black dots indicate the numbers of pups in each litter at the day of birth or recovered by Caesarian section at E18.5. Red lines indicate the mean litter size. p value was calculated by two-tailed t test. ∗∗ p < 0.01. The percentages of progeny that displayed craniofacial or limb dysmorphism are shown below each plot. (C) Neonatal Mettl4 KO pups from a single litter of incrossed Mettl4 KO parents. White arrow indicates mandibular malformation. Scale bar, 10 mm. (D) Representative image of spleens recovered from individual adult (10–12 weeks old) WT or Mettl4 KO mice. Rulers show scale in cm. Separate images for WT and KO spleens were juxtaposed at the dashed line. (E) Bar graph representation of spleen mass divided by total body mass for adult WT and Mettl4 KO mice (n = 6 mice per group). Error bars indicate SD ∗∗ p < 0.01, two-tailed t test. (F) (Left panel) Peripheral blood count of white cells (WBC) and lymphocytes (left-hand scale) and red blood cells (RBC, right-hand scale) in adult WT and Mettl4 KO mice. (Right panel) Hematocrit (HCT) analysis. Each point corresponds to values from an individual mouse. Brackets indicate normal range and mean values in WT mice. p values were calculated using unpaired, two-tailed t test. ∗ p < 0.05. See also and .

Article Snippet: Additional KO lines were generated by transfecting Mettl4 heterozygous ESC with Mettl4 CRISPR/Cas9 KO Plasmid and HDR vector (Santa Cruz Biotechnology) containing a puromycin resistance cassette.

Techniques: Two Tailed Test

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: 6mA Deposition Triggers Proteolysis of the Sensor Proteins ASXL1 and MPND (A) Domain structures of ASXL1, showing the positions of the HARE-HTH and PHD domains and the central proline-rich region (PRR), and the MYSM1 and MPND deubiquitinases, with the percentage amino acid identity and similarity between RAMA domains. JAMM/DUB denotes the deubiquitinase catalytic domain. (B) In vitro DNA pull-down assays. Purified recombinant HARE-HTH and RAMA domains were incubated in the presence of untreated resin or resin coated with duplex DNA containing unmodified adenine (A) or 6mA. After extensive washing, bound protein was resolved by SDS-PAGE and detected by immunoblotting using the indicated antisera (n = 3 experiments). (C) HEK293T cells expressing Flag-ASXL1.591 and either empty vector, Flag-Mettl4, or Flag-Dam were harvested and proteins in whole-cell extracts resolved by SDS-PAGE and immunoblotting with the indicated antisera (upper panels). Dot blot analysis shows relative 6mA levels in genomic DNA recovered from the same samples (n = 3 experiments). (D) Cells expressing Flag-RAMA, Flag-Mettl4, or Flag-Dam were harvested and proteins in whole-cell extracts resolved by SDS-PAGE and immunoblotting (upper panels). Dot blot analysis shows relative 6mA levels in genomic DNA recovered from the same samples (n = 2 experiments). (E) Accumulation of Asxl1 and reduction of H2A-K119Ub and H3K27me3 in Mettl4 KO cells. Whole-cell extracts (upper panels) and purified histones (lower panels) were prepared from spleens isolated from individual WT or Mettl4 KO mice and proteins resolved by SDS-PAGE and immunoblotting (n = 2 experiments). (F) Accumulation of Mpnd in Mettl4 KO cells. Protein extracts were prepared from spleens isolated from individual WT or Mettl4 KO mice and resolved by SDS-PAGE and immunoblotting (n = 2 experiments). See also and .

Article Snippet: Additional KO lines were generated by transfecting Mettl4 heterozygous ESC with Mettl4 CRISPR/Cas9 KO Plasmid and HDR vector (Santa Cruz Biotechnology) containing a puromycin resistance cassette.

Techniques: In Vitro, Purification, Recombinant, Incubation, SDS Page, Western Blot, Expressing, Plasmid Preparation, Dot Blot, Isolation

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: The E3 Ubiquitin Ligase TRIP12 Mediates Proteolysis of ASXL1 (A) Lysates prepared from HEK293T cells expressing the indicated combinations of Flag-ASXL1.591, Flag-Mettl4, or GFP-TRIP12 were immunoprecipitated using TRIP12 antisera or isotype-matched control IgG. Inputs and immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotting with Flag or TRIP12 antisera (n = 2 experiments). (B) HEK293T cells expressing Flag-ASXL1.591 and either a control shRNA targeting GFP or independent shRNAs (sh1 or sh2) targeting TRIP12 were lysed and protein extracts resolved by SDS-PAGE and immunoblotting. Tubulin, loading control (n = 3 experiments). (C) Protein extracts prepared from cells expressing Flag-ASXL1.591, in the absence or presence of Flag-Mettl4 or sh1- TRIP12 , were resolved by SDS-PAGE and immunoblotting with the indicated antisera (n = 3 experiments). (D) Model depicting 6mA deposition by Mettl4, recruitment of ASXL1/PR-DUB to 6mA, and engagement of PR-DUB with the E3 ubiquitin ligase TRIP12. These steps are proposed to stimulate ASXL1 proteolysis, inactivating PR-DUB and thereby preserving the Polycomb repressive mark H2A-K119Ub in chromatin.

Article Snippet: Additional KO lines were generated by transfecting Mettl4 heterozygous ESC with Mettl4 CRISPR/Cas9 KO Plasmid and HDR vector (Santa Cruz Biotechnology) containing a puromycin resistance cassette.

Techniques: Ubiquitin Proteomics, Expressing, Immunoprecipitation, Control, SDS Page, Western Blot, shRNA, Preserving

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: Ectopic Asxl1 and Mpnd Correspond with Loss of Polycomb Silencing in Mettl4 KO ESCs (A) Pie chart illustrating the distribution of called 6mA peaks (n = 4,922) across promoter (−2 kb to TSS), TSS downstream (0–2 kb downstream of TSS), 5′ and 3′ UTR, coding exon, intron, and intergenic regions. Red numbers indicate the fold enrichment or depletion of 6mA at each feature relative to a random distribution. (B) Venn diagram showing overlap of 6mA peaks identified by MeDIP and called 6mA bases identified by PacBio SMRT sequencing analysis ( Wu et al., 2016 ). p value, Fisher’s exact test. (C) Normalized 6mA tag density plotted 1 kb upstream of the TSS, across the first 3 kb of a metagene, and 1 kb downstream of the TES for all genes (black), the top 10% of highly expressed genes (blue), and the bottom 10% of least expressed genes in WT ESCs (red). (D) Venn diagrams showing the number and relative distribution of Asxl1, Bap1, O-GlcNAc, Mpnd, and H2A-K119Ub (H2A-Ub) peaks in WT and Mettl4 KO ESCs. (E) Normalized tag density of ectopic Asxl1 peaks induced in Mettl4 KO ESCs (left) with heatmap representation of peaks ranked-ordered by the mean signal (right), each plotted across a window centered on the TSS. (F) Genome browser view showing profiles of 6mA or isotype-matched control IgG in WT ESCs (top) and Asxl1, Mpnd, H2A-Ub, Bap1, and O-GlcNAc in WT and Mettl4 KO ESCs at two representative loci. Shaded vertical bars highlight regions containing ectopic Asxl1 or Mpnd and depletion of H2A-Ub in Mettl4 KO ESCs. RefSeq exon structures (blue) for each annotated gene are shown at the bottom. (G) ChIP-qPCR analysis of the Rpl13 and Dvl3 genes in WT and Mettl4 KO ESCs. Antisera specific for Asxl1, H2A-K119Ub002C or Bap1 were used for chromatin immunoprecipitation. The mean fold enrichments normalized to isotype-matched IgG control are shown for each condition. Error bars indicate SEM (n = 2 experiments). (H) RT-qPCR analysis of Rpl13 and Dvl3 transcript levels in WT and Mettl4 KO ESCs. The mean value of WT control samples is set as 1. Error bars indicate SEM (n = 2 experiments). (I) Volcano plot presentation of transcript levels for genes expressed in WT and Mettl4 KO ESCs as determined by RNA-seq. Genes strongly up- or downregulated (FC > 2.0, FDR-adjusted p < 0.05) in Mettl4 KO cells are identified and indicated in red or green, respectively. (J) Gene ontology analysis of differentially expressed genes induced in Mettl4 KO cells showing involvement in embryonic development and tissue patterning. The yellow vertical line indicates the threshold for significance. See also and .

Article Snippet: Additional KO lines were generated by transfecting Mettl4 heterozygous ESC with Mettl4 CRISPR/Cas9 KO Plasmid and HDR vector (Santa Cruz Biotechnology) containing a puromycin resistance cassette.

Techniques: Methylated DNA Immunoprecipitation, Sequencing, Control, ChIP-qPCR, Chromatin Immunoprecipitation, Quantitative RT-PCR, RNA Sequencing

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet:

Article Snippet: Additional KO lines were generated by transfecting Mettl4 heterozygous ESC with Mettl4 CRISPR/Cas9 KO Plasmid and HDR vector (Santa Cruz Biotechnology) containing a puromycin resistance cassette.

Techniques: Luciferase, Virus, Recombinant, SYBR Green Assay, cDNA Synthesis, Sequencing, Methylated DNA Immunoprecipitation, RNA Sequencing, Control, Plasmid Preparation, CRISPR, Software

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: Primer Sequences used for qRT-PCR.

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques:

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: Inhibition of m 6 A modification confers resistance to HSC ferroptosis. METTL4 shRNA or FTO plasmid were stably transfected into HSC-LX2 cells followed by erastin (10 μM) treatment for 24 h. ( A ) The m 6 A levels were detected by m 6 A RNA Methylation Quantitative kit (**, p < 0.01, ***, p < 0.001, n = 3 in every group). ( B ) METTL4 shRNA or FTO plasmid were transfected into HSC-LX2 and HSC-T6 cells followed by sorafenib (10 μM) or erastin (10 μM) treatment for 24 h. Cell Counting Kit-8 kit was used to determine the Cell viability (*, p < 0.05, n = 3 in every group). ( C–F ) Iron accumulation, GSH depletion, lipid ROS level and MDA production were assayed (*, p < 0.05, n = 3 in every group). ( G ) FTO plasmid transfected into HSC-LX2 and HSC-T6 cells were treated with erastin (10 μM) with or without the indicated inhibitors (Liproxstatin-1, 100 nM; Ferrostatin-1, 1 μM; Necrostatin-1, 10 μM; ZVAD-FMK, 10 μM; Necrosulfonamide, 0.5 μM) for 24 h. Cell viability was assayed by Cell Counting Kit-8. (***, p < 0.001, n = 3 in every group). ( H ) Control vector or FTO plasmid were transfected into HSC-LX2 cells and treated with erastin (10 μM) for 24 h. Transmission electron microscopy was used to examin the typical changes of ferroptotic cells. Scale bars: 0.2 μm. Representative photographs were showed.

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques: Inhibition, Modification, shRNA, Plasmid Preparation, Stable Transfection, Transfection, Methylation, Cell Counting, Control, Transmission Assay, Electron Microscopy

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: Reduced ferroptosis by m 6 A modification inhibition is associated with autophagy inactivation. (A) FTO plasmid was transfected into HSC-LX2 cells and treated with erastin (10 μM) for 24 h. Total RNA was isolated for RNA-Seq. Clustering of HSC-LX2 cells were demonstrated by microarray heat map. The significantly differentially expressed mRNAs were analysied by hierarchical cluster: gray, no change; bright blue, underexpression; bright red, overexpression (FTO plasmid, n = 3; Control vector, n = 3). ( B ) Differentially expressed mRNAs were enriched by KEGG enrichment analysis in FTO plasmid group (Control vector, n = 3; FTO plasmid, n = 3). ( C ) The levels of m 6 A modification in autophagy-related gene were determined by MeRIP qPCR (*, p < 0.05, **, p < 0.01, ***, p < 0.001, n = 3 in every group). ( D ) METTL4 shRNA or FTO plasmid transfected into HSC-T6 and HSC-LX2 cells were treated with erastin (10 μM) for 24 h. Western blot showed the protein expression of BECN1, ATG3, ATG4A, ATG7, ATG9A, ATG5-ATG12 and ATG16L1 (n = 3 in every group). ( E ) Western blot was used to determine the expression of LC3-I/II and p62 (n = 3 in every group). ( F ) METTL4 shRNA or FTO plasmid with CMV-TurboRFP- EGFP-LC3-PGK-Puro plasmid were transferred into HSC-LX2 cells by erastin (10 μM) treatment for 24 h. The fluorescence spots were detected. Representative photographs were showed. Scale bars: 50 μm. ( G ) HSC-LX2 cells transfected with FTO plasmid or control vector by erastin (10 μM) treatment for 24 h. Transmission electron microscopy was used to examine the autolysosomes or autophagosomes. Representative photographs were showed. Scale bars: 0.2 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques: Modification, Inhibition, Plasmid Preparation, Transfection, Isolation, RNA Sequencing, Microarray, Over Expression, Control, shRNA, Western Blot, Expressing, Fluorescence, Transmission Assay, Electron Microscopy

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: Induction of autophagy by BECN1 plasmid impairs m 6 A modification inhibition-induced resistance to HSC ferroptosis. ( A ) BECN plasmid and METTL4 shRNA or FTO plasmid were transfected into HSC-T6 and HSC-LX2 cells and trested with erastin (10 μM) for 24 h. Real-time PCR were used to measure the mRNA levels of BECN1, MAPILC3B and SQSTM1 (*, p < 0.05, **, p < 0.01, compared with Control group, ## , p < 0.01, compared with BECN1 plasmid group, n = 3 in every group). ( B ) Western blot showed the protein expression of p62 and LC3-I/II (n = 3 in every group). ( C ) The endogenous LC3 levels were measured by immunofluorescence (n = 3 in every group). ( D ) HSC-LX2 and HSC-T6 cells transfected with BECN plasmid and METTL4 shRNA or FTO plasmid were treated with erastin (10 μM) or sorafenib (10 μM) for 24 h. Cell viability was assayed by Cell Counting Kit-8 (n = 3 in every group, *, p < 0.05). ( E, F ) MDA production, Iron accumulation, GSH depletion and lipid ROS level were assayed by commercial kits (*, p < 0.05, n = 3 in every group).

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques: Plasmid Preparation, Modification, Inhibition, shRNA, Transfection, Real-time Polymerase Chain Reaction, Control, Western Blot, Expressing, Immunofluorescence, Cell Counting

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: HSC-specific inhibition of m 6 A modification impairs erastin-induced HSC ferroptosis in murine liver fibrosis. Mice of 6 groups were treated with Vehicle, CCl 4, CCl 4 +VA-Lip-control-vector + Erastin, CCl 4 +VA-Lip-Mettl4-shRNA + Erastin, CCl 4 +VA-Lip-Fto-Plasmid + Erastin, CCl 4 +VA-Lip-Ythdf1-shRNA + Erastin. ( A ) Macroscopic examination was used to observe the pathological changes of the livers. Scale bars: 1 cm. Histopathological study was performed by H&E, Masson, and Sirius Red staining. Representative photographs were showed. Scale bars: 50 μm. (***, p < 0.001, n = 6 in every group). ( B ) Immunohistochemical staining of α-SMA was determined. Representative photographs were showed. Scale bars: 50 μm.(***, p < 0.001, n = 6 in every group). ( C ) Real-time PCR was measured to determine the mRNA expression of liver fibrosis markers (Acta2, Col1a1, Fn1, and Des) (*, p < 0.05, **, p < 0.01, ***, p < 0.001, n = 6 in every group). ( D ) The m 6 A levels were determined by m 6 A RNA Methylation Quantitative kit (n = 6 in every group, ***, p < 0.001, N.S., not significant). ( E, F ) Real-time PCR was used to determine the mRNA expression of autophagy markers (Becn1, Map1lc3b, Sqstm1, and Fth1) (*, p < 0.05, ***, p < 0.001, N.S., not significant, n = 6 in every group). ( G ) The mRNA expression of Ptgs2, iron accumulation and MDA production were determined (*, p < 0.05, **, p < 0.01, ***, p < 0.001, N.S., not significant, n = 6 in every group). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques: Inhibition, Modification, Control, Plasmid Preparation, shRNA, Staining, Immunohistochemical staining, Real-time Polymerase Chain Reaction, Expressing, Methylation

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: m 6 A modification upregulation, autophagy activation, and ferroptosis induction occur in human HSCs receiving sorafenib monotherapy. ( A, B ) laser capture microdissection (LCM) was used to isolated the primary human HSCs from the collected liver tissue. ACTA2, FN1, COL1A1, METTL4, FTO, and YTHDF1 mRNA expression were determined by real-time PCR (No treatment, n = 10; Sorafenib treatment, n = 10, **, p < 0.01, ***, p < 0.001). ( C ) The m 6 A levels were determined by m 6 A RNA Methylation Quantitative kit (No treatment, n = 10; Sorafenib treatment, n = 10, ***, p < 0.001). ( D ) Real-time PCR was used to determine the mRNA expression of autophagy markers BECN1, MAP1LC3B, SQSTM1, and FTH1 (No treatment, n = 10; Sorafenib treatment, n = 10, ***, p < 0.001). ( E ) The PTGS2 mRNA expression, iron accumulation, MDA production and GSH depletion were determined (No treatment, n = 10; Sorafenib treatment, n = 10, ***, p < 0.001).

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques: Modification, Activation Assay, Laser Capture Microdissection, Isolation, Expressing, Real-time Polymerase Chain Reaction, Methylation

Journal: Redox Biology

Article Title: N 6 -methyladenosine modification regulates ferroptosis through autophagy signaling pathway in hepatic stellate cells

doi: 10.1016/j.redox.2021.102151

Figure Lengend Snippet: m 6 A modification induces HSC ferroptosis by regulating autophagy signaling pathway. The upregulation of methylase METTL4 and the downregulation of demethylase FTO increased the levels of m 6 A modifications in BECN1 mRNA. m 6 A reader YTHDF1 promoted BECN1 mRNA stability via recognizing the m 6 A binding site, thus triggering autophagy activation, and eventually leading to HSC ferroptosis.

Article Snippet: The pcDNA3.1-FTO plasmid, pcDNA3.1-BECN1 plasmid, METTL4 shRNA (sc-75777-SH, sc-149388-SH), YTHDF1 shRNA (sc-76945-SH, sc-155423-SH) and control vector were obtained from KeyGEN BioTECH (KG20200903-10) and Santa Cruz Biotechnology, respectively.

Techniques: Modification, Binding Assay, Activation Assay

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: Mettl4 and Alkbh4 Catalyze Deposition and Erasure, Respectively, of 6mA (A) Schematic presentation of Mettl4 protein structure and linear arrangement of conserved motifs in methyltransferase domain (amino acids 257–471), showing detailed alignment to motif IV sequence logo in catalytic site of MT-A70 adenine N6-methyltransferases. (B) Clustering of proteins within the MT-A70 family. RNA-specific methyltransferases form a tight cluster of closely related sequences, while Mettl4 and the DNA adenine N6-methyltransferases DAMT-1 ( C. elegans ) and M. MunI (Mycoplasma) (red circles) are positioned apart. (C) HEK293T cells transfected with empty vector (EV) or expressing Flag-Dam or the wild-type or DPPW catalytic-site mutant (PPmut) variants of Flag-Mettl4 were harvested and proteins in whole-cell extracts resolved by SDS-PAGE and immunoblotting with Flag antisera. Actin, loading control. 6mA and 5mC in the same samples were detected by dot blot of genomic DNA (n = 3 experiments), and mean 6mA levels relative to dA were quantified by LC-MS/MS analysis of genomic DNA hydrolysates (n = 2 experiments). (D) Levels of 6mA relative to total dA in genomic DNA (left panel) and m6A relative to total adenosine (A) in mRNA (right panel), purified from WT and Mettl4 KO ESCs, as determined by quantitative LC-MS/MS. Dashed line represents limit of detection. n.d., not detected. Data are plotted as mean with SD (n = 2 experiments). (E) Overlaid extracted LC-MS chromatograms of dA and 6mA in genomic DNA hydrolysates prepared from WT and Mettl4 KO spleens. (F) In vitro 6mA demethylation assay. Recombinant His-Alkbh4 was purified from bacterial cell extracts (left panel). Irrelevant lanes were omitted from the gel. Human genomic DNA containing 6mA was incubated in buffer supplemented with Fe 2+ and 2-oxoglutarate cofactors in the absence or presence of His-Alkbh4 or vitamin C (VitC) for the indicated times. 6mA in each sample was detected by dot blot analysis (n = 3 experiments). (G) Alkbh4 demethylates 6mA in double-stranded (ds) DNA. Single-stranded (ss) or double-stranded DNA oligonucleotide containing unmodified adenine or 6mA was incubated in the absence or presence of His-Alkbh4 for the indicated times. Following the reactions, 6mA in 10 pmol of each DNA sample was detected by dot blot analysis (n = 3 experiments). See also and .

Article Snippet: Additional KO lines were generated by transfecting Mettl4 heterozygous ESC with Mettl4 CRISPR/Cas9 KO Plasmid and HDR vector (Santa Cruz Biotechnology) containing a puromycin resistance cassette.

Techniques: Sequencing, Transfection, Plasmid Preparation, Expressing, Mutagenesis, SDS Page, Western Blot, Control, Dot Blot, Liquid Chromatography with Mass Spectroscopy, Purification, In Vitro, Demethylation Assay, Recombinant, Incubation

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: Embryonic Sublethality and Craniofacial Dysmorphism in Mettl4 KO Incross Progeny (A) Genotypic analysis of progeny from intercrossed Mettl4 ± heterozygous (Het) mice, showing expected and observed frequencies for each genotype. n.s., not significant, Chi-square test. (B) Mating scores of strain-matched wild-type (WT) controls, intercrossed Mettl4 ± heterozygotes, and incrossed Mettl4 KO mice. Black dots indicate the numbers of pups in each litter at the day of birth or recovered by Caesarian section at E18.5. Red lines indicate the mean litter size. p value was calculated by two-tailed t test. ∗∗ p < 0.01. The percentages of progeny that displayed craniofacial or limb dysmorphism are shown below each plot. (C) Neonatal Mettl4 KO pups from a single litter of incrossed Mettl4 KO parents. White arrow indicates mandibular malformation. Scale bar, 10 mm. (D) Representative image of spleens recovered from individual adult (10–12 weeks old) WT or Mettl4 KO mice. Rulers show scale in cm. Separate images for WT and KO spleens were juxtaposed at the dashed line. (E) Bar graph representation of spleen mass divided by total body mass for adult WT and Mettl4 KO mice (n = 6 mice per group). Error bars indicate SD ∗∗ p < 0.01, two-tailed t test. (F) (Left panel) Peripheral blood count of white cells (WBC) and lymphocytes (left-hand scale) and red blood cells (RBC, right-hand scale) in adult WT and Mettl4 KO mice. (Right panel) Hematocrit (HCT) analysis. Each point corresponds to values from an individual mouse. Brackets indicate normal range and mean values in WT mice. p values were calculated using unpaired, two-tailed t test. ∗ p < 0.05. See also and .

Article Snippet: Additional KO lines were generated by transfecting Mettl4 heterozygous ESC with Mettl4 CRISPR/Cas9 KO Plasmid and HDR vector (Santa Cruz Biotechnology) containing a puromycin resistance cassette.

Techniques: Two Tailed Test

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: 6mA Deposition Triggers Proteolysis of the Sensor Proteins ASXL1 and MPND (A) Domain structures of ASXL1, showing the positions of the HARE-HTH and PHD domains and the central proline-rich region (PRR), and the MYSM1 and MPND deubiquitinases, with the percentage amino acid identity and similarity between RAMA domains. JAMM/DUB denotes the deubiquitinase catalytic domain. (B) In vitro DNA pull-down assays. Purified recombinant HARE-HTH and RAMA domains were incubated in the presence of untreated resin or resin coated with duplex DNA containing unmodified adenine (A) or 6mA. After extensive washing, bound protein was resolved by SDS-PAGE and detected by immunoblotting using the indicated antisera (n = 3 experiments). (C) HEK293T cells expressing Flag-ASXL1.591 and either empty vector, Flag-Mettl4, or Flag-Dam were harvested and proteins in whole-cell extracts resolved by SDS-PAGE and immunoblotting with the indicated antisera (upper panels). Dot blot analysis shows relative 6mA levels in genomic DNA recovered from the same samples (n = 3 experiments). (D) Cells expressing Flag-RAMA, Flag-Mettl4, or Flag-Dam were harvested and proteins in whole-cell extracts resolved by SDS-PAGE and immunoblotting (upper panels). Dot blot analysis shows relative 6mA levels in genomic DNA recovered from the same samples (n = 2 experiments). (E) Accumulation of Asxl1 and reduction of H2A-K119Ub and H3K27me3 in Mettl4 KO cells. Whole-cell extracts (upper panels) and purified histones (lower panels) were prepared from spleens isolated from individual WT or Mettl4 KO mice and proteins resolved by SDS-PAGE and immunoblotting (n = 2 experiments). (F) Accumulation of Mpnd in Mettl4 KO cells. Protein extracts were prepared from spleens isolated from individual WT or Mettl4 KO mice and resolved by SDS-PAGE and immunoblotting (n = 2 experiments). See also and .

Article Snippet: Additional KO lines were generated by transfecting Mettl4 heterozygous ESC with Mettl4 CRISPR/Cas9 KO Plasmid and HDR vector (Santa Cruz Biotechnology) containing a puromycin resistance cassette.

Techniques: In Vitro, Purification, Recombinant, Incubation, SDS Page, Western Blot, Expressing, Plasmid Preparation, Dot Blot, Isolation

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: The E3 Ubiquitin Ligase TRIP12 Mediates Proteolysis of ASXL1 (A) Lysates prepared from HEK293T cells expressing the indicated combinations of Flag-ASXL1.591, Flag-Mettl4, or GFP-TRIP12 were immunoprecipitated using TRIP12 antisera or isotype-matched control IgG. Inputs and immunoprecipitated proteins were resolved by SDS-PAGE and immunoblotting with Flag or TRIP12 antisera (n = 2 experiments). (B) HEK293T cells expressing Flag-ASXL1.591 and either a control shRNA targeting GFP or independent shRNAs (sh1 or sh2) targeting TRIP12 were lysed and protein extracts resolved by SDS-PAGE and immunoblotting. Tubulin, loading control (n = 3 experiments). (C) Protein extracts prepared from cells expressing Flag-ASXL1.591, in the absence or presence of Flag-Mettl4 or sh1- TRIP12 , were resolved by SDS-PAGE and immunoblotting with the indicated antisera (n = 3 experiments). (D) Model depicting 6mA deposition by Mettl4, recruitment of ASXL1/PR-DUB to 6mA, and engagement of PR-DUB with the E3 ubiquitin ligase TRIP12. These steps are proposed to stimulate ASXL1 proteolysis, inactivating PR-DUB and thereby preserving the Polycomb repressive mark H2A-K119Ub in chromatin.

Article Snippet: Additional KO lines were generated by transfecting Mettl4 heterozygous ESC with Mettl4 CRISPR/Cas9 KO Plasmid and HDR vector (Santa Cruz Biotechnology) containing a puromycin resistance cassette.

Techniques: Ubiquitin Proteomics, Expressing, Immunoprecipitation, Control, SDS Page, Western Blot, shRNA, Preserving

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet: Ectopic Asxl1 and Mpnd Correspond with Loss of Polycomb Silencing in Mettl4 KO ESCs (A) Pie chart illustrating the distribution of called 6mA peaks (n = 4,922) across promoter (−2 kb to TSS), TSS downstream (0–2 kb downstream of TSS), 5′ and 3′ UTR, coding exon, intron, and intergenic regions. Red numbers indicate the fold enrichment or depletion of 6mA at each feature relative to a random distribution. (B) Venn diagram showing overlap of 6mA peaks identified by MeDIP and called 6mA bases identified by PacBio SMRT sequencing analysis ( Wu et al., 2016 ). p value, Fisher’s exact test. (C) Normalized 6mA tag density plotted 1 kb upstream of the TSS, across the first 3 kb of a metagene, and 1 kb downstream of the TES for all genes (black), the top 10% of highly expressed genes (blue), and the bottom 10% of least expressed genes in WT ESCs (red). (D) Venn diagrams showing the number and relative distribution of Asxl1, Bap1, O-GlcNAc, Mpnd, and H2A-K119Ub (H2A-Ub) peaks in WT and Mettl4 KO ESCs. (E) Normalized tag density of ectopic Asxl1 peaks induced in Mettl4 KO ESCs (left) with heatmap representation of peaks ranked-ordered by the mean signal (right), each plotted across a window centered on the TSS. (F) Genome browser view showing profiles of 6mA or isotype-matched control IgG in WT ESCs (top) and Asxl1, Mpnd, H2A-Ub, Bap1, and O-GlcNAc in WT and Mettl4 KO ESCs at two representative loci. Shaded vertical bars highlight regions containing ectopic Asxl1 or Mpnd and depletion of H2A-Ub in Mettl4 KO ESCs. RefSeq exon structures (blue) for each annotated gene are shown at the bottom. (G) ChIP-qPCR analysis of the Rpl13 and Dvl3 genes in WT and Mettl4 KO ESCs. Antisera specific for Asxl1, H2A-K119Ub002C or Bap1 were used for chromatin immunoprecipitation. The mean fold enrichments normalized to isotype-matched IgG control are shown for each condition. Error bars indicate SEM (n = 2 experiments). (H) RT-qPCR analysis of Rpl13 and Dvl3 transcript levels in WT and Mettl4 KO ESCs. The mean value of WT control samples is set as 1. Error bars indicate SEM (n = 2 experiments). (I) Volcano plot presentation of transcript levels for genes expressed in WT and Mettl4 KO ESCs as determined by RNA-seq. Genes strongly up- or downregulated (FC > 2.0, FDR-adjusted p < 0.05) in Mettl4 KO cells are identified and indicated in red or green, respectively. (J) Gene ontology analysis of differentially expressed genes induced in Mettl4 KO cells showing involvement in embryonic development and tissue patterning. The yellow vertical line indicates the threshold for significance. See also and .

Article Snippet: Additional KO lines were generated by transfecting Mettl4 heterozygous ESC with Mettl4 CRISPR/Cas9 KO Plasmid and HDR vector (Santa Cruz Biotechnology) containing a puromycin resistance cassette.

Techniques: Methylated DNA Immunoprecipitation, Sequencing, Control, ChIP-qPCR, Chromatin Immunoprecipitation, Quantitative RT-PCR, RNA Sequencing

Journal: Molecular Cell

Article Title: An Adversarial DNA N 6 -Methyladenine-Sensor Network Preserves Polycomb Silencing

doi: 10.1016/j.molcel.2019.03.018

Figure Lengend Snippet:

Article Snippet: Additional KO lines were generated by transfecting Mettl4 heterozygous ESC with Mettl4 CRISPR/Cas9 KO Plasmid and HDR vector (Santa Cruz Biotechnology) containing a puromycin resistance cassette.

Techniques: Luciferase, Virus, Recombinant, SYBR Green Assay, cDNA Synthesis, Sequencing, Methylated DNA Immunoprecipitation, RNA Sequencing, Control, Plasmid Preparation, CRISPR, Software

Journal: Molecular cell

Article Title: N 6 -Deoxyadenosine Methylation in Mammalian Mitochondrial DNA

doi: 10.1016/j.molcel.2020.02.018

Figure Lengend Snippet: (A) Multiple alignments of METTL4 to METTL3/METTL14 and DAMT-1.

Article Snippet: METTL4 Antibody (C-term) , Abgent , Cat # AP10377b, RRID:AB_10821964.

Techniques:

Journal: Molecular cell

Article Title: N 6 -Deoxyadenosine Methylation in Mammalian Mitochondrial DNA

doi: 10.1016/j.molcel.2020.02.018

Figure Lengend Snippet: (A) Metabolic phenotype plot of oxygen consumption rate (OCR) vs. extracellular acidification rate (ECAR). METTL4 knockdown increased mitochondrial activity under both basal and stressed conditions (n = 4, mean ± SEM).

Article Snippet: METTL4 Antibody (C-term) , Abgent , Cat # AP10377b, RRID:AB_10821964.

Techniques: Activity Assay

Journal: Molecular cell

Article Title: N 6 -Deoxyadenosine Methylation in Mammalian Mitochondrial DNA

doi: 10.1016/j.molcel.2020.02.018

Figure Lengend Snippet: (A) Heatmap summarizing the mtDNA encoded transcripts from RNA-seq analysis. METTL4 knockdown increased the expression level of most mtDNA encoded mRNAs and rRNA.

Article Snippet: METTL4 Antibody (C-term) , Abgent , Cat # AP10377b, RRID:AB_10821964.

Techniques: RNA Sequencing Assay, Expressing

Journal: Molecular cell

Article Title: N 6 -Deoxyadenosine Methylation in Mammalian Mitochondrial DNA

doi: 10.1016/j.molcel.2020.02.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: METTL4 Antibody (C-term) , Abgent , Cat # AP10377b, RRID:AB_10821964.

Techniques: Recombinant, Isolation, Sample Prep, Imaging, Sequencing, shRNA, Plasmid Preparation, Software