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: 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:

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: 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: BMC Cancer

Article Title: N6-methyladenosine RNA modification (m6A) is of prognostic value in HPV-dependent vulvar squamous cell carcinoma

doi: 10.1186/s12885-022-10010-x

Figure Lengend Snippet: Summary of the analyzed m6A proteins as indicated and their correlation with overall survival (indicated as %alive) for the entire study cohort, HPV-independent, and HPV-dependent VSCC. The HPV-status was not available for 24 patients. Samples were grouped according to high and low expression based on the staining intensities. p -values for the group comparisons are based on log-rank tests (significance threshold p < 0.5). q -values are based on multiple hypotheses testing using the method of Benjamini and Hochberg with a significance threshold of q < 0.1

Article Snippet: Immunostaining of METTL3, METTL4, METTL14, WTAP, KIAA1429, FTO, ALKBH5, HNRNPA2B1, HNRNPC, YTHDC1, YTHDF1,YTHDF2, and YTHDF3 was performed on the TMAs using an automated staining system (BenchMark ULTRA; Ventana Medical Systems) which performed deparaffinization, pretreatment with cell conditioning buffer (CC1 buffer, pH8), and incubation with primary antibodies (FTO (1:50; Atlas Antibodies #HPA041086), ALKBH5 (1:200; Novus #NBP1-82,188), METTL3 (1:1000; Biorbyt #orb374082), METTL4 (1:40; Atlas Antibodies #HPA040061), METTL14 (1:100; Atlas Antibodies #HPA038002), WTAP (1:100; Atlas Antibodies #HPA010550), KIAA1429 (1:25; Atlas Antibodies #HPA031530), HNRNPC (1:25; Atlas Antibodies #HPA051075), HNRNPA2B1 (1:100; Atlas Antibodies #HPA001666), YTHDC1 (1:25; Atlas Antibodies #HPA036462), YTHDF1 (1:10; Biorbyt #orb179018), YTHDF2 (1:200; Biorbyt #orb39199), YTHDF3 (1:200; Biorbyt #orb374095) at 4 °C overnight.

Techniques: Expressing, Staining

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: Genome Biology

Article Title: METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets

doi: 10.1186/s13059-022-02819-3

Figure Lengend Snippet: Increase in the nuclear 6mA levels through nuclear activation of METTL4 expression under hypoxia. a An increase in nuclear 6mA levels was observed in BFTC909 and FADU cells under hypoxia (see “Methods”). The collected results were summarized as the ratios of 6mA/dA in the bar graph (lower panel). Corresponding 6mA dot blots with methyl blue loading controls are shown together with bar graphs. N, normoxia; H, hypoxia. Normoxic condition was used as a control. b Immunofluorescence staining shows the increased nuclear staining of 6mA in FADU cells under hypoxia. Staining of cell nuclei by DAPI and mitochondria by MitoTracker was used as controls. Bar graph indicated the percentage of cells containing nuclear 6mA signals. c Detected 6mA and dA in METTL4-induced gDNAs were verified by product ion conformation spectra (PICS) fit to the spectrum generated from each standard. Parental ion of 6mA was m/z 266 with major daughter ion m/z 150. Parental ion of dA was m/z 252 with major daughter ion m/z 136. d Western blot analysis shows the more prominent nuclear activation of METTL4 levels through nuclear fractionation in two different cell lines. Histone H3 and GAPDH was used as a nuclear and cytoplasmic control, respectively. e Immunofluorescence staining shows the increased nuclear METTL4 expression in cells under hypoxia in BFTC909 and FADU cells. Mitotracker: mitochondria DNA. Cell nuclei were stained by DAPI. f Knockdown of METTL4 abolished the increase in nuclear 6mA levels induced by hypoxia in BFTC909 and FADU cells. Corresponding 6mA dot blots are shown. g In vitro DNA methylation assays show an increase in the 6mA levels by incubating METTL4 with genomic DNAs from BFTC909 or FADU cells. The METTL4 mutant and incubation without SAM were used as controls. Corresponding 6mA dot blots are shown. N, normoxia; H, hypoxia. Normoxic condition was used as a control. The asterisk (*) indicated statistical significance ( P <0.05) between experimental and control groups

Article Snippet: For double staining of mouse tissues, the tissue sections were incubated with anti-METTL4 (1:200 dilution; Abnova), anti-6mA (1:500 dilution; Synaptic Systems), or anti-HIF-1α (1:200 dilution; Abcam) antibodies.

Techniques: Activation Assay, Expressing, Control, Immunofluorescence, Staining, Generated, Western Blot, Fractionation, Knockdown, In Vitro, DNA Methylation Assay, Mutagenesis, Incubation

Journal: Genome Biology

Article Title: METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets

doi: 10.1186/s13059-022-02819-3

Figure Lengend Snippet: METTL4 is essential in hypoxia-induced EMT and in vitro / in vivo metastatic activity. a Overexpression of METTL4 induced EMT in BFTC909 and FADU cells by Western blot analysis. The cell clone transfected with the control vector was used as a control. b Knockdown of METTL4 reversed the expression of EMT markers regulated by hypoxia in BFTC909 and FADU cells. c Overexpression of METTL4 induced the expression of a set of EMT transcriptional regulators and knockdown of METTL4 abolished the activation of these EMT regulators induced by hypoxia. d Overexpression of METTL4 induced increased numbers of metastatic lung nodules in mice in tail vein and orthotopic implantation experiments. Representative gross anatomy and histology are shown on the left; measurement of metastatic lung nodules is shown on the right. e Knockdown of METTL4 significantly decreased the increased metastatic lung nodules in mice injected with cells overexpressing a HIF-1α constitutively active mutant. Representative gross anatomy and histology are shown on the left; measurement of metastatic lung nodules is shown on the right. f Hypoxic tumor cells sorted from xenografted tumors from BFTC909 and FADU cells show the increased HIF-1α and METTL4 protein levels together with increased HIF-1α target gene expression. Glut1 activation was used as a positive control. N, normoxia; H, hypoxia. Normoxic cells were used as a control. g Hypoxic tumor cells sorted from xenografted tumors from BFTC909 and FADU cells show an increase in the 6mA levels. Corresponding 6mA dot blots with methyl blue loading controls are shown together with bar graphs. N, normoxia; H, hypoxia. h Immunofluorescence staining shows the co-staining of 6mA and METTL4 in xenografted tumors from BFTC909, FADU, and KTCC28M cells. Green fluorescence: 6mA staining (RNase treatment); red fluorescence: METTL4 staining. Cell nuclei were stained by DAPI. H, highly colocalized area; L, less colocalized area. The asterisk (*) indicated statistical significance ( P <0.05) between experimental and control groups

Article Snippet: For double staining of mouse tissues, the tissue sections were incubated with anti-METTL4 (1:200 dilution; Abnova), anti-6mA (1:500 dilution; Synaptic Systems), or anti-HIF-1α (1:200 dilution; Abcam) antibodies.

Techniques: In Vitro, In Vivo, Activity Assay, Over Expression, Western Blot, Transfection, Control, Plasmid Preparation, Knockdown, Expressing, Activation Assay, Injection, Mutagenesis, Targeted Gene Expression, Positive Control, Immunofluorescence, Staining, Fluorescence

Journal: Genome Biology

Article Title: METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets

doi: 10.1186/s13059-022-02819-3

Figure Lengend Snippet: The essential role of the enzymatic activity of METTL4 in hypoxia-induced phenotypes and clinical implications. a Mutation of the enzymatic site of METTL4 by a prime-cutting CRISPR-Cas9 approach in BFTC909 cells abolished the induction of EMT by hypoxia and significantly decreased the RNA expression of RP11-390F4.3 and Glut1 . The induction of 6mA levels was abolished in enzymatically inactive METTL4 mutant BFTC909 cells. N, normoxia; H, hypoxia. The normoxic condition for METTL4 wild type BFTC909 cells was used as a control. A corresponding 6mA dot blot with methyl blue loading control is shown together with the bar graph. The asterisk (*) indicated statistical significance ( P <0.05) between experimental and control groups. b Immunofluorescence staining shows the abolishment of EMT induction by hypoxia in the enzymatically inactive METTL4 mutant FADU and BFTC909 cells. Green fluorescence represented staining of E-cadherin; red fluorescence represented staining of vimentin. Cell nuclei were stained by DAPI. N, normoxia; H, hypoxia. The normoxic condition for METTL4 wild type BFTC909 and FADU cells were used as a control. c Increased 6mA levels in UTUC, but not in bladder cancer (BC), patient samples. d Increased METTL4, 6mA, and HIF-1α levels by immunohistochemistry staining in the tumor part (T) (vs. the normal part (N)) of UTUC patient samples are shown by H-score measurement. The error bars represented the standard deviation (SD). Student’s t test was used to compare two groups of independent samples. e A representative case of immunohistochemistry staining of UTUC patient samples using antibodies against METTL4, 6mA, and HIF-1α between normal and tumor tissues. f Co-expression of METTL4 and 6mA predicted a poor prognosis of UTUC patients in either overall survival or disease-free survival by Kaplan-Meier analysis. Subgroup analysis of overall survival and disease-free survival of UTUC cases according to the expression profile of METTL4 low/6mA low (Group 1), METTL4 high/6mA high (Group 2), and others (Group 3) in tumors. P values of the comparison between each group are shown

Article Snippet: For double staining of mouse tissues, the tissue sections were incubated with anti-METTL4 (1:200 dilution; Abnova), anti-6mA (1:500 dilution; Synaptic Systems), or anti-HIF-1α (1:200 dilution; Abcam) antibodies.

Techniques: Activity Assay, Mutagenesis, CRISPR, RNA Expression, Control, Dot Blot, Immunofluorescence, Staining, Fluorescence, Immunohistochemistry, Standard Deviation, Expressing, Comparison

Journal: Genome Biology

Article Title: METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets

doi: 10.1186/s13059-022-02819-3

Figure Lengend Snippet: Analysis of RNA-seq and 6mA-ChIP-exo-seq datasets shows hypoxia/METTL4 co-regulated genes and 6mA signals-regulated genes. a The expression heatmap of EMT-related genes in FADU and BFTC909 cells, whose expression increased under hypoxia and decreased in the hypoxic status undergoing METTL4 knockdown. b Venn diagram shows the overlapping set of genes ( n =220) co-regulated by hypoxia and METTL4 through overlapping of upregulated genes under hypoxia and downregulated genes in the hypoxic status undergoing METTL4 knockdown in both FADU and BFTC909 cells. c Pie chart shows the different percentage of hypoxia and METTL4 co-regulated genes according to the classification of gene feature (protein coding, 75%; lncRNA, 15%; others, 10%). d KEGG analysis shows the top 10 enriched pathways in the class of protein-coding genes co-regulated by hypoxia and METTL4. e Heatmap shows the expression of top ten lncRNAs co-regulated by hypoxia and METTL4 using RNA-seq datasets from FADU cells. f Western blot analysis shows that overexpression of lncRNA RP11-390F4.3 activated a set of EMT transcriptional regulators. g Pie chart shows the annotation of hypoxia-induced/METTL4 dependent gain-of-6mA regions located in different genomic regions. h LncRNA RP11-390F4.3 was used as an example of hypoxia-induced/METTL4-dependent 6mA regulated gene that contained hypoxia-induced/METTL4-dependent 6mA signals on its promoter region. Different 6mA motifs were calculated by HOMER. Only motif-10 was indicated on the lncRNA RP11-390F4.3 promoter. A magnified window around the motif-10 area from 3 gene tracks is shown. Y -axis denotes the scale of the number of reads

Article Snippet: For double staining of mouse tissues, the tissue sections were incubated with anti-METTL4 (1:200 dilution; Abnova), anti-6mA (1:500 dilution; Synaptic Systems), or anti-HIF-1α (1:200 dilution; Abcam) antibodies.

Techniques: RNA Sequencing Assay, Expressing, Knockdown, Western Blot, Over Expression

Journal: Genome Biology

Article Title: METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets

doi: 10.1186/s13059-022-02819-3

Figure Lengend Snippet: Characterizations of lncRNA RP11-390F4.3 using different in vitro and in vivo metastatic assays. a Immunofluorescence staining shows the nuclear localization of lncRNA RP11-390F4.3. Cell nuclei were stained by DAPI. b Measurement of the copy number of lncRNA RP11-390F4.3 in FADU cells (normoxia vs. hypoxia). Titration standard curve was used for measurement of the copy number of lncRNA RP11-390F4.3 per 500,000 cells. The red point represents the qRT-PCR value from a standard sample of 500,000 FADU cells under hypoxia. c Knockdown of lncRNA RP11-390F4.3 significantly decreased the induction of various HIF-1α target genes using qRT-PCR analysis. Knockdown using the scrambled control siRNA was used as a control. N, normoxia; H, hypoxia. The asterisk (*) indicated statistical significance ( P <0.05) between experimental and control clones ( n =3). d qChIRP assays show the significantly decreased lncRNA RP11-390F4.3 binding to the promoter regions of EMT regulators and Glut1 gene in hypoxic FADU cells under knockdown of lncRNA RP11-390F4.3 vs. the hypoxic control knockdown cells. e Heatmap analysis of the EMT transcription regulator genes induced by lncRNA RP11-390F4.3 or METTL4 overexpression using RNA-seq datasets. f Western blot analysis shows that knockdown of lncRNA RP11-390F4.3 abolished the EMT phenotypes induced by METTL4 overexpression in FADU and BFTC909 cells. g Overexpression of lncRNA RP11-390F4.3 in BFTC909 cells significantly increased metastatic lung nodules in mice after injection of these cells into mice. Representative gross anatomy and histology are shown on the left, and measurement of metastatic lung nodules is shown on the right. BFTC control-transfected clone was used as a control. h Knockdown of lncRNA RP11-390F4.3 in BFTC909 cells overexpressing METTL4 significantly decreased the metastatic lung nodules in mice. Representative gross anatomy and histology are shown on the left, and measurement of metastatic lung nodules is shown on the right. BFTC control-transfected clone was used as a control. The asterisk (*) indicates statistical significance ( P <0.05) between experimental and control groups

Article Snippet: For double staining of mouse tissues, the tissue sections were incubated with anti-METTL4 (1:200 dilution; Abnova), anti-6mA (1:500 dilution; Synaptic Systems), or anti-HIF-1α (1:200 dilution; Abcam) antibodies.

Techniques: In Vitro, In Vivo, Immunofluorescence, Staining, Titration, Quantitative RT-PCR, Knockdown, Control, Clone Assay, Binding Assay, Over Expression, RNA Sequencing Assay, Western Blot, Injection, Transfection

Journal: Genome Biology

Article Title: METTL4-mediated nuclear N6-deoxyadenosine methylation promotes metastasis through activating multiple metastasis-inducing targets

doi: 10.1186/s13059-022-02819-3

Figure Lengend Snippet: Analysis of overlapping datasets regulated by METTL4/6mA, lncRNA RP11-390F4.3 , and HIF-1α, and a summary model. a Venn diagram shows the number of genes that was co-regulated by METTL4/6mA, lncRNA RP11-390F4.3 , and HIF-1α. b KEGG analysis of the 92 co-regulated genes, in which glycolysis and HIF-1 signaling pathways are on the top of the list. c Categorization of the 92 genes shows that they mainly belonged to the main hypoxia-induced phenotypes (angiogenesis, stemness, cancer metabolism). d A model to summarize the results and the mechanisms from this report

Article Snippet: For double staining of mouse tissues, the tissue sections were incubated with anti-METTL4 (1:200 dilution; Abnova), anti-6mA (1:500 dilution; Synaptic Systems), or anti-HIF-1α (1:200 dilution; Abcam) antibodies.

Techniques: