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Addgene inc pcmv6 xl4 asxl1 p y591x 3x flag

Figure 1. <t>ASXL1</t> binds the BRD4-ET domain. (A) Schematic representing domain structure of ASXL1. Indicated on ASXL1 is the proposed BRD4 binding site (538–578; dark red) and the relative positions of commonly mutated residues in cancer that produce a truncated protein either by frameshift (black circle) or nonsense (grey circle). (B) Multiple sequence alignment of the BRD4 binding motif in ASXL1 compared with known BRD4-ET domain interactors, highlighting a conserved ﬁve residue motif. These residues we propose bind BRD4 in the same groove of the BRD4-ET as known interactors (PDB: 6BGG; ET bound by CHD4) (C/D) Coomassie-stained gels showing pulldown of His6BRD4600–678 by GST-ASXL1537–587 (C), and the reciprocal pulldown; His6MBP-ASXL1537–587 by GST-BRD4600–678 (D).

Pcmv6 Xl4 Asxl1 P Y591x 3x Flag, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Oncogenic Truncations of ASXL1 Enhance a Motif for BRD4 ET-Domain Binding."

Article Title: Oncogenic Truncations of ASXL1 Enhance a Motif for BRD4 ET-Domain Binding.

Journal: Journal of molecular biology

doi: 10.1016/j.jmb.2021.167242

Figure 1. ASXL1 binds the BRD4-ET domain. (A) Schematic representing domain structure of ASXL1. Indicated on ASXL1 is the proposed BRD4 binding site (538–578; dark red) and the relative positions of commonly mutated residues in cancer that produce a truncated protein either by frameshift (black circle) or nonsense (grey circle). (B) Multiple sequence alignment of the BRD4 binding motif in ASXL1 compared with known BRD4-ET domain interactors, highlighting a conserved ﬁve residue motif. These residues we propose bind BRD4 in the same groove of the BRD4-ET as known interactors (PDB: 6BGG; ET bound by CHD4) (C/D) Coomassie-stained gels showing pulldown of His6BRD4600–678 by GST-ASXL1537–587 (C), and the reciprocal pulldown; His6MBP-ASXL1537–587 by GST-BRD4600–678 (D).

Figure Legend Snippet: Figure 1. ASXL1 binds the BRD4-ET domain. (A) Schematic representing domain structure of ASXL1. Indicated on ASXL1 is the proposed BRD4 binding site (538–578; dark red) and the relative positions of commonly mutated residues in cancer that produce a truncated protein either by frameshift (black circle) or nonsense (grey circle). (B) Multiple sequence alignment of the BRD4 binding motif in ASXL1 compared with known BRD4-ET domain interactors, highlighting a conserved ﬁve residue motif. These residues we propose bind BRD4 in the same groove of the BRD4-ET as known interactors (PDB: 6BGG; ET bound by CHD4) (C/D) Coomassie-stained gels showing pulldown of His6BRD4600–678 by GST-ASXL1537–587 (C), and the reciprocal pulldown; His6MBP-ASXL1537–587 by GST-BRD4600–678 (D).

Techniques Used: Binding Assay, Sequencing, Residue, Staining

Figure 2. ASXL1 binds the BRD4-ET domain through a core motif. (A) Isothermal titration calorimetry of ASXL1 and BRD4-ET domain. Isothermal titration calorimetry (ITC) of the BRD4 ET domain injected into His6MBP-fused ASXL1537–587 injected into BRD4-ET domain reveals a high afﬁnity interaction, with KD = 0.535 mM. Data points of triplicate titrations, following His6MBP titration subtraction. (B) Introduction of point mutations within the binding sequence of ASXL1 disrupts binding of His6MBP-ASXL1537–587 to GST-BRD4600–678. Coomassie-stained gels showing pulldown of His6MBP-ASXL1537–587 by GST-BRD4600–678. Multiple sequence alignment of the ASXL1 mutations below; the residues colored according to ALSCRIPT Calcons. (C) Alphafold2 model of ASXL1537–587 and the BRD4-ET domain (purple); indicating the ﬁve residues of the core motif. (D) NMR structure of BRD3:NSD3 from PDB 7jyn. The inset box indicates the additional hydrophobic pocket occupied by Phe168 of NSD3, and the equivalent region from the BRD4:ASXL1 model in panel C.

Figure Legend Snippet: Figure 2. ASXL1 binds the BRD4-ET domain through a core motif. (A) Isothermal titration calorimetry of ASXL1 and BRD4-ET domain. Isothermal titration calorimetry (ITC) of the BRD4 ET domain injected into His6MBP-fused ASXL1537–587 injected into BRD4-ET domain reveals a high afﬁnity interaction, with KD = 0.535 mM. Data points of triplicate titrations, following His6MBP titration subtraction. (B) Introduction of point mutations within the binding sequence of ASXL1 disrupts binding of His6MBP-ASXL1537–587 to GST-BRD4600–678. Coomassie-stained gels showing pulldown of His6MBP-ASXL1537–587 by GST-BRD4600–678. Multiple sequence alignment of the ASXL1 mutations below; the residues colored according to ALSCRIPT Calcons. (C) Alphafold2 model of ASXL1537–587 and the BRD4-ET domain (purple); indicating the ﬁve residues of the core motif. (D) NMR structure of BRD3:NSD3 from PDB 7jyn. The inset box indicates the additional hydrophobic pocket occupied by Phe168 of NSD3, and the equivalent region from the BRD4:ASXL1 model in panel C.

Techniques Used: Isothermal Titration Calorimetry, Injection, Titration, Binding Assay, Sequencing, Staining

Figure 3. ASXL1-BRD4-ET interaction is conserved. (A) ASXL1 binds to the highly conserved ET domain of BRD2, BRD3 and BRD4. Coomassie-stained gels showing pulldown of His6MBP-ASXL1537–587 by GST-BRD2632–710, GST-BRD3562–640 and GST-BRD4600–678. (B) Multiple sequence alignment of ASXL1, ASXL2 and ASXL3; the residues colored according to ALSCRIPT Calcons, demonstrates the conservation of the binding site; the core motif is highlighted (red box). (C) BRD4 binds ASXL1, ASXL2 and ASXL3. Coomassie-stained gels showing pulldown of His6MBP-ASXL1537–587, His6MBP-ASXL2582–634 and His6MBP-ASXL3977–1027 by GST-BRD4600–678. (D) Isothermal titration calorimetry of ASXL1 (green), ASXL2 (red) and ASXL3 (blue) injected into BRD4-ET domain reveals all ASXL homologues tightly bind the BRD4-ET domain. Data points of triplicate titrations, following buffer titration subtraction.

Figure Legend Snippet: Figure 3. ASXL1-BRD4-ET interaction is conserved. (A) ASXL1 binds to the highly conserved ET domain of BRD2, BRD3 and BRD4. Coomassie-stained gels showing pulldown of His6MBP-ASXL1537–587 by GST-BRD2632–710, GST-BRD3562–640 and GST-BRD4600–678. (B) Multiple sequence alignment of ASXL1, ASXL2 and ASXL3; the residues colored according to ALSCRIPT Calcons, demonstrates the conservation of the binding site; the core motif is highlighted (red box). (C) BRD4 binds ASXL1, ASXL2 and ASXL3. Coomassie-stained gels showing pulldown of His6MBP-ASXL1537–587, His6MBP-ASXL2582–634 and His6MBP-ASXL3977–1027 by GST-BRD4600–678. (D) Isothermal titration calorimetry of ASXL1 (green), ASXL2 (red) and ASXL3 (blue) injected into BRD4-ET domain reveals all ASXL homologues tightly bind the BRD4-ET domain. Data points of triplicate titrations, following buffer titration subtraction.

Techniques Used: Staining, Sequencing, Binding Assay, Isothermal Titration Calorimetry, Injection, Titration

Figure 4. Enhanced BRD4-ET domain binding by ASXL1Y591X (A) Schematic showing the approach used for targeted analysis. TurboID was fused to the BRD4-ET domain and used to investigate the interaction with different lengths of ASXL1 (B) Proximity based labelling assays indicate that BRD4 interacts with ASXL1Y591X and neither the full-length, nor ASXL11–472 which lacks the ET binding site. HEK293T cells were transiently co-transfected with either TurboID-BRD4-ET domain or TurboID alone and varying lengths of ASXL1. Cells were treated with biotin for ten minutes prior to lysis. Immunoprecipitation and Western blotting were performed using to determine if ASXL1 was biotinylated. (C) Representative ion intensities of the strongest y-ion (y6) of the GQAEVTQDPAPLLR peptide in overlaid spectra of the endogenous peptide (blue trace) and heavy stable isotope labelled internal standard peptide (red trace). (D) Quantiﬁcation of ASXL1 enrichment using parallel reaction-monitoring (PRM) mass spectrometry with isotopically-labelled peptide to determine fold enrichment of ASXL1 after one hour treatment with biotin and immunoprecipitation.

Figure Legend Snippet: Figure 4. Enhanced BRD4-ET domain binding by ASXL1Y591X (A) Schematic showing the approach used for targeted analysis. TurboID was fused to the BRD4-ET domain and used to investigate the interaction with different lengths of ASXL1 (B) Proximity based labelling assays indicate that BRD4 interacts with ASXL1Y591X and neither the full-length, nor ASXL11–472 which lacks the ET binding site. HEK293T cells were transiently co-transfected with either TurboID-BRD4-ET domain or TurboID alone and varying lengths of ASXL1. Cells were treated with biotin for ten minutes prior to lysis. Immunoprecipitation and Western blotting were performed using to determine if ASXL1 was biotinylated. (C) Representative ion intensities of the strongest y-ion (y6) of the GQAEVTQDPAPLLR peptide in overlaid spectra of the endogenous peptide (blue trace) and heavy stable isotope labelled internal standard peptide (red trace). (D) Quantiﬁcation of ASXL1 enrichment using parallel reaction-monitoring (PRM) mass spectrometry with isotopically-labelled peptide to determine fold enrichment of ASXL1 after one hour treatment with biotin and immunoprecipitation.

Techniques Used: Binding Assay, Transfection, Lysis, Immunoprecipitation, Western Blot, Targeted Proteomics, Mass Spectrometry

Figure 5. Global Protein Proﬁle Analysis of BRD4- ET domain co-expressed withASXL1. Protein identi- ﬁcation following streptavidin enrichment of cells treated with TurboID-BRD4-ET domain alone or TurboID- BRD4-ET domain with either Full-length ASXL1 or ASXL1Y591X. A volcano plot was generated to demon- strate changes between proteins detected with FL ASXL1 (A) or ASXL1Y591X (B) compared with the control (BRD4 alone). The fold-change (ASXL1/BRD4 alone) is plotted against the statistical signiﬁcance (log10p- value). Horizontal dashed line indicates where p = 0.05/Proteins with signiﬁcant p-values are high- lighted (red) and labelled. (C) Comparative plot of FL ASXL1 and ASXL1Y591X. The fold-change (ASXL1 FL/ BRD4 alone) is plotted against the fold-change (ASXL1Y591X /BRD4 alone). Proteins which are signiﬁ- cantly changed in either data set are highlighted (red).

Figure Legend Snippet: Figure 5. Global Protein Proﬁle Analysis of BRD4- ET domain co-expressed withASXL1. Protein identi- ﬁcation following streptavidin enrichment of cells treated with TurboID-BRD4-ET domain alone or TurboID- BRD4-ET domain with either Full-length ASXL1 or ASXL1Y591X. A volcano plot was generated to demon- strate changes between proteins detected with FL ASXL1 (A) or ASXL1Y591X (B) compared with the control (BRD4 alone). The fold-change (ASXL1/BRD4 alone) is plotted against the statistical signiﬁcance (log10p- value). Horizontal dashed line indicates where p = 0.05/Proteins with signiﬁcant p-values are high- lighted (red) and labelled. (C) Comparative plot of FL ASXL1 and ASXL1Y591X. The fold-change (ASXL1 FL/ BRD4 alone) is plotted against the fold-change (ASXL1Y591X /BRD4 alone). Proteins which are signiﬁ- cantly changed in either data set are highlighted (red).

Techniques Used: Generated, Control

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Journal: Journal of molecular biology

Article Title: Oncogenic Truncations of ASXL1 Enhance a Motif for BRD4 ET-Domain Binding.

doi: 10.1016/j.jmb.2021.167242

Figure Lengend Snippet: Figure 1. ASXL1 binds the BRD4-ET domain. (A) Schematic representing domain structure of ASXL1. Indicated on ASXL1 is the proposed BRD4 binding site (538–578; dark red) and the relative positions of commonly mutated residues in cancer that produce a truncated protein either by frameshift (black circle) or nonsense (grey circle). (B) Multiple sequence alignment of the BRD4 binding motif in ASXL1 compared with known BRD4-ET domain interactors, highlighting a conserved ﬁve residue motif. These residues we propose bind BRD4 in the same groove of the BRD4-ET as known interactors (PDB: 6BGG; ET bound by CHD4) (C/D) Coomassie-stained gels showing pulldown of His6BRD4600–678 by GST-ASXL1537–587 (C), and the reciprocal pulldown; His6MBP-ASXL1537–587 by GST-BRD4600–678 (D).

Article Snippet: ASXL1 truncations were purchased from Addgene: pCMV6-XL4 ASXL1 (1-1304) (Addgene # 74244); pCMV6-XL4 ASXL1 (p.Y591X) 3x FLAG (Addgene # 74261); pCMV6-XL4 ASXL1 (1-479) 3x FLAG (Addgene # 74262) were a gift from Anjana Rao.

Techniques: Binding Assay, Sequencing, Residue, Staining

Journal: Journal of molecular biology

Article Title: Oncogenic Truncations of ASXL1 Enhance a Motif for BRD4 ET-Domain Binding.

doi: 10.1016/j.jmb.2021.167242

Figure Lengend Snippet: Figure 2. ASXL1 binds the BRD4-ET domain through a core motif. (A) Isothermal titration calorimetry of ASXL1 and BRD4-ET domain. Isothermal titration calorimetry (ITC) of the BRD4 ET domain injected into His6MBP-fused ASXL1537–587 injected into BRD4-ET domain reveals a high afﬁnity interaction, with KD = 0.535 mM. Data points of triplicate titrations, following His6MBP titration subtraction. (B) Introduction of point mutations within the binding sequence of ASXL1 disrupts binding of His6MBP-ASXL1537–587 to GST-BRD4600–678. Coomassie-stained gels showing pulldown of His6MBP-ASXL1537–587 by GST-BRD4600–678. Multiple sequence alignment of the ASXL1 mutations below; the residues colored according to ALSCRIPT Calcons. (C) Alphafold2 model of ASXL1537–587 and the BRD4-ET domain (purple); indicating the ﬁve residues of the core motif. (D) NMR structure of BRD3:NSD3 from PDB 7jyn. The inset box indicates the additional hydrophobic pocket occupied by Phe168 of NSD3, and the equivalent region from the BRD4:ASXL1 model in panel C.

Techniques: Isothermal Titration Calorimetry, Injection, Titration, Binding Assay, Sequencing, Staining

Journal: Journal of molecular biology

Article Title: Oncogenic Truncations of ASXL1 Enhance a Motif for BRD4 ET-Domain Binding.

doi: 10.1016/j.jmb.2021.167242

Figure Lengend Snippet: Figure 3. ASXL1-BRD4-ET interaction is conserved. (A) ASXL1 binds to the highly conserved ET domain of BRD2, BRD3 and BRD4. Coomassie-stained gels showing pulldown of His6MBP-ASXL1537–587 by GST-BRD2632–710, GST-BRD3562–640 and GST-BRD4600–678. (B) Multiple sequence alignment of ASXL1, ASXL2 and ASXL3; the residues colored according to ALSCRIPT Calcons, demonstrates the conservation of the binding site; the core motif is highlighted (red box). (C) BRD4 binds ASXL1, ASXL2 and ASXL3. Coomassie-stained gels showing pulldown of His6MBP-ASXL1537–587, His6MBP-ASXL2582–634 and His6MBP-ASXL3977–1027 by GST-BRD4600–678. (D) Isothermal titration calorimetry of ASXL1 (green), ASXL2 (red) and ASXL3 (blue) injected into BRD4-ET domain reveals all ASXL homologues tightly bind the BRD4-ET domain. Data points of triplicate titrations, following buffer titration subtraction.

Techniques: Staining, Sequencing, Binding Assay, Isothermal Titration Calorimetry, Injection, Titration

Journal: Journal of molecular biology

Article Title: Oncogenic Truncations of ASXL1 Enhance a Motif for BRD4 ET-Domain Binding.

doi: 10.1016/j.jmb.2021.167242

Figure Lengend Snippet: Figure 4. Enhanced BRD4-ET domain binding by ASXL1Y591X (A) Schematic showing the approach used for targeted analysis. TurboID was fused to the BRD4-ET domain and used to investigate the interaction with different lengths of ASXL1 (B) Proximity based labelling assays indicate that BRD4 interacts with ASXL1Y591X and neither the full-length, nor ASXL11–472 which lacks the ET binding site. HEK293T cells were transiently co-transfected with either TurboID-BRD4-ET domain or TurboID alone and varying lengths of ASXL1. Cells were treated with biotin for ten minutes prior to lysis. Immunoprecipitation and Western blotting were performed using to determine if ASXL1 was biotinylated. (C) Representative ion intensities of the strongest y-ion (y6) of the GQAEVTQDPAPLLR peptide in overlaid spectra of the endogenous peptide (blue trace) and heavy stable isotope labelled internal standard peptide (red trace). (D) Quantiﬁcation of ASXL1 enrichment using parallel reaction-monitoring (PRM) mass spectrometry with isotopically-labelled peptide to determine fold enrichment of ASXL1 after one hour treatment with biotin and immunoprecipitation.

Techniques: Binding Assay, Transfection, Lysis, Immunoprecipitation, Western Blot, Targeted Proteomics, Mass Spectrometry

Journal: Journal of molecular biology

Article Title: Oncogenic Truncations of ASXL1 Enhance a Motif for BRD4 ET-Domain Binding.

doi: 10.1016/j.jmb.2021.167242

Figure Lengend Snippet: Figure 5. Global Protein Proﬁle Analysis of BRD4- ET domain co-expressed withASXL1. Protein identi- ﬁcation following streptavidin enrichment of cells treated with TurboID-BRD4-ET domain alone or TurboID- BRD4-ET domain with either Full-length ASXL1 or ASXL1Y591X. A volcano plot was generated to demon- strate changes between proteins detected with FL ASXL1 (A) or ASXL1Y591X (B) compared with the control (BRD4 alone). The fold-change (ASXL1/BRD4 alone) is plotted against the statistical signiﬁcance (log10p- value). Horizontal dashed line indicates where p = 0.05/Proteins with signiﬁcant p-values are high- lighted (red) and labelled. (C) Comparative plot of FL ASXL1 and ASXL1Y591X. The fold-change (ASXL1 FL/ BRD4 alone) is plotted against the fold-change (ASXL1Y591X /BRD4 alone). Proteins which are signiﬁ- cantly changed in either data set are highlighted (red).

Techniques: Generated, Control

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 .

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: pCMV6-XL4 ASXL1 (p.Y591X) 3x Flag , , Addgene plasmid 74261.

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

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.

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: pCMV6-XL4 ASXL1 (p.Y591X) 3x Flag , , Addgene plasmid 74261.

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

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 ( <xref ref-type=

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 . " width="100%" height="100%">

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: pCMV6-XL4 ASXL1 (p.Y591X) 3x Flag , , Addgene plasmid 74261.

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: pCMV6-XL4 ASXL1 (p.Y591X) 3x Flag , , Addgene plasmid 74261.

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:

Article Snippet: Plasmid: pCMV6-XL4 ASXL1 (p.Y591X) 3x Flag , , Addgene plasmid 74261.

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

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1) Product Images from "Oncogenic Truncations of ASXL1 Enhance a Motif for BRD4 ET-Domain Binding."

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