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

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.

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: 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.

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: 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.

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, 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).

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: Generated, Control