Journal: Oncogene

Article Title: Multiple interactions of the oncoprotein transcription factor MYC with the SWI/SNF chromatin remodeler

doi: 10.1038/s41388-021-01804-7

Figure Lengend Snippet: (a) Flag-HA-epitope tagged central portion MYC (amino acids 151–319) fused to the Gal4 DNA binding domain and Flag-tagged full-length MYC were expressed into HEK293T cells, along with an empty Flag vector control. Immunoprecipitation of the central portion compared to full-length MYC reveals that SNF5 binding is specific for full-length MYC, while BRD9 co-precipitates with the central portion of MYC and full-length MYC. (b) HEK cells engineered to express a version of HA-tagged SNF5 that can be removed by the dTAG method were treated with dTAG47 molecule and a BAF155 IP performed. Upon depletion of SNF5 over time, MYC remains bound to BAF155. (c) Endogenous BAF155 was immunoprecipitated with an anti-BAF155 antibody in G401 cells. Co-immunoprecipitated material was then probed for the SWI/SNF subunit BAF170 and MYC. (d) As in (c) except the MRT cell line A204 was used and the SWI/SNF subunit BRD9 was probed as a control BAF155 interaction partner. (e) Proximity ligation assay (PLA) was performed in A204 cells and results were quantified. PLA foci detected per nucleus were counted and each data point is shown. Red bar indicates mean value per sample and n of nuclei counted appears below sample. ( n = 3 biological replicates, error bars are standard error; *** P < 0.001 using unpaired t -test , two-tailed between “BAF155” and “MYC/BAF155”) (f) Representative images obtained for PLA assay in A204 cells. DAPI was used as the nuclear stain and scale bar is 10 μm. (g) Same as in (e) for G401 cells. ( n = 3 biological replicates, error bars are standard error; *** P < 0.001 using unpaired t -test , two-tailed between “BAF155” and “MYC/BAF155”). (h) Representative images obtained for PLA assay as in (f) but for G401 cells.

Article Snippet: To create similar constructs for BAF170, the plasmid pFastBac1 Flag BAF170 ( ) was used as the source for all amplified sequences. pFastBac1 Flag BAF155 b was a gift from Robert Kingston (Addgene plasmid # 1954; http://n2t.net/addgene:1954 ; RRID:Addgene_1954). pFastBac1 Flag Baf170 was a gift from Robert Kingston (Addgene plasmid # 1955 ; http://n2t.net/addgene:1955 ; RRID:Addgene_1955).

Techniques: Binding Assay, Plasmid Preparation, Control, Immunoprecipitation, Proximity Ligation Assay, Two Tailed Test, Staining

Journal: Oncogene

Article Title: Multiple interactions of the oncoprotein transcription factor MYC with the SWI/SNF chromatin remodeler

doi: 10.1038/s41388-021-01804-7

Figure Lengend Snippet: (a) T7-tagged BAF170, BAF155, or BAF155 domain mutants were expressed in HEK293T cells, together with the MYC central portion used in . T7-tagged proteins were recovered by T7-IP and samples probed for HA to detect MYC or SNF5 to detect endogenous SNF5. Domains present in each BAF155 construct are denoted and listed in detail within . AC = acidic domain, SW = swirm domain, SA = SANT domain, CTERM = C-terminal domain. (b) Recombinant Flag-tagged MYC central portion was mixed with recombinant BAF155 SWIRM domain (amino acids 417–580 of BAF155) or SUMO control protein and a Flag-IP performed. IP samples were eluted with 3X Flag peptide and all products, and inputs, were resolved by SDS-PAGE. Proteins visualized by Coomassie staining. Light chain of antibody used for IP shows up inconsistently in IP samples and is marked. (c) Recombinant Flag-tagged MYC central portion and Flag-tagged SNF5 were mixed with a recombinant protein containing 4 repeating sequences of the region of BAF155 corresponding to amino acids 533–546 (“4CT”). This region of BAF155 is not sufficient for binding to either MYC central portion or SNF5. Flag-IP of MYC and SNF5 with the SWIRM domain (amino acids 417–546 of BAF155: “Δ546”) are shown as a control for the IP. (d) Recombinant Flag-tagged MYC central portion or Flag-tagged SNF5 were mixed with wild-type recombinant SWIRM domain (“Δ580 WT) or a mutated SWIRM domain (“Δ580AA”; double alanine :L534A, N536A) and Flag-IP performed as in (b). (e) Recombinant Flag-tagged SWIRM domain (amino acids 417–564 of BAF155, “546-F”), HA-tagged MYC central portion, or untagged SNF5, were mixed in the indicated molar combinations. FLAG IP was performed to recover the BAF155 SWIRM domain. Proteins were visualized by western blotting with the indicated antibodies. Beginning at a 1:1 molar ratio of SNF5:MYC, SNF5 attenuates binding of MYC central portion to the BAF155 SWIRM. (f) T7-tagged WT SWIRM domain or the “AA” mutant (amino acid mutations L534A and N536A) were expressed in HEK293T cells and T7-IP performed. (g) T7-tagged BAF155 (amino acids 1–580) or truncations targeting the SWIRM domain, were expressed in HEK293T cells along with Flag-tagged MYC and T7-IP performed. When SNF5 binding is impacted by the SWIRM truncations, Flag-tagged MYC binding increases.

Article Snippet: To create similar constructs for BAF170, the plasmid pFastBac1 Flag BAF170 ( ) was used as the source for all amplified sequences. pFastBac1 Flag BAF155 b was a gift from Robert Kingston (Addgene plasmid # 1954; http://n2t.net/addgene:1954 ; RRID:Addgene_1954). pFastBac1 Flag Baf170 was a gift from Robert Kingston (Addgene plasmid # 1955 ; http://n2t.net/addgene:1955 ; RRID:Addgene_1955).

Techniques: Construct, Recombinant, Control, SDS Page, Staining, Binding Assay, Western Blot, Mutagenesis