Fig 1: Cohesin AML mutations affect the interaction with U-70.(A) Top: Immunoblots showing pull-down analysis of recombinant WT SMC3 and mutant SMC3 L316P and SMC3 T1174I with recombinant U1-70. SMC3 (0.90 µg) and mutants were incubated with 0.75 µg of U1-70 (molar ratio of 1:2.5) followed by FLAG pull-down. Recombinant SMC3 and mutants were FLAG tagged on the C-terminus. Recombinant U1-70 was HIS tagged. (B) Top: Immunoblots showing pull-down analysis of recombinant WT SMC1 and SMC1 K384E with recombinant U1-70. SMC1 (0.92 µg) and mutant were incubated with 0.75 µg of U1-70 (molar ratios of 1:2.5) followed by anti-SMC1 pull-down. Recombinant SMC1, K384E mutant, and U1-70 all were HIS tagged. (A and B) Bottom: Quantitation of respective pull-downs, data of three indivdual replicates with standard mean deviation is plotted; variables of significance *P < 0.05, ns P > 0.05. (C) Table showing the number of differential splice events/genes in mESCs with R586W SMC1a mutations compared with WT mESCs (FDR < 0.05). (D) Quantification of PLA shown in insets comparing the RAD21-U170 proximity between the two mES SMC1a R586W clones with WT mESCs (see fig. S15B). Statistics were performed with unpaired t test with Welch’s correction, where n = 431 and ****P < 0.0001. (E) A possible mechanistic model by which cohesin mutations affect alternative splicing in AML. Cohesin colocalizes with RNAPII at the TSS and in gene bodies. Cohesin directly interacts with splicing factors at a subset of genes, ensuring the proper splicing of their transcripts. The cohesin mutations occurring in AML affect cohesin’s interaction with splicing factors, resulting in altered patterns of splicing of metabolic pathway genes.
Fig 2: Proximity of cohesin to U1-70 increases in the absence of BRD4.(A) PLAs between anti-RAD21/anti–U1-70 antibodies following the indicated treatment conditions. (B) Quantification of PLA shown in (A). Statistics were performed with unpaired t test with Welch’s correction, where n = ~201 and ****P < 0.0001.(C) PLAs between anti-BRD4/anti–U1-70 antibodies following the indicated treatment conditions. (D) Quantification of PLA shown in (C). Statistics were performed with Unpaired t test with Welch’s correction, where n = ~175 and ns, P > 0.05 and ****P < 0.0001. (E) Model showing the potential mechanism by which cohesin and BRD4 affect alternative splicing. Both cohesin and BRD4 colocalize with RNAPII at the TSS and in gene bodies. Both interact with a subset of splicing factors where BRD4 is juxtaposed to cohesin in such a manner that it limits the access of cohesin to splicing factors. Overall, this assembly ensures proper splicing. The targeted degradation of cohesin and BRD4 affects the association of splicing factors resulting in changes in alternative splicing. WT, wild-type.
Fig 3: Cohesin colocalizes and directly interacts with splicing factors.(A and B) PLAs between anti-RAD21/anti–U1-70, anti-RAD21/anti-FUS (A), anti-RAD21/anti-HNRNPM, and anti-RAD21/anti-nucleolin (B) antibodies. (C) Quantification of PLA shown in Fig. 5 (A and B). Statistics were performed with unpaired t test with Welch’s correction, where n = 35 and ****P < 0.0001 and ns, P > 0.05. (D) The binding profiles of FUS, RAD21, and H3K27Ac around FUS binding peak summits (top) and FUS binding peaks (bottom). (E) Immunoprecipitates from HCT116 whole-cell extracts (WCE) with anti-RAD21 antibody were immunoblotted with the indicated antibodies to RAD21, SMC3, and U1-70. Twenty-five percent of the WCE was used in the input lanes, and immunoglobulin G (IgG; mock) was used as a negative control for the immunoprecipitation (IP) reactions. (F to H) Immunoblots (IB) showing pull-down analysis of recombinant SMC3 with recombinant U1-70 (F), FUS (G), and HNRNPM (H). rSMC3 (0.90 µg) was incubated with 0.6 and 1.2 µg of U1-70 (molar ratios of 1:2 and 1:4, respectively), 1.6 and 2.6 µg of FUS (molar ratios of 1:3 and 1:5, respectively), and 0.6 and 1.2 µg of HNRNPM (molar ratios of 1:1.5 and 1:3, respectively), followed by pull-down using FLAG beads. Recombinant SMC3 was FLAG tagged; recombinant U1-70 and HNRNPM were HIS tagged, and FUS was glutathione S-transferase (GST) tagged. (I to K) Immunoblots of pull-down assay of recombinant SMC1 (0.92 µg) with 0.6 and 1.2 µg of U1-70 (molar ratios of 1:2 and 1:4, respectively) (I), 1.6 and 2.6 µg of FUS (molar ratios of 1:2 and 1:4, respectively) (J), and 0.6 and 1.2 µg of HNRNPM (molar ratios of 1:1.5 and 1:3, respectively) (K). (I and J) Because SMC1, U1-70, and HNRNPM were HIS tagged, pull-down assays were performed by anti-SMC1 antibody immobilized on protein A Magna beads and (J) pull-down assay of recombinant FUS with SMC1 immobilized on Ni-NTA (nitrilotriacetic acid) beads. DAPI, 4',6-diamidino-2-phenylindole.
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