Fig. 4: The regular and alternative BRD9 isoforms display different interactomes.

A Scheme illustrating BRD9 splice variants and predicted protein isoforms according to NCBI RefSeq (Annotation release GCF_000001405.40-RS_2023_03). The zoom-in view highlights the predicted protein sequence stemming from exon 15 inclusion in BRD9. Upon translation of exon 15, a stop codon emerges near the end of the exon, resulting in a shorter splice isoform with an alternative C-terminus. B Scheme depicting lentiviral constructs for overexpression of the regular and alternative BRD9 splice variants. The corresponding DNA and protein sequences are available in Supplementary Information Appendix 1. C Heatmap illustrating the detected ncBAF complex subunits upon co-immunoprecipitation and subsequent mass spectrometry analysis with the regular or alternative BRD9 isoforms stably overexpressed in the HEK293T cell line. The relative protein levels are based on the total number of identified peptide-spectrum matches (PSMs) for the corresponding protein from the mass spectrometry analysis. The ncBAF complex model to the right of the heatmap illustrates that the alternative BRD9 isoform precipitated a majority of the ncBAF complex subunits. ACTB was not detected among the co-immunoprecipitates. The control cells expressing FLAG-V5-tagged YFP were used. D Volcano plot displaying differential interaction analysis of proteins that selectively interacted more with the regular or alternative BRD9 isoforms (multiple Student’s t-tests with Benjamini–Hochberg multiple testing correction). The top 5 candidates are highlighted in red. E Venn diagram showing a comparison of the significant differentially interacting proteins with the regular or alternative BRD9 isoforms and BRD9-interacting proteins reported by Gaudio et al. [46]. The intersecting proteins are SPEN, BRCA2, and CHD9. F Western blot analysis of V5-tag and BICRA co-immunoprecipitates in the HEK293T cell line with stable overexpression of the regular and alternative BRD9 isoforms. In the V5-tag-immunoprecipitations, both overexpressed BRD9 isoforms precipitated BICRA together with SMARCA4 and SMARCC1. Reciprocal BICRA co-immunoprecipitations precipitated both overexpressed BRD9 isoforms, SMARCA4 and SMARCC1. The control cells were transduced with an empty lentiviral vector. For complete blot images see Supplementary Fig. 13A. G Bar plots showing V5-tag and BICRA co-immunoprecipitation efficiency with BRD9, BICRA, SMARCA4, and SMARCC1. Results are expressed as fold differences between the regular and alternative BRD9 isoforms with corresponding P values (Student’s t-test). The bar plots display the mean values from three repeated experiments, with error bars representing the standard deviation. For detailed quantification and calculation see Supplementary Fig. 13B. PSM: peptide-spectrum matches; FDR: false discovery rate; IP: immunoprecipitation.