The phenylalanine-and-glycine repeats of NUP98 oncofusions form condensates that selectively partition transcriptional coactivators

Cancer-associated genetic aberrations, such as the nucleoporin 98 (NUP98) gene rearrangement detected in human leukemias, often produce condensates, a type of membrane-less biomolecular assemblies. How exactly the cancer-related condensation contributes to oncogenesis remains far from clear. Here, we investigate NUP98-PHF23, a leukemia-causing chimeric protein that fuses NUP98's sequence enriched in phenylalanine-and-glycine repeats (FG repeats, also known as intrinsically disordered region [IDR]) in-frame with PHF23's PHD finger, a domain that specifically 'reads' and binds H3K4 trimethylation (H3K4me3). Our integrated analyses using protein module mutagenesis, cell imaging, genomic profiling and condensation reconstitution collectively demonstrate a multifaced role for NUP98's FG repeats in driving fusion condensation while recruiting and co-mixing with a set of histone modifiers and chromatin remodelers, notably the MLL family of H3K4 methylation-'writing' enzymes. The H3K4me3-'reading' PHD finger and NUP98's IDR are cooperative in mediating efficient targeting of NUP98-PHF23 and its coactivators onto leukemic genes, leading to active transcription. Together, we show that NUP98-PHF23 coordinates a set of homotypic and heterotypic interactions (IDR:IDR, IDR:coactivator and PHD:histone) to organize formation of the chromatin-bound multi-component condensates, wherein a feedforward loop involving the 'reading' and 'writing' of H3K4 methylation acts to enforce an open chromatin landscape at leukemogenic loci, thereby driving oncogenic transformation. Overall design: In order to dissect requirement of NUP98-PHF23's protein modules for its biological functions, we generated two function-disruptive mutants — one containing the substitution of Phe in NUP98's FG repeats with Ala (the “FA” mutant), which was known to disrupt FG repeats-mediated LLPS, and the other containing a W362A mutation at PHF23's PHD finger, which was reported to abolish the H3K4me3 binding and suppresses leukemogenic transformation. Next, we generated HEK293FT cells with stable expression of NUP98-PHF23 (GFP-FLAG tagged), either wild-type (WT) or mutant. Total RNAs were purified using RNeasy Plus kit (Qiagen, 74136). For RNA-seq, the RNA samples were processed using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490) and NEBNext Ultra II RNA library Prep kit (NEB, E7770) as per the manufacturer's instructions.