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. 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.