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

Recurrent cancer-causing fusions of NUP98 produce higher-order assemblies known as condensates. How NUP98 oncofusion-driven condensates activate oncogenes remainspoorly understood. Here, we investigate NUP98-PHF23, a leukemogenic chimera of the FG-repeat region of NUP98 and the H3K4me3-binding PHD finger of PHF23. Our integrated analyses using mutagenesis, proteomics, genomics, and condensate reconstitution demonstrate that the PHD finger targets condensates to H3K4me3-demarcated developmental genes and the FG repeats determine condensate composition and gene activation. The FG repeats are necessary to form condensates that partition a specific set of transcriptional regulators, notably the MLL family of H3K4 methylation-writing enzymes and BRD4. The FG repeats are sufficient, when tethered to the genome, to partition these transcriptional regulators and activate genes. NUP98-PHF23 assembles chromatin-bound condensates that partition multiple positive regulators, initiating a feed-forward loop of reading-and-writing active histone modifications. This network of interactions enforces an open chromatin landscape at proto-oncogenes, thereby driving cancerous transcriptional programs. 748T cells, which were derived from V5-tagged NUP98-PHF23 (NP23)-induced primary murine T-cell leukemias, were subject to treatment with a WDR5-specific PROTAC degrader (MS67), in comparison to DMSO, to remove WDR5, followed by CUT&Tag for V5-NP23, as well as the associated cofactors and chromatin modifications. Spikein controls were added to all samples for normalization before CUT&Tag.