Myo-differentiation reporter screen reveals NF-Y as an activator of PAX3–FOXO1 in rhabdomyosarcoma

Recurrent chromosomal rearrangements found in rhabdomyosarcoma (RMS) produce the PAX3–FOXO1 fusion protein, which is an oncogenic driver and a dependency in this disease. One important function of PAX3–FOXO1 is to arrest myogenic differentiation, which is linked to the ability of RMS cells to gain an unlimited proliferation potential. Here, we developed a phenotypic screening strategy for identifying factors that collaborate with PAX3–FOXO1 to block myo-differentiation in RMS. Unlike most genes evaluated in our screen, we found that loss of any of the three subunits of the Nuclear Factor Y (NF-Y) complex leads to a myo-differentiation phenotype that resembles the effect of inactivating PAX3–FOXO1. While the transcriptomes of NF-Y- and PAX3–FOXO1-deficient RMS cells bear remarkable similarity to one another, we found that these two transcription factors occupy nonoverlapping sites along the genome: NF-Y preferentially occupies promoters, whereas PAX3–FOXO1 primarily binds to distal enhancers. By integrating multiple functional approaches, we map the PAX3 promoter as the point of intersection between these two regulators. We show that NF-Y occupies CCAAT motifs present upstream of PAX3 to function as a transcriptional activator of PAX3–FOXO1 expression in RMS. These findings reveal a critical upstream role of NF-Y in the oncogenic PAX3–FOXO1 pathway, highlighting how a broadly essential transcription factor can perform tumor-specific roles in governing cellular state.