Phase separation of Myc differentially regulates gene transcription

Abstract

AbstractDysregulation and enhanced expression of MYC transcription factors including MYC and MYCN contribute to majority of human cancers. For example, MYCN is amplified up to several hundred-fold in high-risk neuroblastoma. One potential consequence of elevated expression is liquid-liquid phase separation (LLPS), occurring when the concentration of certain macromolecules and biopolymers is above a threshold. Here, we show that in MYCN-amplified human neuroblastoma cells, N-myc protein forms condensate-like structures. Using MYCN-nonamplified neuroblastoma cells that have no or little endogenous N-myc protein expression, we found that exogenously expressed N-myc undergoes LLPS in a concentration-dependent manner, and determined its threshold concentration for LLPS in the cellular context. Biophysically, N-myc condensates in live cells exhibit liquid-like behavior. The intrinsically disordered transactivation domain (TAD) of N-myc is indispensable for LLPS. Functionally, the N-myc condensates contain its obligatory DNA-binding and dimerization partner, genomic DNA, transcriptional machinery, and nascent RNA. These condensates are dynamically regulated during cell mitosis, correlated with chromosomal condensation and de-condensation. We further show that the TAD and the DNA-binding domain are both required for transcriptional activity of N-myc condensates. Most importantly, using a chemogenetic tool that decouples the role of phase separation from changes in protein abundance level in the nucleus, we discovered that N-myc phase separation regulates gene transcription and promotes SH-EP cell proliferation. Interestingly, LLPS of N-myc only modulates a small proportion of N-myc-regulated genes. Taken together, our results demonstrate that N-myc undergoes LLPS, and that its phase separation differentially modulates the transcriptome, partially contributes to gene transcription, and promotes cell proliferation. Our work opens a new direction in understanding Myc-related cancer biology that has been studied for several decades.