PT  - JOURNAL ARTICLE
AU  - Junjiao Yang
AU  - Chan-I Chung
AU  - Jessica Koach
AU  - Hongjiang Liu
AU  - Qian Zhao
AU  - Xiaoyu Yang
AU  - Yin Shen
AU  - William A. Weiss
AU  - Xiaokun Shu
TI  - Phase separation of Myc differentially regulates gene transcription
AID  - 10.1101/2022.06.28.498043
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.06.28.498043
4099  - http://biorxiv.org/content/early/2022/09/07/2022.06.28.498043.short
4100  - http://biorxiv.org/content/early/2022/09/07/2022.06.28.498043.full
AB  - Dysregulation 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.Competing Interest StatementX.S. and W.A.W. are co-founders of Granule Therapeutics.