Fig 2: W135 is essential for MYC:TRRAP.(A) Luminescence measurements of cells transfected with TRRAP 2033-2283-LgBiT and empty vector, SmBiT-MYC 1-190 (+/− MB2), and with a W135E substitution. These results indicate the importance of W135 for the MYC:TRRAP interaction. An unpaired Student’s t-test was performed to determine standard deviation and statistical significance. P-value ≤ 0.05 was considered statistically significant. Error bars represent SD and ns: p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. (B) Western blot analysis of a coimmunoprecipitation experiment where the native TRRAP and MYC proteins or the indicated MYC mutants were co-transfected into Expi293F cells. PYO-tagged full-length MYC was co-expressed with FLAG-tagged full-length TRRAP and then MYC was IP’d with anti-PYO beads. High/low MAX refers to exposure time. These results validate the MYC:TRRAP NanoBiT platform and indicate that it can provide quantifiable measurements that recapitulate the binding interaction of the native MYC:TRRAP complex.

Fig 3: Proposed model of MYC S146L activity in transcription and cancer development.(A) Wild-type MYC likely exists in an equilibrium of TRRAP-bound and unbound states. Upon TRRAP recruitment, MYC directs the acetylation of proximal histones to promote transcription. Expression of oncogenic levels of WT MYC promotes non-adherent growth and cellular transformation. In contrast, MYC S146L promotes increased TRRAP affinity, likely leading to enhanced histone acetylation with a concomitant increase in the transcription of target genes. As a result, MYC S146L promotes increased fitness and growth in low attachment and likely augments both cell proliferation and apoptosis in a context-dependent manner. (B) Normal cells under oncogenic transformation following the deleterious genomic alterations, such as the loss of tumor suppressor function and the activation of oncogenes. Following this process, mutation of MYC S146L further activates transcription of MYC target genes. Subsequently, subclones tolerating this mutation are established and are endowed with increased fitness because of heightened MYC activity.

Fig 4: S146L enhances TRRAP binding to MYC.(A) Luminescence measurements of complementation binding assay in Expi293F cells for the indicated amino acid substitutions within MYC. The reduced-function MB2 substitution W135E served as a control for assay sensitivity. Error bars represent ±SEM (Standard Error of the Mean). (B) The S146L mutation enhances binding to TRRAP approximately two-fold. (B) Immunoprecipitation of stably expressed V5-MYC from MCF10A cells. TRRAP co-immunoprecipitation was determined by western blot. (C) Representative images of proximity ligation assay (PLA) results between V5 epitope (MYC) and TRRAP for MCF10A cells expressing the indicated MYC substitutions. (D) Quantification of results of three independent experiments (=30 nuclei per field). PLA foci for each group were calculated as a percentage relative to WT. Quantification indicates ratio of IP TRRAP intensity to IP MYC intensity. Statistics: Holm-Šidák test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Fig 5: TRRAP 2033-2283 is the minimal MYC-binding domain.(A) Western blot analysis of a co-immunoprecipitation experiment where the indicated regions of TRRAP were cloned into a CMV-FLAG expression vector and transfected into HEK293T cells. Proteins were co-expressed with PYO-tagged full-length MYC and then MYC was IP’d with anti-PYO beads. Co-IP was assessed by western blot with anti-FLAG. The most critical binding domain is within residues 2033-2088, consistent with previous results [5], and the minimal domain TRRAP 2033-2283 is established. (B) Western blot analysis of a co-immunoprecipitation experiment where the indicated regions of TRRAP were cloned into a CMV-FLAG expression vector and transfected into HEK293T cells. Endogenous MYC was IP’d with C-33 beads (Santa Cruz Biotechnology). The TRRAP and FLAG blots show TRRAP 2033-2283 can compete off the endogenous native MYC:TRRAP complex in cells, but the same domain of TRRAP lacking the critical region 2033-2088 does not show the same level of competition.