The non-canonical role of DOT1L in regulating c-MYC-driven transcription

Sepulveda, Gian Paolo Bagarino

The non-canonical role of DOT1L in regulating c-MYC-driven transcription

Date

2024

Authors

Sepulveda, Gian Paolo Bagarino

URI

https://hdl.handle.net/2144/51139

Abstract

The proto-oncogene c-MYC is a key representative of the MYC transcription factor network regulating growth and metabolism. MML-1 (Myc- and Mondo-like) is its homolog in C. elegans. The functional and molecular cooperation between c-MYC and the histone 3 lysine 79 (H3K79) methyltransferase DOT1L was demonstrated in several human cancer types, and we have earlier discovered the connection between C. elegans MML-1 and DOT-1.1. Here, we demonstrate the critical role of DOT1L in regulating c-MYC target genes genome-wide in triple-negative breast cancer cells, wherein both acute (siRNA treatment) and permanent (CRISPR/Cas9-based) knock-down of DOT1L led to reduced expression of c-MYC target genes (assessed by RNA-seq, proteomics, and RT-qPCR). Surprisingly, this downregulation of c-MYC target genes is accompanied by elevated occupancy of c-MYC at their promoters, suggesting an accumulation of inactive or “spent” c-MYC. The nuclear proteasome plays an important role in removing transcription factors from chromatin. In the case of c-MYC, it was shown that its timely removal by the ubiquitin-proteasome system (UPS) was critical for target gene activation. Here, we demonstrate the non-canonical role of DOT1L in ensuring the removal of “spent” c-MYC from chromatin. We recapitulated reduced expression of c-MYC target genes upon proteasome inhibitor treatment in control cells. However, the inhibitor effect was dampened in DOT1L KO/+ cells already deficient in c-MYC target gene expression indicating that DOT1L and nuclear proteasome function in the same pathway. VCP/p97 is an ATPase extracting proteins from the membranes and chromatin to promote their degradation and is known to regulate c-MYC. Indeed, specific inhibition of the ATPase activity of VCP led to reduced expression of c-MYC target genes in our system but had no effect on these genes in DOT1L KO/+ cells. Depletion of VCP also led to delayed clearance of ubiquitinated proteins upon inhibition of ubiquitination. Finally, we uncover a previously unrecognized proteolytic cleavage of c-MYC performed by DOT1L, which may facilitate c-MYC turnover. This new mechanism of c-MYC regulation by DOT1L incorporates transcription factor dynamics at promoters with gene expression, thereby challenging the common perception of transcription factor activation. Elucidating this novel mechanism may lead to the development of new approaches for cancer treatment.