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. 2022 Dec 27;2(12):1693–1710. doi: 10.1158/2767-9764.CRC-22-0255

FIGURE 2.

FIGURE 2

Proteasome inhibitors repress c-MYC gene expression and induce chromatin condensation at the c-MYC super-enhancer. A, Western blot analysis of c-MYC protein levels in MOLP-8 cells shows the biphasic effect of proteasome inhibitor treatment (12.5 μmol/L Lactacystin; 30 nmol/L Bortezomib) on c-MYC protein levels over time (3, 6, and 24 hours). GAPDH was used as an internal control. The relative expression of c-MYC protein was quantified in ImageJ and normalized to that of GAPDH protein. The levels of c-MYC protein at each timepoint were densitometrically compared and expressed as percent of the untreated 0-hour time point. Degradation of c-MYC at later timepoints can be attributed to incomplete inhibition of the proteasome and a bortezomib half-life of about 12 hours. B, qRT-PCR measurement of MYC mRNA levels in MM.1S, MOLP-8 and U266.B1 cells demonstrates that lactacystin or bortezomib transcriptionally repress the c-MYC oncogene after 6-hour treatment. ***, P < 0.001 determined by unpaired Student two-tailed t test. C, Representative ChIP-seq tracks of H3K27ac sites on MYC gene following exposure of MM.1S cells to different proteasome inhibitors show that H3K27 acetylation at the c-MYC promoter was rapidly repressed after treatment. The gene structure is shown in black at the bottom of the panel. IGV tracks in a given comparison are represented at the same scale (numbers in brackets at the y-axis). The genomic region on the x-axis spans 5 kb of the MYC gene. IGV snapshots are representative of two independent experiments. D, Gene tracks of H3K27ac ChIP-seq occupancy at c-MYC super-enhancer in MM.1S cells show that exposure to lactacystin, bortezomib, and carfilzomib reduces H3K27 acetylation of the super-enhancer, especially at its 5′-end (red dashed box). IGV tracks in a given comparison are represented at the same scale (numbers in brackets at the y-axis). The genomic region on the x-axis spans 9 kb of the c-MYC super-enhancer. IGV snapshots are representative of two independent experiments. E, CUT & RUN gene tracks of c-MYC–binding sites in MM.1S cells. The genomic region on the x-axis spans 2.5 kb for all the genes. IGV snapshots were the result of a single CUT & RUN experiment.