MYC overexpression leads to increased chromatin interactions at superenhancers and c-Myc binding sites

The MYC oncogene encodes for the c-Myc protein and is frequently dysregulated across multiple cancer cell types, making it an attractive target for cancer therapy. There have been many difficulties in targeting c-Myc, due to its complex network of regulators and the unstructured nature of its protein. Thus, we are interested in looking at the downstream cancer-specific functions of c-Myc. Overexpression of MYC leads to c-Myc binding at active enhancers, resulting in a global transcriptional amplification of active genes. However, the mechanism underlying this c-Myc enhancer invasion has not been well studied. To that end, we performed ChIP-seq, RNA-seq, 4C-seq and SIQHiC (Spike-in Quantitative Hi-C) on the U2OS osteosarcoma cell line with tetracycline-inducible MYC. MYC overexpression in U2OS cells modulated histone acetylation and increased c-Myc binding at superenhancers. SIQHiC analysis revealed increased global chromatin contact frequency, particularly at chromatin interactions connecting c-Myc binding sites. Our results suggest that c-Myc molecules are recruited to and accumulates within zones of high transcription activity, binding first at stable promoter binding sites at low expression levels, then at superenhancer binding sites when overexpressed. At the same time, the recruitment of c-Myc and other transcription factors may stabilize chromatin interactions to increase chromatin contact frequency. The accumulation of c-Myc at cancer-type specific superenhancers may then drive the expression of interacting oncogenes that each cancer is highly reliant on. By elucidating the chromatin landscape of c-Myc driven cancers, we can potentially target these chromatin interactions for cancer therapy, without affecting physiological c-Myc signaling.

Dissecting transcriptional amplification by MYC

Supraphysiological MYC levels are oncogenic. Originally considered a typical transcription factor recruited to E-boxes (CACGTG), another theory posits MYC a global amplifier increasing output at all active promoters. Both models rest on large-scale genome-wide ”-omics’. Because the assumptions, statistical parameter and model choice dictates the ‘-omic’ results, whether MYC is a general or specific transcription factor remains controversial. Therefore, an orthogonal series of experiments interrogated MYC’s effect on the expression of synthetic reporters. Dose-dependently, MYC increased output at minimal promoters with or without an E-box. Driving minimal promoters with exogenous (glucocorticoid receptor) or synthetic transcription factors made expression more MYC-responsive, effectively increasing MYC-amplifier gain. Mutations of conserved MYC-Box regions I and II impaired amplification, whereas MYC-box III mutations delivered higher reporter output indicating that MBIII limits over-amplification. Kinetic theory and experiments indicate that MYC activates at least two steps in the transcription-cycle to explain the non-linear amplification of transcription that is essential for global, supraphysiological transcription in cancer.

Transcription amplification by nuclear speckle association

A significant fraction of active chromosome regions and genes reproducibly position near nuclear speckles, but the functional significance of this positioning is unknown. Here we show that Hsp70 BAC transgenes and endogenous genes turn on 2-4 mins after heat shock irrespective of their distance to nuclear speckles. However, we observe 12-56-fold and 3-7-fold higher transcription levels for speckle-associated Hsp70 transgenes and endogenous genes, respectively, after 1-2 hrs heat shock. Several fold higher transcription levels for several genes flanking the Hsp70 locus also correlate with speckle-association at 37 °C. Live-cell imaging reveals this modulation of Hsp70 transcription temporally correlates with speckle association/disassociation. Our results demonstrate stochastic gene expression dependent on positioning relative to a liquid-droplet nuclear compartment through a “transcriptional amplification” mechanism distinct from transcriptional bursting.

Development of a Transcriptional Amplification System Based on the PEG3 Promoter to Target Androgen Receptor-Positive and -Negative Prostate Cancer Cells

Localized prostate cancer (PCa) is often curable, whereas metastatic disease treated by castration inevitably progresses toward castration-resistant PCa (CRPC). Most CRPC treatments target androgen receptor (AR) signaling. However, not all CRPC cells rely on AR activity for survival and proliferation. With advances in immunotherapy and fluid biopsies for cancer management, expression systems specific for both AR-positive and -negative PCa are required for virus-based vaccines and cell imaging. To target both AR-responsive and non-responsive cells, we developed a three-step transcriptional amplification (3STA) system based on the progression elevated gene-3 (PEG3) promoter named PEG3AP1-3STA. Notably, we report on different genetic modifications that significantly improved PEG3 promoter’s strength in PCa cells. Adenoviruses incorporating PEG3 promoter with and without transcriptional amplification systems were generated. The potential of PEG3AP1-3STA to target PCa cells was then evaluated in vitro and in vivo in androgen-responsive and non-responsive PCa cell lines. PEG3AP1-3STA was shown to be active in all PCa cell lines and not regulated by androgens, and its activity was amplified 97-fold compared to that of a non-amplified promoter. The PEG3AP1-3STA system can thus be used to target advanced AR+ and AR− cells for imaging or immunovirotherapy in advanced PCa.