Abstract:
Core regulatory transcription factors (CR TFs) orchestrate the placement of super enhancers (SEs) to activate transcription of cell-identity specifying gene networks and are critical in promoting cancer. We defined the core regulatory circuitry of fusion positive rhabdomyosarcoma (FP-RMS, a cancer of childhood) in primary tumors and cell lines, which includes PAX3-FOXO1 (P3F), MYOD1, SOX8, MYCN and others. To find chemical probes able to selectively inhibit CR TF transcription, we screened the Structural Genomics Consortium epigenetic probe set by RNA-seq. We found that chemical probes along the acetylation-axis, and not the methylation-axis, are able to cause selective disruption of CR TF transcription. Inhibitors of HDACs (acetylation erasers), BRD4 (acetylation readers) and CBP/p300 (acetylation writers) were all able to selectively halt CR TF transcription.
For HDACs, this raised a conundrum: why would too much histone acetylation, an active chromatin mark, stop transcription at CR TFs? ChIP-seq showed that CR TFs build SEs that have the largest quantities of histone acetylation and the enzymes that write acetylation (i.e., p300), yet paradoxically also harbor the highest amounts of the opposing histone deacetylases (HDACs). To investigate the architectural effects of disabling HDACs and causing hyper acetylation, we developed Absolute Quantification of Architecture (AQuA) HiChIP, revealing erosion of native SE contacts at CR TFs, and extensive aberrant contacts. This did not cause an elongation defect, but rather removed RNA Pol2 from core regulatory genetic elements and eliminated RNA-Pol2 phase condensates in 20 minutes. We further dissected the contribution of HDAC isoforms using a set of HDAC selective inhibitors, finding HDAC1/2/3 are co-essential to CR transcription.
Using HAT inhibitors/degraders, we discovered a profound dependence on CBP/p300 for clustering of Pol2 loops that connect P3F to its target genes. In the absence of CBP/p300, Pol2 long range enhancer loops collapse, Pol2 accumulates in CpG islands and fails to exit the gene body. These results reveal a potential novel axis for therapeutic interference with P3F in FP-RMS and clarify the molecular relationship of P3F and CBP/p300 in sustaining active Pol2 clusters essential for oncogenic transcription.
In multiple contexts, we propose Pol2 Un-Loading Ratio (PULR) as a new key metric to quantify and explain the drug induced defects in CR TF transcription. Overall, our data reveals a SE-specific need for balancing histone acetylation states to maintain SE architecture, Pol2 clustering in 3D, and CR TF transcription.

https://umich-health.zoom.us/j/93929606089?pwd=SHh6R1FOQm8xMThRemdxTEFMWWpVdz09

Short bio:
Dr. Berkley Gryder is a chemist who retrained as a molecular biologist and computer scientist. He is passionate about understanding how cancer cells control their genes, and developing new chemical strategies to stop cancer cell’s addiction to gene transcription. Along the way, he has proposed paradigm shifts and surprises that are explaining old conundrums. Recognizing that the “right” answer to a tough question is often out of reach with current tools, Dr. Gryder is always innovating techniques with an ultra-high level of spatial and temporal precision to study gene control/epigenetics.