Rajiv Kumar Jha

Dysregulated MYC provokes profound changes in cancer gene expression. Although usually considered an E-box-dependent transcription activator, an alternate model is that MYC is a universal amplifier of transcription. Although MYC participates in pause release, its full role in the transcription cycle remains poorly illuminated. MYC also stimulates topoisomerases to resolve DNA topological issues complicating its transactions; how this stimulation is coordinated with its transcriptional role has not been examined. We have developed a system that discriminates between MYC-driven activation versus amplification in any cell. This system enables the interrogation of genes, cofactors, and compounds that execute or modulate MYC activity. Using a combination of biochemical and cellular assays, we reveal dynamic interplay between MYC-driven transcription amplification and DNA supercoiling. Because the early stages of transcription are sensitive to DNA supercoiling, Reducing TOP1 activity 1 either through genetic knockdown or low-dose inhibition, potentiates transcription amplification by MYC. By increasing promoter melting and RNA polymerase loading, negative supercoiling provides the early transcription complexes upon which MYC acts to increase output.

MYC dysregulation occurs in most cancers provoking, profound changes in gene expression. Although sometimes considered to be an E-box-dependent transcription factor, an alternate model posits MYC to be a universal amplifier of active genes. Although MYC is associated with accelerated pause release, the full extent of its participation in the transcription cycle remains poorly illuminated. MYC also stimulates topoisomerases to resolve topological issues that complicate DNA transactions; whether and how this stimulation is coordinated with or independent of its transcriptional role has not been examined. We have developed a genetic tool that discriminates between MYC’s ability to activate versus amplify reporter gene expression in any cell. This system enables the interrogation of genes, cofactors, and compounds that execute or modulate MYC activity. Using a combination of biochemical and cellular assays, we reveal the dynamic interplay between MYC-driven transcription amplification and DNA supercoiling. The early stages of transcription are highly sensitive to the level of DNA supercoiling. Reducing the activity of TOP1 either through genetic knockdown or low-dose inhibition potentiates transcription amplification by MYC. This enhancement is associated with pre-initiation complex (PIC) stabilization by DNA supercoiling. MYC helps to mobilize these stabilized PICs. Significance Statement: MYC is dysregulated in most cancers, yet its precise functions in transcription are poorly understood. Using a genetic system that distinguishes transcriptional activation from amplification, we reveal a functional interplay between MYC-driven amplification and DNA supercoiling. Reduced topoisomerase I activity increases DNA-supercoiling that in turn stabilizes PICs to provide more substrate for MYC-dependent transcription amplification. Thus, DNA topology as a critical regulator of MYC-dependent transcription.

Transcription in the nucleus occurs in a concentrated, dense environment, and no reasonable biochemical facsimile of this milieu exists. Such a biochemical environment would be important for further understanding transcriptional regulation. We describe here the formation of dense, transcriptionally active bodies in vitro with only nuclear extracts and promoter DNA. These biomolecular condensates (BMCs) are 0.5 to 1 μm in diameter, have a macromolecular density of approximately 100 mg/ml, and are a consequence of a phase transition between promoter DNA and nuclear extract proteins. BMCs are physically associated with transcription as any disruption of one compromised the other. The BMCs contain RNA polymerase II and elongation factors, as well as factors necessary for BMC formation in vivo. We suggest that BMCs are representative of the in vivo nuclear environment and a more physiologically relevant manifestation of the preinitiation complex/elongation machinery.