Brian A. Lewis

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.

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.

Transcription factor TAF7 links transcription and translation by delivering its transcripts to polysomes for translation.

Paused RNA polymerase II is the major regulated step of transcription in metazoans. We describe here a unique human cell-free transcription system that recapitulates RNA pol II pausing and assemble paused pol IIs on the human CMV IE, SV40, and heat shock promoters, all is the case in vivo. We then use the system to show that PARP-1 and CDK12/13 inhibitors directly affect pausing and elongation. We then show that O-GlcNAcylation is required for the establishment of a paused pol II: inhibition of OGT allowed pol II to bypass pausing and begin to elongate. Addback of rOGT or the pausing factor NELF re-established the pausing. In vivo nascent RNA measurements showed that OGA inhibition blocks elongation. These data show that cell-free systems can recapitulate RNA pol II pausing, that PARP-1 and CDK12/13 directly regulate RNA pol II elongation, and identify O-GlcNAc cycling regulating both pausing and pause release.