David Levens

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.

DNA supercoiling can be accommodated through changes in DNA conformation, from global duplex rearrangements to localized non-B DNA structures. These alternative states are predicted to compete for shared superhelical stress, producing long-range torsional coupling between distant DNA elements, but direct evidence for such coupling has been lacking. Here, we present Permanganate Pore-Seq (P2-seq), which integrates potassium permanganate footprinting with Oxford Nanopore native long-read sequencing to map supercoiling-dependent DNA conformations along individual molecules at near-nucleotide resolution. To detect permanganate oxidation products, we developed Rembo, a generalizable computational workflow based on raw signal comparison. Using P2-seq on supercoiled plasmids across a range of superhelical densities, we resolve multiple non-B DNA structural transitions and identify rare structural intermediates. Importantly, we find that distant non-B DNA structures within the same molecule are inversely coupled, providing direct single-molecule evidence of competition among alternative torsional sinks. P2-seq also detects distributed duplex destabilization associated with residual superhelical stress, enabling simultaneous analysis of localized and distributed torsional stress accommodation. Together, these results establish P2-seq as a platform for single-molecule, sequence-resolved analysis of topology-dependent DNA structural states and their coupling, providing a framework for studying how DNA topology shapes genome function.

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.

Spatially resolved transcriptomics (SRT) technology has enabled a new level of knowledge about tumors. Many critical tumor properties, such as invasiveness and growth, depend on both specific transcriptomic changes in tumor cells and the tumor microenvironment. However, computational methods to study clinical phenotypes of spatial regions, such as hazard and drug response, have not yet been developed. Since clinical phenotypes are measured at the patient level and not at the level of spatial regions, such a method would require transferring knowledge from the patient level domain to the spot level domain. To overcome this challenge, we developed SpacePhenotyper. Our approach uses algebraic spectral techniques to transfer the predictive relationship between gene expression and clinical phenotypes from bulk gene expression data to SRT data. Our approach captures a gene expression pattern that is predictive of the phenotype of interest in the form of a vector, the Eigen-Patient, which is then used to quantify the phenotype in spatial spots. After extensively validating SpacePhenotyper on simulated and real data, we utilize it to study how the spatial heterogeneity of breast cancer tumors influences residual cancer burden after treatment. By assigning relative quantities of clinical phenotypes to spatial locations, SpacePhenotyper has proven a powerful tool for the identification and interpretation of transcriptional changes over spatial regions and of spatially-regulated patterns of cellular states.

The MYC proto-oncogene is crucial for neoplasia in most tumors. Overexpressed, oncogenic MYC amplifies the flux through most major processes but does not specify a unique carcinogenic pathway. This “amplifier” model suggests that MYC must exceed an expression threshold to become oncogenic. We designed a genetic test of this model, using the mouse Trp53 null mutant ( p53KO ) as a highly robust tumor generator to examine the effect of a modest change in the endogenous Myc level ( Myc ^+/- ). Strikingly, tumor-free survival is greatly extended in p53KO mice with haploid Myc gene-dosage, yet in the tumors that do develop (mainly hemangiosarcomas and thymic lymphomas), their Myc deficit has been invariably compensated either by increasing Myc genomic dosage (hemangiosarcomas) or expression (lymphomas). Furthermore, acutely halving the endogenous Myc gene-dosage in established tumor allografts curtails growth rates. These results indicate that even an incremental reduction of MYC activity can be salutary in cancer and that one of the major tumor suppressor functions of p53 derives from its ability to prevent MYC overexpression. Myc generates acute DNA damage by several mechanisms and accordingly, p53’s anti-Myc function may be inextricably linked to its role in genome integrity surveillance.

A new method for mapping torsion provides insights into the ways that the genome responds to the torsion generated by RNA polymerase II.

MYC, a key member of the Myc-proto-oncogene family, is a universal transcription amplifier that regulates almost every physiological process in a cell including cell cycle, proliferation, metabolism, differentiation, and apoptosis. MYC interacts with several cofactors, chromatin modifiers, and regulators to direct gene expression. MYC levels are tightly regulated, and deregulation of MYC has been associated with numerous diseases including cancer. Understanding the comprehensive biology of MYC under physiological conditions is an utmost necessity to demark biological functions of MYC from its pathological functions. Here we review the recent advances in biological mechanisms, functions, and regulation of MYC. We also emphasize the role of MYC as a global transcription amplifier.

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.