Biology subjects

Levens, D.

Publications and source records attributed to Levens, D..

6 recordsLinked to original sources

bioRxiv2026

Dynamic Supercoiling Sponsors Transcription Amplification by MYC

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 MYCs 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 StatementMYC 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.

molecular biology↗

bioRxiv2025

Connecting spatial regions to clinical phenotypes by transferring knowledge from bulk patient data

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. SpacePhenotyper is implemented in Python. The source code and data sets used for and generated during this study are available at https://github.com/ncbi/SpacePhenotyper

cancer biology↗

bioRxiv2025

A tumorigenesis threshold for endogenous Myc revealed by dosage-compensation for Myc haploinsufficiency in the absence of p53.

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, p53s anti-Myc function may be inextricably linked to its role in genome integrity surveillance.

cancer biology↗

bioRxiv2020

O-GlcNAc Transferase Activity is Essential for RNA Pol II Pausing in a Human Cell-Free Transcription System

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.

biochemistry↗

bioRxiv2019

Single-molecule visualization of the effects of ionic strength and crowding on structure-mediated interactions in supercoiled DNA molecules

DNA unwinding is an important cellular process involved in DNA replication, transcription and repair. In cells, molecular crowding caused by the presence of organelles, proteins, and other molecules affects numerous internal cellular structures. Here, we visualize plasmid DNA unwinding and binding dynamics to an oligonucleotide probe as functions of ionic strength, crowding agent concentration, and crowding agent species using single-molecule CLiC microscopy. We demonstrate increased probe-plasmid interaction over time with increasing concentration of 8 kDa polyethylene glycol (PEG), a crowding agent. We show decreased probe-plasmid interactions as ionic strength is increased without crowding. However, when crowding is introduced via 10% 8 kDa PEG, interactions between plasmids and oligos are enhanced. This is beyond what is expected for normal in vitro conditions, and may be a critically important, but as of yet unknown, factor in DNAs proper biological function in vivo. Our results show that crowding has a strong effect on the initial concentration of unwound plasmids. In the dilute conditions used in these experiments, crowding does not impact probe-plasmid interactions once the site is unwound.

biophysics↗

bioRxiv2018

Visualizing structure-mediated interactions in supercoiled DNA molecules

We directly visualize the topology-mediated interactions between an unwinding site on a supercoiled DNA plasmid and a specific probe molecule designed to bind to this site, as a function of DNA supercoiling and temperature. The visualization relies on containing the DNA molecules within an enclosed array of glass nanopits using the Convex Lens-induced Confinement (CLiC) imaging method. This method traps molecules within the focal plane while excluding signal from out-of-focus probes. Simultaneously, the molecules can freely diffuse within the nanopits, allowing for accurate measurements of exchange rates, unlike other methods which could introduce an artifactual bias in measurements of binding kinetics. We demonstrate that the plasmids structure influences the binding of the fluorescent probes to the unwinding site through the presence, or lack, of other secondary structures. With this method, we observe an increase in the binding rate of the fluorescent probe to the unwinding site with increasing temperature and negative supercoiling. This increase in binding is consistent with the results of our numerical simulations of the probability of site-unwinding. The temperature dependence of the binding rate has allowed us to distinguish the effects of competing higher order DNA structures, such as Z-DNA, in modulating local site-unwinding, and therefore binding.

biophysics↗