About

Jennifer Kugel is a Research Professor in the Department of Biochemistry at the University of Colorado Boulder. She earned her PhD from the University of Colorado Boulder in 2001. Her areas of expertise include gene expression and regulation, molecular biophysics, nucleic acids, and single molecule biology. Her research focuses on mammalian mRNA transcription and its regulation, aiming to uncover the molecular mechanisms governing how mammalian gene expression is regulated. Her work primarily investigates two points of control: transcription of messenger RNA (mRNA) and post-transcriptional regulation of mRNAs by microRNAs (miRNAs). She employs a combination of biochemistry, molecular biology, cell-based techniques, molecular genetics, and single molecule studies to explore gene expression across various biochemical and cellular systems.

Research topics

• Genetics
• Biology
• Cell biology
• Computational biology
• Chemistry
• Neuroscience
• Virology
• Molecular biology

Selected publications

• Multi-omics and biochemical reconstitution reveal CDK7-dependent mechanisms controlling RNA polymerase II function at gene 5’- and 3’-ends

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-01-09

  preprintOpen access

  CDK7 regulates RNA polymerase II (RNAPII) initiation, elongation, and termination through incompletely understood mechanisms. Because contaminating kinases precluded CDK7 analysis with nuclear extracts, we completed biochemical assays with purified factors. Reconstitution of RNAPII transcription initiation showed CDK7 inhibition slowed and/or paused RNAPII promoter-proximal transcription, which reduced re-initiation. These CDK7-regulatory functions were Mediator- and TFIID-dependent. Similarly in human cells, CDK7 inhibition reduced transcription by suppressing RNAPII activity at promoters, consistent with reduced initiation and/or re-initiation. Moreover, widespread 3'-end readthrough transcription was observed in CDK7-inhibited cells; mechanistically, this occurred through rapid nuclear depletion of RNAPII elongation and termination factors, including high-confidence CDK7 targets. Collectively, these results define how CDK7 governs RNAPII function at gene 5'-ends and 3'-ends, and reveal that nuclear abundance of elongation and termination factors is kinase-dependent. Because 3'-readthrough transcription is commonly induced during stress, our results further suggest regulated suppression of CDK7 activity may enable this RNAPII transcriptional response.

  PublisherOA PDFDOI

• Single-molecule Microscopy Reveals That TFIIE Subunits Dynamically Interact With Preinitiation Complexes in a Manner Impacted by TFIIH

  Journal of Molecular Biology · 2025-11-01

  articleSenior authorCorresponding
  PublisherDOI

• Single-molecule microscopy reveals that TFIIE subunits dynamically interact with preinitiation complexes in a manner controlled by TFIIH

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-04-20

  preprintOpen accessSenior authorCorresponding

  Abstract Transcription by RNA polymerase II (Pol II) requires general transcription factors that bind with Pol II at the promoters of protein-coding genes to form preinitiation complexes (PICs). Among these is TFIIE, which recruits TFIIH to the PIC and stimulates the kinase and translocase activities of TFIIH, thereby regulating the fate of formed PICs. In this study, we used a purified reconstituted human Pol II transcription system and single molecule total internal reflection fluorescence (smTIRF) microscopy to monitor TFIIE binding dynamics in PICs under different conditions in real time. We observed highly dynamic interactions of the two subunits of TFIIE (TFIIEα and TFIIEβ) with PICs. Measurement of rate constants for on/off binding of each subunit suggest they behave asynchronously. TFIIH exclusion increased the rates of association and dissociation for both subunits, with the strongest effect on TFIIEα. Despite stabilization of TFIIE by TFIIH the TFIIE subunits remain dynamic in PICs. Additionally, two disease-related TFIIEβ point mutations destabilized TFIIEβ and altered its kinetic behaviors within PICs. Our results contribute to an emerging model that PICs are not static assemblies and highlight important connections between the structural arrangement and kinetic behaviors of GTFs in PICs.

  PublisherOA PDFDOI

• Multi-omics and biochemical reconstitution reveal CDK7-dependent mechanisms controlling RNA polymerase II function at gene 5′- and 3′ ends

  Cell Reports · 2025-06-25 · 3 citations

  articleOpen access

  CDK7 regulates RNA polymerase II (RNAPII) initiation, elongation, and termination through incompletely understood mechanisms. Because contaminating kinases prevent reliable CDK7 analysis with nuclear extracts, we reconstitute RNAPII transcription with purified factors. We show that CDK7 inhibition slows and/or pauses RNAPII promoter-proximal transcription and suppresses re-initiation, and these effects are Mediator and TFIID dependent. Similarly in human cells, CDK7 inhibition reduces transcriptional output by suppressing RNAPII initiation and/or re-initiation. Moreover, widespread 3' end readthrough transcription occurs in CDK7-inhibited cells; mechanistically, this results from rapid nuclear depletion of RNAPII elongation and termination factors (e.g., DSIF, Integrator, NELF, SPT6, PPP1R10/PNUTS, and SCAF8), including high-confidence CDK7 kinase targets. Collectively, these results define how CDK7 governs RNAPII function at gene 5' ends and 3' ends and reveal that nuclear abundance of elongation and termination factors is kinase dependent. Because 3'-readthrough transcription is commonly induced during stress, our results further suggest that regulated suppression of CDK7 activity enables this transcriptional response.

  PublisherOA PDFDOI

• Mechanisms and Functions of the RNA Polymerase II General Transcription Machinery during the Transcription Cycle

  Biomolecules · 2024-02-01 · 25 citations

  articleOpen accessSenior authorCorresponding

  Central to the development and survival of all organisms is the regulation of gene expression, which begins with the process of transcription catalyzed by RNA polymerases. During transcription of protein-coding genes, the general transcription factors (GTFs) work alongside RNA polymerase II (Pol II) to assemble the preinitiation complex at the transcription start site, open the promoter DNA, initiate synthesis of the nascent messenger RNA, transition to productive elongation, and ultimately terminate transcription. Through these different stages of transcription, Pol II is dynamically phosphorylated at the C-terminal tail of its largest subunit, serving as a control mechanism for Pol II elongation and a signaling/binding platform for co-transcriptional factors. The large number of core protein factors participating in the fundamental steps of transcription add dense layers of regulation that contribute to the complexity of temporal and spatial control of gene expression within any given cell type. The Pol II transcription system is highly conserved across different levels of eukaryotes; however, most of the information here will focus on the human Pol II system. This review walks through various stages of transcription, from preinitiation complex assembly to termination, highlighting the functions and mechanisms of the core machinery that participates in each stage.

  PublisherOA PDFDOI

• Single molecule studies characterize the kinetic mechanism of tetrameric p53 binding to different native response elements

  PLoS ONE · 2023-08-15

  articleOpen accessSenior authorCorresponding

  The transcriptional activator p53 is a tumor suppressor protein that controls cellular pathways important for cell fate decisions, including cell cycle arrest, senescence, and apoptosis. It functions as a tetramer by binding to specific DNA sequences known as response elements (REs) to control transcription via interactions with co-regulatory complexes. Despite its biological importance, the mechanism by which p53 binds REs remains unclear. To address this, we have used an in vitro single molecule fluorescence approach to quantify the dynamic binding of full-length human p53 to five native REs in real time under equilibrium conditions. Our approach enabled us to quantify the oligomeric state of DNA-bound p53. We found little evidence that dimer/DNA complexes form as intermediates en route to binding or dissociation of p53 tetramer/DNA complexes. Interestingly, however, at some REs dimers can rapidly exchange from tetramer/DNA complexes. Real time kinetic measurements enabled us to determine rate constants for association and dissociation at all five REs, which revealed two kinetically distinct populations of tetrameric p53/RE complexes. For the less stable population, the rate constants for dissociation were larger at REs closest to consensus, showing that the more favorable binding sequences form the least kinetically stable complexes. Together our single molecule measurements provide new insight into mechanisms by which tetrameric p53 forms complexes on different native REs.

  PublisherOA PDFDOI

• Evaluating two steps in transcription using a fluorescence‐based electrophoretic mobility shift assay

  Biochemistry and Molecular Biology Education · 2023-01-04 · 1 citations

  reviewSenior authorCorresponding

  Transcription is the critical first step in expressing a gene, during which an RNA polymerase (RNAP) synthesizes an RNA copy of one strand of the DNA that encodes a gene. Here we describe a laboratory experiment that uses a single assay to probe two important steps in transcription: (1) RNAP binding to DNA, and (2) the transcriptional activity of the polymerase. Students probe both these steps in a single experiment using a fluorescence-based electrophoretic mobility shift assay (EMSA) and commercially available Escherichia coli RNAP. As an inquiry-driven component, students add the transcriptional inhibitor rifampicin to reactions and draw conclusions about its mechanism of inhibition by determining whether it blocks polymerase binding to DNA or transcriptional activity. Depending on the curriculum and learning goals of individual courses, this experimental module could be easily expanded to include additional experimentation that mimics a research environment more closely. After completing the experiment students understand basic principles of transcription, mechanisms of inhibition, and the use of EMSAs to probe protein/DNA interactions.

  PublisherDOI

• Mechanical memory stored through epigenetic remodeling reduces cell therapeutic potential

  Biophysical Journal · 2023 · 39 citations

  • Cell biology
  • Chemistry
  • Neuroscience
  PublisherOA PDFDOI

• Short‐term exposure to ethanol induces transcriptional changes in nontumorigenic breast cells

  FEBS Open Bio · 2023-08-12 · 1 citations

  articleOpen accessSenior authorCorresponding

  Breast cancer is a leading cause of cancer-related deaths in women. Many genetic and behavioral risk factors can contribute to the initiation and progression of breast cancer, one being alcohol consumption. Numerous epidemiological studies have established a positive correlation between alcohol consumption and breast cancer; however, the molecular basis for this link remains ill defined. Elucidating ethanol-induced changes to global transcriptional programming in breast cells is important to ultimately understand how alcohol and breast cancer are connected mechanistically. We investigated induced transcriptional changes in response to a short cellular exposure to moderate levels of alcohol. We treated the nontumorigenic breast cell line MCF10A and the tumorigenic breast cell lines MDA-MB-231 and MCF7, with ethanol for 6 h, and then captured the changes to ongoing transcription using 4-thiouridine metabolic labeling followed by deep sequencing. Only the MCF10A cell line exhibited statistically significant changes in newly transcribed RNA in response to ethanol treatment. Further experiments revealed that some ethanol-upregulated genes are sensitive to the dose of alcohol treatment, while others are not. Gene Ontology and biochemical pathway analyses revealed that ethanol-upregulated genes in MCF10A cells are enriched in biological functions that could contribute to cancer development.

  PublisherOA PDFDOI

• Doxorubicin Impacts the Chromatin Binding of Hmgb1, Histone H1 and Retinoic Acid Receptor

  Research Square · 2022-01-05

  preprintOpen access

  Abstract Doxorubicin (Dox), a widely used anticancer DNA-binding drug, affects chromatin in multiple ways, and these effects contribute to both its efficacy and dose-limiting side-effects, especially cardiotoxicity. Here we studied the Dox effects on the chromatin binding of the architectural proteins high mobility group B1 (HMGB1) and the linker histone H1, and the transcription factor retinoic acid receptor (RARα) by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS), in live cells. At lower drug concentrations, Dox increased the binding of HMGB1 to DNA while decreasing the binding of the linker histone H1. At higher doses that correspond to the peak plasma concentrations reached in chemotherapy, Dox reduced the binding of HMGB1 as well. This biphasic effect is interpreted in terms of a hierarchy of competition between the ligands involved and Dox-induced local conformational changes of nucleosome-free DNA. When combined, FRAP and FCS mobility data suggest that Dox decreases the overall binding of RARα to DNA, an effect that was only partially overcome by agonist binding. The intertwined interactions described likely contribute to the effects as well as side-effects of Dox.

  PublisherOA PDFDOI

Recent grants

• Investigating Mechanisms of RNA Polymerase II Transcription and Regulation Using Single Molecule Fluorescence

  NSF · $958k · 2018–2023

• Mechanism and Regulation of Core Promoter Recognition and Promoter Escape during Transcription by RNA Polymerase II

  NSF · $520k · 2009–2013

• Investigating mechanisms of RNA polymerase II transcription and regulation using single molecule fluorescence

  NSF · $1.1M · 2023–2027

• Mechanism and Regulation of Early Trasncription by RNA polymerase II

  NSF · $420k · 2005–2009

• Investigating Mechanisms of RNA Polymerase II Transcription and Regulation Using Single-molecule Fluorescence

  NSF · $796k · 2013–2018

Frequent coauthors

• James A. Goodrich

  Environmental Protection Agency

  63 shared

• Ryan D. Walters

  15 shared

• James A. Goodrich

  Environmental Protection Agency

  8 shared

• Elina Ly

  University of Colorado Boulder

  7 shared

• David T. McSwiggen

  7 shared

• Steven L. Ponicsan

  University of Colorado Boulder

  7 shared

• David R. Hinton

  University of Southern California

  6 shared

• James Goodrich

  6 shared

Education

• Ph.D., Gene Expression and Regulation, Molecular Biophysics, Nucleic Acids, and Single Molecule Biology

  University of Colorado Boulder

  2001