Diana Stavreva | National Institutes of Health (original) (raw)

Papers by Diana Stavreva

Scientific Reports, Dec 6, 2012

Contamination of the environment with endocrine disrupting chemicals (EDCs) is a major health con... more Contamination of the environment with endocrine disrupting chemicals (EDCs) is a major health concern. The presence of estrogenic compounds in water and their deleterious effect are well documented. However, detection and monitoring of other classes of EDCs is limited. Here we utilize a high-throughput live cell assay based on sub-cellular relocalization of GFP-tagged glucocorticoid and androgen receptors (GFP-GR and GFP-AR), in combination with gene transcription analysis, to screen for glucocorticoid and androgen activity in water samples. We report previously unrecognized glucocorticoid activity in 27%, and androgen activity in 35% of tested water sources from 14 states in the US. Steroids of both classes impact body development, metabolism, and interfere with reproductive, endocrine, and immune systems. This prevalent contamination could negatively affect wildlife and human populations.

Trends in Cell Biology, Jun 1, 2021

Mechanotransduction is the ability of a cell to sense mechanical cues from its microenvironment a... more Mechanotransduction is the ability of a cell to sense mechanical cues from its microenvironment and convert them into biochemical signals to elicit adaptive transcriptional and other cellular responses. Here, we describe recent advances in the field of mechanical regulation of transcription, highlight mechanical regulation of the epigenome as a key novel aspect of mechanotransduction, and describe recent technological advances that could further elucidate the link between mechanical stimuli and gene expression. In this review, we emphasize the importance of mechanotransduction as one of the governing principles of cancer progression, underscoring the need to conduct further studies of the molecular mechanisms involved in sensing mechanical cues and coordinating transcriptional responses.

Representative datasets associated with article "Transcriptional Bursting and Co-bursting Re... more Representative datasets associated with article "Transcriptional Bursting and Co-bursting Regulation by Steroid Hormone Release Pattern and Transcription Factor Mobility."

Biophysical Journal, Jun 1, 2004

Fluorescence recovery after photobleaching (FRAP) is now widely used to investigate binding inter... more Fluorescence recovery after photobleaching (FRAP) is now widely used to investigate binding interactions in live cells. Although various idealized solutions have been identified for the reaction-diffusion equations that govern FRAP, there has been no comprehensive analysis or systematic approach to serve as a guide for extracting binding information from an arbitrary FRAP curve. Here we present a complete solution to the FRAP reaction-diffusion equations for either single or multiple independent binding interactions, and then relate our solution to the various idealized cases. This yields a coherent approach to extract binding information from FRAP data which we have applied to the question of transcription factor mobility in the nucleus. We show that within the nucleus, the glucocorticoid receptor is transiently bound to a single state, with each molecule binding on average 65 sites per second. This rapid sampling is likely to be important in finding a specific promoter target sequence. Further we show that this predominant binding state is not the nuclear matrix, as some studies have suggested. We illustrate how our analysis provides several self-consistency checks on a FRAP fit. We also define constraints on what can be estimated from FRAP data, show that diffusion should play a key role in many FRAP recoveries, and provide tools to test its contribution. Overall our approach establishes a more general framework to assess the role of diffusion, the number of binding states, and the binding constants underlying a FRAP recovery.

Endocrinology and Metabolic Syndrome, Nov 19, 2014

Molecular and Cellular Endocrinology, 2022

Sex hormones, such as androgens, estrogens and progestins are naturally occurring compounds that ... more Sex hormones, such as androgens, estrogens and progestins are naturally occurring compounds that tightly regulate endocrine systems in a variety of living organisms. Uncontrolled environmental exposure to these hormones or their biological and synthetic mimetics has been widely documented. Furthermore, water contaminants penetrate soil to affect flora, fauna and ultimately humans. Because endocrine systems evolved to respond to very small changes in hormone levels, the low levels found in the environment cannot be ignored. The combined actions of sex hormones with glucocorticoids and other nuclear receptors disruptors creates additional level of complexity including the newly described "dynamic assisted loading" mechanism. We reviewed the extensive literature pertaining to world-wide detection of these disruptors and created a detailed Table on the development and current status of methods used for their analysis.

Mutation Research, Apr 1, 2001

The advantage of using the tobacco (Nicotiana tabacum var. xanthi) mutagenicity assay is the abil... more The advantage of using the tobacco (Nicotiana tabacum var. xanthi) mutagenicity assay is the ability to analyze and compare on the same plants under identical treatment conditions both the induced acute DNA damage in somatic cells as measured by the Comet assay and the yield of induced leaf somatic mutations. Gamma-irradiation of tobacco seedlings induced a dose-dependent increase in somatic mutations from 0.5 (control) to 240 per leaf (10 Gy). The increased yield of somatic mutations was highly correlated (r = 0.996) with the increased DNA damage measured by the Comet assay immediately after irradiation. With increased dose of gamma-irradiation, the averaged median tail moment values (±S.E.) significantly increased from 1.08 ± 0.10 (control) to 20.26 ± 1.61 m (10 Gy). Nuclei isolated from leaves 24 h after irradiation expressed tail moment values that were not significantly different from the control (2.08 ± 0.11). Thus a complete repair of DNA damage induced by gamma-irradiation and measurable by the Comet assay was observed, whereas the yield of somatic mutations increased in relation to the radiation dose. Data on the kinetics of DNA repair and of DNA damage induced by gamma-radiation on isolated tobacco nuclei, and on nuclei isolated from irradiated leaves and roots are presented.

EMBO Reports, Oct 12, 2007

Nucleic Acids Research, Sep 28, 2007

Molecular and Cellular Biology, Jul 1, 2006

We have investigated the possible involvement of the ubiquitin-proteasome system (UPS) in ribosom... more We have investigated the possible involvement of the ubiquitin-proteasome system (UPS) in ribosome biogenesis. We find by immunofluorescence that ubiquitin is present within nucleoli and also demonstrate by immunoprecipitation that complexes associated with pre-rRNA processing factors are ubiquitinated. Using short proteasome inhibition treatments, we show by fluorescence microscopy that nucleolar morphology is disrupted for some but not all factors involved in ribosome biogenesis. Interference with proteasome degradation also induces the accumulation of 90S preribosomes, alters the dynamic properties of a number of processing factors, slows the release of mature rRNA from the nucleolus, and leads to the depletion of 18S and 28S rRNAs. Together, these results suggest that the UPS is probably involved at many steps during ribosome biogenesis, including the maturation of the 90S preribosome.

Nucleus, Nov 2, 2015

L ocalized chromatin organization is now recognized as an important determinant of cell identity ... more L ocalized chromatin organization is now recognized as an important determinant of cell identity and developmental pathways. Recent studies have demonstrated that these epigenetic states are unexpectedly dynamic and malleable. In this Extra view we will highlight the transient nature of stimulus-induced enhancer accessibility and its importance for transcription regulation. Using glucocorticoid receptor (GR) as a model system we will discuss spatiotemporal relationships between receptor/chromatin interactions, lifetimes of the DNase I hypersensitivity sites (DHSs), long-range interactions, and gene regulation. We propose that differential temporal activation and utilization of distal regulatory elements plays a role in directing divergent stimulus-induced transcriptional programs. Dynamic Interactions of TFs with Regulatory Elements It is becoming apparent that transcription regulation is comprised of many rapid and time-sensitive molecular processes. 11-15 To integrate various temporal signals in coherent transcriptional responses, transcription factors (TFs), target chromatin sites, and the transcriptional machinery must detect, adapt, and respond to these changes. It is well established that in vivo the majority of TFs interact with chromatin targets only transiently. 12,16-18 Several implications for these dynamic interactions were proposed: (i) regulation of the transcriptional output, 19,20 (ii) "sensing" fluctuations in the

Mutation Research, Aug 1, 2001

Of the three isomers of the aromatic amine phenylenediamine (PDA), only o-PDA, but not m-and p-PD... more Of the three isomers of the aromatic amine phenylenediamine (PDA), only o-PDA, but not m-and p-PDA, induced DNA damage (as measured by the Comet assay), and somatic mutations in the leaves of the chlorophyll-deficient tester strain Nicotiana tabacum var. xanthi. With increasing light intensity (0, 30, 80 or 140 mol m −2 s −1 photosynthetic photon fluence rate) during a 72 h mutagenic treatment of tobacco seedlings, o-PDA-induced DNA damage and the yield of somatic mutations were significantly increased. The peroxidase inhibitor diethyldithiocarbamate (DEDTC) repressed o-PDA-induced DNA damage. The effect of light is caused by the light-dependent increase of peroxidase activity and the accumulation of hydrogen peroxide, which participate in the metabolic activation of the promutagen o-PDA to mutagenic product(s). In contrast, DNA damage induced by the direct-acting alkylating mutagen ethyl methanesulphonate was the same whether treatment was in the light or in the dark, and was not repressed by the peroxidase inhibitor DEDTC.

Journal of Biological Chemistry, Dec 1, 2009

Biophysical Journal, Aug 1, 2006

Cells contain many subcellular structures in which specialized proteins locally cluster. Binding ... more Cells contain many subcellular structures in which specialized proteins locally cluster. Binding interactions within such clusters may be analyzed in live cells using models for fluorescence recovery after photobleaching (FRAP). Here we analyze a threedimensional FRAP model that accounts for a single spatially localized cluster of binding sites in the presence of both diffusion and impermeable boundaries. We demonstrate that models completely ignoring the spatial localization of binding yield poor estimates for the binding parameters within the binding site cluster. In contrast, we find that ignoring only the restricted axial height of the bindingsite cluster is far less detrimental, thereby enabling the use of computationally less expensive models. We also identify simplified solutions to the FRAP model for limiting behaviors where either diffusion or binding dominate. We show how ignoring a role for diffusion can sometimes produce serious errors in binding parameter estimation. We illustrate application of the method by analyzing binding of a transcription factor, the glucocorticoid receptor, to a tandem array of mouse mammary tumor virus promoter sites in live cells, obtaining an estimate for an in vivo binding constant (10 ÿ7 M), and a first approximation of an upper bound on the transcriptionfactor residence time at the promoter (;170 ms). These FRAP analysis tools will be important for measuring key cellular binding parameters necessary for a complete and accurate description of the networks that regulate cellular behavior.

Annual Review of Cell and Developmental Biology, Aug 4, 2023

Cells must tightly regulate their gene expression programs and yet rapidly respond to acute bioch... more Cells must tightly regulate their gene expression programs and yet rapidly respond to acute biochemical and biophysical cues within their environment. This information is transmitted to the nucleus through various signaling cascades, culminating in the activation or repression of target genes. Transcription factors (TFs) are key mediators of these signals, binding to specific regulatory elements within chromatin. While live-cell imaging has conclusively proven that TF–chromatin interactions are highly dynamic, how such transient interactions can have long-term impacts on developmental trajectories and disease progression is still largely unclear. In this review, we summarize our current understanding of the dynamic nature of TF functions, starting with a historical overview of early live-cell experiments. We highlight key factors that govern TF dynamics and how TF dynamics, in turn, affect downstream transcriptional bursting. Finally, we conclude with open challenges and emerging technologies that will further our understanding of transcriptional regulation. Expected final online publication date for the Annual Review of Cell and Developmental Biology, Volume 39 is October 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Histochemistry and Cell Biology, Nov 17, 2005

Photobleaching technology has demonstrated in live cells that the glucocorticoid receptor (GR) ex... more Photobleaching technology has demonstrated in live cells that the glucocorticoid receptor (GR) exchanges rapidly at the mouse mammary tumor virus (MMTV) promoter. GR rapid exchange at MMTV depends on chaperone and proteasome activity, and as suggested by several in vitro and in vivo biochemical approaches, may also involve chromatin remodeling activity. Inhibition of H1 phosphorylation, chromatin remodeling and transcription from MMTV can be accomplished by long-term blocking of Cdk2 protein kinase activity. We find that Cdk2 is recruited by a tandem array of MMTV promoters, strengthening the model that this kinase has a specific role in MMTV transcription. We also demonstrate that following a brief Cdk2 inhibition by a selective cyclin-dependent kinase inhibitor (Roscovitine), transcription from MMTV drops and GR exchange at MMTV becomes slower, with a fraction of GR molecules now tightly bound at the promoter. This immobile fraction is absent elsewhere in the nucleus, suggesting a specific effect of Cdk2 inhibition on GR-MMTV interactions. These are the first live cell data suggesting a role for H1 phosphorylation, and by implication chromatin remodeling, in rapid exchange of GR at MMTV.

Mutation Research, Feb 1, 2002

The level of hydrogen peroxide (H 2 O 2)-induced genomic DNA damage measured by the Comet assay i... more The level of hydrogen peroxide (H 2 O 2)-induced genomic DNA damage measured by the Comet assay in tobacco suspension cells (TX1) increased as a function of the age of the culture. After treatment of TX1 cells with 15 mM H 2 O 2 , the average (±S.E.) median tail moment value was only 4.85 ± 1.00 m in nuclei isolated from 2-day-old cells compared to 72.33 ± 1.40 m in nuclei isolated from 12-day-old cells. By contrast, nuclei first isolated from 2 and 12-day-old cells and then treated with H 2 O 2 , expressed the same level of DNA damage. The activity of catalases was markedly higher in 2-day-old TX1 cells compared to 12-day-old cells. The results indicate that the reaction of the H 2 O 2 with nuclear DNA is modified by the presence of the plant cell wall, and enzymes and macromolecules present in the cytosol, and is not connected with changes in the nuclear DNA sensitivity during cell suspension growth.

Mutation Research, Oct 1, 2000

We employed single cell gel electrophoresis to analyze the kinetics of DNA repair in nuclei isola... more We employed single cell gel electrophoresis to analyze the kinetics of DNA repair in nuclei isolated from tobacco plants exposed to ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU) and ␥-radiation. DNA repair was measured as the reduction of the tail moment values as a function of time after the mutagen treatment ended. DNA damage in leaf nuclei of EMS-or ENU-treated tobacco plants persisted over a 72 h recovery period. However, a reduction of the SCGE tail moment values in nuclei isolated from leaves was observed over a 4-week period of recovery. Newly emerged leaves expressed a lower level of DNA damage due to more efficient repair and/or dilution of initial DNA lesions during cell division. After 24 h recovery, leaf nuclei from cells exposed to 20 or 40 Gy of ␥-radiation expressed complete DNA repair. These data indicate that DNA lesions induced by alkylating agents are not readily repaired and persist beyond 4 weeks. Enzymes necessary to repair ␥-induced DNA lesions are fully functional in non-replicating leaf cells and single and double strand breaks are rapidly repaired.

Biophysical Journal, Feb 1, 2021

pre-dimeric b 2-AR was almost completely disrupted by inverse agonists, ICI-118,551 and carazolol... more pre-dimeric b 2-AR was almost completely disrupted by inverse agonists, ICI-118,551 and carazolol. We confirmed that the pre-dimeric b 2-AR transiently interacts with a Gas protein, which indicates that the b 2-AR dimer controls the basal activity of b 2-AR signaling. Finally, we demonstrated that cholesterol on the plasma membrane is a key to the production of the basal activity by directly modulating the formation of b 2-AR dimers. The effect on b 2-AR dimers made by cholesterol depletion was similar to that by inverse agonists, suggesting that the inverse agonists restore the abnormal activity of b 2-AR by an increased cholesterol level on the plasma membrane. Our results not only provide the molecular basis of the inverse agonism of GPCRs, but also implicate the inverse agonists in the effective treatment to the GPCR-derived diseases under an increased cholesterol level.

Environmental health perspectives, Aug 17, 2016

Introduction: Some natural and anthropogenic substances in drinking water sources are known or su... more Introduction: Some natural and anthropogenic substances in drinking water sources are known or suspected endocrine disrupting chemicals (EDC), but few are measured in U.S. public water supplies (PW...