Diana A. Stavreva

Ultradian hormone stimulation induces glucocorticoid receptor-mediated pulses of gene transcription.

Studies on glucocorticoid receptor (GR) action typically assess gene responses by long-term stimulation with synthetic hormones. As corticosteroids are released from adrenal glands in a circadian and high-frequency (ultradian) mode, such treatments may not provide an accurate assessment of physiological hormone action. Here we demonstrate that ultradian hormone stimulation induces cyclic GR-mediated transcriptional regulation, or gene pulsing, both in cultured cells and in animal models. Equilibrium receptor-occupancy of regulatory elements precisely tracks the ligand pulses. Nascent RNA transcripts from GR-regulated genes are released in distinct quanta, demonstrating a profound difference between the transcriptional programs induced by ultradian and constant stimulation. Gene pulsing is driven by rapid GR exchange with response elements and by GR recycling through the chaperone machinery, which promotes GR activation and reactivation in response to the ultradian hormone release, thus coupling promoter activity to the naturally occurring fluctuations in hormone levels. The GR signalling pathway has been optimized for a prompt and timely response to fluctuations in hormone levels, indicating that biologically accurate regulation of gene targets by GR requires an ultradian mode of hormone stimulation.

Rapid glucocorticoid receptor exchange at a promoter is coupled to transcription and regulated by chaperones and proteasomes.

ABSTRACT Exchange of the glucocorticoid receptor (GR) at promoter target sites provides the only known system in which transcription factor cycling at a promoter is fast, occurring on a time scale of seconds. The mechanism and function of this rapid exchange are unknown. We provide evidence that proteasome activity is required for rapid GR exchange at a promoter. We also show that chaperones, specifically hsp90, stabilize the binding of GR to the promoter, complicating models in which the associated chaperone, p23, has been proposed to induce GR removal. Our results are the first to connect chaperone and proteasome functions in setting the residence time of a transcription factor at a target promoter. Moreover, our results reveal that longer GR residence times are consistently associated with greater transcriptional output, suggesting a new paradigm in which the rate of rapid exchange provides a means to tune transcriptional levels.

Potential roles for ubiquitin and the proteasome during ribosome biogenesis.

ABSTRACT 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.

Falling off the academic bandwagon. Women are more likely to quit at the postdoc to principal investigator transition

Women constitute approximately 45% of the postdoctoral fellows in the biomedical sciences at universities and research institutions in the uSa, but a much lower percent age of women hold faculty positions. in the uS national institutes of Health (niH; Bethesda, MD) intramural research program, for example, women make up only 29% of the tenure-track investiga tors and hold just 19% of the tenured sen ior investigator appointments. a similar disparity between the ratio of men and women in independent faculty positions exists in most academic institutions across the uSa (nelson, 2005; nSF, 2004, 2006), and statistics from Europe show a similar trend of women disappearing from the higher echelons of academia (E c, 2006). the transition from postdoctoral fellow to faculty is a period during which a wor rying number of women leave academic research. Several recent surveys have tried to identify factors that lead to the attrition of women from the life sciences and engi

Trans-regulation of Histone Deacetylase Activities through Acetylation

HDAC1 and -2 are highly conserved enzymes and often coexist in the same coregulator complexes. Understanding the regulation of histone deacetylase activities is extremely important because these enzymes play key roles in epigenetic regulation in normal and cancer cells. We previously showed that HDAC1 is required for glucocorticoid receptor-mediated transcription activation and that its activity is regulated through acetylation by p300 during the induction cycle. Here, we showed that HDAC2 is also required for glucocorticoid receptor-mediated gene activation. HDAC2, however, is regulated through a different mechanism from that of HDAC1. HDAC2 is not acetylated by p300, although 5 of 6 acetylated lysine residues in HDAC1 are also present in HDAC2. More importantly, the activity of HDAC2 is inhibited by acetylated HDAC1. Additionally, we showed that acetylated HDAC1 can trans-regulate HDAC2 through heterodimerization. Thus, this study uncovered fundamental differences between HDAC1 and HDAC2. It also unveiled a new mechanism of collaborative regulation by HDAC1/2 containing coregulator complexes.

Dynamics of chromatin accessibility and long-range interactions in response to glucocorticoid pulsing

Although physiological steroid levels are often pulsatile (ultradian), the genomic effects of this pulsatility are poorly understood. By utilizing glucocorticoid receptor (GR) signaling as a model system, we uncovered striking spatiotemporal relationships between receptor loading, lifetimes of the DNase I hypersensitivity sites (DHSs), long-range interactions, and gene regulation. We found that hormone-induced DHSs were enriched within ± 50 kb of GR-responsive genes and displayed a broad spectrum of lifetimes upon hormone withdrawal. These lifetimes dictate the strength of the DHS interactions with gene targets and contribute to gene regulation from a distance. Our results demonstrate that pulsatile and constant hormone stimulations induce unique, treatment-specific patterns of gene and regulatory element activation. These modes of activation have implications for corticosteroid function in vivo and for steroid therapies in various clinical settings.

Fluorescence recovery after photobleaching (FRAP) methods for visualizing protein dynamics in living mammalian cell nuclei.

Publisher Summary Fluorescence recovery after photobleaching (FRAP) is an optical technique used to measure the temporal dynamics of fluorescently tagged molecules. FRAP is a relatively old technique. This renaissance is driven largely by the advent of confocal microscopy and the introduction of green fluorescent protein (GFP) as an endogenous protein marker. After GFP (or another fluorophore) is covalently attached to the protein of interest, its cellular distribution can be visualized at low light intensities that do not damage cellular processes. Photobleaching is the irreversible destruction of fluorescence in a region within the sample by brief exposure to high light intensities. After bleaching a region, the recovery of fluorescence over time there can be recorded to measure the rate of redistribution of fluorescent molecules. This rate of fluorescence redistribution provides information about the processes involved in the movement of the molecule. The movement reflects diffusion, which can be retarded if the fluorescently tagged molecule binds to other molecules. In the latter case, the rate of redistribution of bleached and unbleached molecules contain information about the strength of the binding interaction. Thus, FRAP is a valuable technique to study molecular dynamics in live cells.

Prevalent Glucocorticoid and Androgen Activity in US Water Sources

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.

Complex Dynamics of Transcription Regulation

Transcription is a tightly regulated cellular function which can be triggered by endogenous (intrinsic) or exogenous (extrinsic) signals. The development of novel techniques to examine the dynamic behavior of transcription factors and the analysis of transcriptional activity at the single cell level with increased temporal resolution has revealed unexpected elements of stochasticity and dynamics of this process. Emerging research reveals a complex picture, wherein a wide range of time scales and temporal transcription patterns overlap to generate transcriptional programs. The challenge now is to develop a perspective that can guide us to common underlying mechanisms, and consolidate these findings. Here we review the recent literature on temporal dynamics and stochastic gene regulation patterns governed by intrinsic or extrinsic signals, utilizing the glucocorticoid receptor (GR)-mediated transcriptional model to illustrate commonality of these emerging concepts. This article is part of a Special Issue entitled: Chromatin in time and space.

Dynamic Interaction of HDAC1 with a Glucocorticoid Receptor-regulated Gene Is Modulated by the Activity State of the Promoter

Although histone deacetylases (HDACs) are normally considered as co-repressors, HDAC1 has been identified as a coactivator for the glucocorticoid receptor (GR) (Qiu, Y., Zhao, Y., Becker, M., John, S., Parekh, B. S., Huang, S., Hendarwanto, A., Martinez, E. D., Chen, Y., Lu, H., Adkins, N. L., Stavreva, D. A., Wiench, M., Georgel, P. T., Schiltz, R. L., and Hager, G. L. (2006) Mol. Cell 22, 669–679). Furthermore, HDAC1 is acetylated, and its acetylation level is linked to the transcription state of a GR-induced promoter (mouse mammary tumor virus). GR is also known to interact dynamically with regulatory elements in living cells (McNally, J. G., Müller, W. G., Walker, D., Wolford, R., and Hager, G. L. (2000) Science 287, 1262–1265). However, HDAC1 dynamics have never been studied. We demonstrate here that HDAC1 also exchanges rapidly with promoter chromatin, and its exchange rate is significantly modulated during the development of promoter activity. Prior to induction, HDAC1 mobility was retarded compared with the exchange rate for GR. HDAC1 mobility then increased substantially, coordinately with the peak of promoter activity. At later time points, promoter activity was severely repressed, and HDAC1 mobility returned to the rate of exchange observed for the uninduced promoter. Thus, alterations of the exchange rates of HDAC1 at the promoter are correlated with the activity state of the promoter. These findings provide direct evidence for the functional role of highly mobile transcription factor complexes in transcription regulation.