Recruitment Timing and Dynamics of Transcription Factors at the Hsp70 Loci in Living Cells

Zobeck, Katie L.; Buckley, Martin S.; Zipfel, Warren R.; Lis, John T.

doi:10.1016/j.molcel.2010.11.022

Cited by 124 publications

(132 citation statements)

References 52 publications

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“…The rate of Pol II escape into elongation rapidly increases after the heat-shock induction. This increase is equal to the initial rate of Pol II recruitment to the activated Hsp70, which was measured previously using a high-temporal-resolution recruitment study of Pol II in living cells (Zobeck et al 2010), and here we estimate this rate to be about six molecules per minute per promoter (Fig. 4A).…”

supporting

confidence: 73%

“…However, the paused Pol II is known to be phosphorylated at Ser5, and it is the unphosphorylated form of Pol II that is used in the reinitiation step (Laybourn and Dahmus 1990). Additionally, we observed previously in FRAP assays that, during the early phases of induction of the Hsp70 gene, Pol II dissociates from the locus rather than recycling and only partially recycles after accumulating at the locus at high concentrations (Yao et al 2007;Zobeck et al 2010). In addition, we further provide orthogonal, biochemical evidence of the stability of paused Pol II using nascent RNA, which can be assessed independently from recycling.…”

Section: Org Downloaded Frommentioning

confidence: 87%

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Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights into its regulation

Buckley¹,

Kwak²,

Zipfel

et al. 2014

Genes Dev.

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The kinetics with which promoter-proximal paused RNA polymerase II (Pol II) undergoes premature termination versus productive elongation is central to understanding underlying mechanisms of metazoan transcription regulation. To assess the fate of Pol II quantitatively, we tracked photoactivatable GFP-tagged Pol II at uninduced Hsp70 on polytene chromosomes and showed that Pol II is stably paused with a half-life of 5 min. Biochemical analysis of short nascent RNA from Hsp70 reveals that this half-life is determined by two comparable rates of productive elongation and premature termination of paused Pol II. Importantly, heat shock dramatically increases elongating Pol II without decreasing termination, indicating that regulation acts at the step of paused Pol II entry to productive elongation.Supplemental material is available for this article.Received September 30, 2013; revised version accepted December 3, 2013. Many metazoan genes have a high occupancy of transcriptionally engaged RNA polymerase II (Pol II) paused near their promoters (Bentley and Groudine 1986;Rougvie and Lis 1988;Muse et al. 2007;Zeitlinger et al. 2007;Core et al. 2008). Increasingly, studies indicate that the transition of this promoter-proximal Pol II into productive elongation is one of the major regulatory checkpoints of gene expression. Previous studies hypothesized that the accumulated Pol II at the promoter is either stably paused or iteratively terminating prematurely during early elongation (Bentley and Groudine 1986;Rougvie and Lis 1988). Interestingly, recent reports indicate that both pausing (DSIF and NELF) (Wu et al. 2003; Rahl et al. 2010) and termination (Dcp1a, Xrn2, and TTF2) (Brannan et al. 2012) factors are enriched at metazoan promoters, and their depletions can alter the distribution of Pol II. Therefore, the relative contribution of pausing or premature termination to promoter Pol II accumulation and its regulation is a central question that has yet to be quantitatively addressed in vivo.The Drosophila Hsp70 heat-shock gene possesses a promoter-proximal Pol II that has been extensively characterized (Fuda et al. 2009). Under basal conditions, Pol II on Hsp70 pauses at sites 20-40 base pairs (bp) downstream from the transcription start site (Rasmussen and Lis 1993;Kwak et al. 2013), producing an accumulation of Pol II at the 59 end of the gene and a basal distribution of Pol II along the gene body (Lis 1998). Upon heat-shock induction, the escape of paused Pol II to productive elongation as well as the initiation rate increase up to 100-fold, leading to the massive production of Hsp70 mRNA.High-resolution live-cell imaging of the polytene chromosomes in Drosophila salivary glands provides one method to analyze the kinetics of Pol II induction and elongation during the heat-shock activation of Hsp70 (Darzacq et al. 2009). The heat-shock-activated endogenous Hsp70 loci (87A/C) can be easily located in living polytene nuclei because they produce a distinct doublet of intense GFP-Pol II (or RFP)-containing puffs after t...

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supporting

confidence: 73%

Section: Org Downloaded Frommentioning

confidence: 87%

Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights into its regulation

Buckley¹,

Kwak²,

Zipfel

et al. 2014

Genes Dev.

Self Cite

View full text Add to dashboard Cite

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“…Furthermore, live-cell imaging and fluorescence recovery after photobleaching (FRAP) analyses in heatshocked Drosophila salivary gland nuclei have captured the progressive recruitment of HSF, RNA polymerase II, and transcription elongation factors to the heat-shock loci to form a so-called ''transcription compartment'' at the Hsp70 locus. dPARP activity is essential for the maintenance of this transcription compartment (Zobeck et al 2010). The PAR-dependent assembly of the transcription compartment provides new perspectives to PARP's function in chromatin-dependent transcriptional regulation.…”

Section: Signaling To Chromatin Through Parmentioning

confidence: 99%

On PAR with PARP: cellular stress signaling through poly(ADP-ribose) and PARP-1

Luo¹,

Kraus²

2012

Genes Dev.

624

607

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Cellular stress responses are mediated through a series of regulatory processes that occur at the genomic, transcriptional, post-transcriptional, translational, and posttranslational levels. These responses require a complex network of sensors and effectors from multiple signaling pathways, including the abundant and ubiquitous nuclear enzyme poly(ADP-ribose) (PAR) polymerase-1 (PARP-1). PARP-1 functions at the center of cellular stress responses, where it processes diverse signals and, in response, directs cells to specific fates (e.g., DNA repair vs. cell death) based on the type and strength of the stress stimulus. Many of PARP-1's functions in stress response pathways are mediated by its regulated synthesis of PAR, a negatively charged polymer, using NAD + as a donor of ADP-ribose units. Thus, PARP-1's functions are intimately tied to nuclear NAD + metabolism and the broader metabolic profile of the cell. Recent studies in cell and animal models have highlighted the roles of PARP-1 and PAR in the response to a wide variety of extrinsic and intrinsic stress signals, including those initiated by oxidative, nitrosative, genotoxic, oncogenic, thermal, inflammatory, and metabolic stresses. These responses underlie pathological conditions, including cancer, inflammation-related diseases, and metabolic dysregulation. The development of PARP inhibitors is being pursued as a therapeutic approach to these conditions. In this review, we highlight the newest findings about PARP-1's role in stress responses in the context of the historical data.Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) is the most abundant and ubiquitous member of a family of 17 related mammalian proteins. Studies over the past two decades, complemented by some exciting recent reports, have begun to crystallize a clearer view of one of the most robust and consistent functions of PARP-1 across biological systems: as a stress sensor and a stress response mediator. In this review, we highlight the newest findings in this area in the context of the historical data. We did not attempt to provide a comprehensive review, but rather opted to focus on key findings that have clarified the role of PARP-1 (and related PARPs) in stress responses. The reader is directed to a variety of other excellent reviews that cover other aspects of PARP-1 biology (D

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“…Effective RNA PolII elongation along the Hsp70 gene is eased by nucleosome remodeling complexes, facilitates transcription (FACT, also known as SSRP1) and suppressor of Ty 6 (SPT6, SPT6H in mammals), as well as by topoisomerase I, which relieves DNA coiling (Andrulis et al, 2000;Gilmour et al, 1986;Kaplan et al, 2000). Finally, poly(ADP-ribose) polymerase (PARP) generates a compartment that selectively retains and recycles transcriptional components (see poster; Petesch and Lis, 2008;Zobeck et al, 2010).…”

Section: Hsf1-mediated Transactivation Of Heat Shock Genesmentioning

confidence: 99%

HSF1 at a glance

Vihervaara

Sistonen

2014

Journal of Cell Science

263

216

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Heat shock factor 1 (HSF1) is an evolutionarily highly conserved transcription factor that coordinates stress-induced transcription and directs versatile physiological processes in eukaryotes. The central position of HSF1 in cellular homeostasis has been well demonstrated, mainly through its strong effect in transactivating genes that encode heat shock proteins (HSPs). However, recent genome-wide studies have revealed that HSF1 is capable of reprogramming transcription more extensively than previously assumed; it is also involved in a multitude of processes in stressed and non-stressed cells. Consequently, the importance of HSF1 in fundamental physiological events, including metabolism, gametogenesis and aging, has become apparent and its significance in pathologies, such as cancer progression, is now evident. In this Cell Science at a Glance article, we highlight recent advances in the HSF1 field, discuss the organismal control over HSF1, and present the processes that are mediated by HSF1 in the context of cell type, cell-cycle phase, physiological condition and received stimuli.

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Recruitment Timing and Dynamics of Transcription Factors at the Hsp70 Loci in Living Cells

Cited by 124 publications

References 52 publications

Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights into its regulation

Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights into its regulation

On PAR with PARP: cellular stress signaling through poly(ADP-ribose) and PARP-1

HSF1 at a glance

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