Integration of the Unfolded Protein and Oxidative Stress Responses through SKN-1/Nrf

The Unfolded Protein Response (UPR) maintains homeostasis in the endoplasmic reticulum (ER) and defends against ER stress, an underlying factor in various human diseases. During the UPR, numerous genes are activated that sustain and protect the ER. These responses are known to involve the canonical UPR transcription factors XBP1, ATF4, and ATF6. Here, we show in C. elegans that the conserved stress defense factor SKN-1/Nrf plays a central and essential role in the transcriptional UPR. While SKN-1/Nrf has a well-established function in protection against oxidative and xenobiotic stress, we find that it also mobilizes an overlapping but distinct response to ER stress. SKN-1/Nrf is regulated by the UPR, directly controls UPR signaling and transcription factor genes, binds to common downstream targets with XBP-1 and ATF-6, and is present at the ER. SKN-1/Nrf is also essential for resistance to ER stress, including reductive stress. Remarkably, SKN-1/Nrf-mediated responses to oxidative stress depend upon signaling from the ER. We conclude that SKN-1/Nrf plays a critical role in the UPR, but orchestrates a distinct oxidative stress response that is licensed by ER signaling. Regulatory integration through SKN-1/Nrf may coordinate ER and cytoplasmic homeostasis

Identification and characterization of a salt stress-inducible zinc finger protein from Festuca arundinacea

<p>Abstract</p> <p>Background</p> <p>Increased biotic and abiotic plant stresses due to climate change together with an expected global human population of over 9 billion by 2050 intensifies the demand for agricultural production on marginal lands. Soil salinity is one of the major abiotic stresses responsible for reduced crop productivity worldwide and the salinization of arable land has dramatically increased over the last few decades. Consequently, as land becomes less amenable for conventional agriculture, plants grown on marginal soils will be exposed to higher levels of soil salinity. Forage grasses are a critical component of feed used in livestock production worldwide, with many of these same species of grasses being utilized for lawns, erosion prevention, and recreation. Consequently, it is important to develop a better understanding of salt tolerance in forage and related grass species.</p> <p>Findings</p> <p>A gene encoding a ZnF protein was identified during the analysis of a salt-stress suppression subtractive hybridization (SSH) expression library from the forage grass species <it>Festuca arundinacea</it>. The expression pattern of <it>FaZnF </it>was compared to that of the well characterized gene for delta 1-pyrroline-5-carboxylate synthetase (<it>P5CS</it>), a key enzyme in proline biosynthesis, which was also identified in the salt-stress SSH library. The <it>FaZnF </it>and <it>P5CS </it>genes were both up-regulated in response to salt and drought stresses suggesting a role in dehydration stress. <it>FaZnF </it>was also up-regulated in response to heat and wounding, suggesting that it might have a more general function in multiple abiotic stress responses. Additionally, potential downstream targets of FaZnF (a MAPK [Mitogen-Activated Protein Kinase], GST [Glutathione-S-Transferase] and lipoxygenase L2) were found to be up-regulated in calli overexpressing <it>FaZnF </it>when compared to control cell lines.</p> <p>Conclusions</p> <p>This work provides evidence that FaZnF is an AN1/A20 zinc finger protein that is involved in the regulation of at least two pathways initiated by the salt stress response, thus furthering our understanding of the mechanisms of cellular action during a stress that is applicable to commercial crops worldwide.</p

Specific SKN-1/Nrf Stress Responses to Perturbations in Translation Elongation and Proteasome Activity

Peer reviewe

Assessing Potential Effects of Highway and Urban Runoff on Receiving Streams in Total Maximum Daily Load Watersheds in Oregon Using the Stochastic Empirical Loading and Dilution Model

The Stochastic Empirical Loading and Dilution Model (SELDM) was developed by the U.S. Geological Survey (USGS) in cooperation with the Federal Highway Administration to simulate stormwater quality. To assess the effects of runoff, SELDM uses a stochastic mass-balance approach to estimate combinations of pre-storm streamflow, stormflow, highway runoff, event mean concentrations (EMCs) and stormwater constituent loads from a site of interest. In addition, SELDM can be used to assess the effects of stormwater Best Management Practices (BMPs), which are designed to mitigate the adverse effects of runoff into a waterbody

Association of SKN-1 with the ER.

<p>(A, B) Interaction between endogenous SKN-1 and HSP-3/4, detected by IP/Western. Lysates were prepared from animals in which proteins had been crosslinked under ChIP conditions. (A) Monoclonal αSKN-1 IP blotted with αHsc3 (HSP-3/4). (B) αHsc3 (HSP-3/4) IP blotted with monoclonal αSKN-1. (C–E) Analyses of ER fractions prepared from whole worms. The fractionation scheme is described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s006" target="_blank">Fig. S6B</a>. (C) Detection of endogenous HSP-3/4 and the cytoplasmic marker GAPDH in ER and Mitochondrial fractions, and total worm lysate. Note the enrichment of the ER marker HSP-3/4 compared to GAPDH in the ER fraction. TM indicates lysates from animals that had been treated with TM. (D) Presence of endogenous SKN-1 in the ER fraction, detected by western and IP/western blotting. Note that TM treatment increased the levels of SKN-1 protein. (E) Association between endogenous SKN-1 and HSP-3/4 within the ER fraction, detected with polyclonal αSKN-1 and αBiP (HSP-3/4), by IP/Western that was performed without crosslinking. Fractionations and analyses were performed independently twice, with similar results. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s006" target="_blank">Figure S6</a>.</p

SKN-1 directly regulates target genes during the UPR.

<p>(A–L) ER stress-induced SKN-1 recruitment and transcriptional activation was analyzed at the SKN-1-regulated genes <i>pcp-2</i> (A–D), <i>atf-5</i> (E–H), and <i>gst-4</i> (I–L). TM treatment leads to SKN-1 recruitment (A, E, I), accumulation of Pol II that is phosphorylated at CTD Ser 2 (P-Ser2) (B, F, J), decreased Histone H3 occupancy (C, G, K), and increased H3-AcK56 density (D, H, L) at the site of transcription. Maps mark qPCR amplicons relative to the predicted transcription start site, with exons marked as black boxes. % ChIP signal is relative to input, and normalized to the highest signal for each run <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701-GloverCutter1" target="_blank">[44]</a>. In (D, H, L), a ratio of acetyl histone to histone signal is presented. For ChIP experiments in this study error bars represent SEM, and * p≤.05, ** p≤.01, *** p≤.001, relative to <i>pL4440</i> Control calculated using one-sided student's t-test. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s002" target="_blank">Figure S2</a>.</p

ER stress activates SKN-1 independently of oxidative stress.

<p>(A) Treatment with TM (16 hrs), thapsigargin (Thap, 2 hrs), or bortezomib (6 hrs) increased <i>skn-1</i> mRNA levels, as determined by qRT-PCR. RNAi knockdown of <i>hsp-4</i> or <i>atf-6</i> also increased <i>skn-1</i> mRNA levels. (B) Increased endogenous SKN-1 protein levels in response to TM-induced ER stress. SKN-1 was detected by Western blotting with the polyclonal antibody, with GAPDH serving as the loading control. (C) Induction of <i>skn-1</i> expression and SKN-1-regulated UPR target genes by reductive ER stress (DTT treatment for 2 hrs), assayed by qRT-PCR. (D) Induction of the UPR, <i>skn-1</i> expression, and SKN-1 target genes by <i>ero-1</i> RNAi, assayed by qRT-PCR. Different primer sets were used to distinguish among mRNAs that correspond to different <i>skn-1</i> isoforms. Error bars represent SEM, and * p≤.05, ** p≤.01, *** p≤.001, relative to <i>pL4440</i> Control calculated using student's t-test. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s004" target="_blank">Figure S4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s010" target="_blank">Table S3</a>.</p

SKN-1 regulates diverse functions in response to ER stress.

<p>(A, B) ER stress induces <i>skn</i>-1-dependent activation of ER- or UPR-associated genes. qRT-PCR was performed after RNAi Control (<i>pL4440</i> in all panels) or <i>skn-1</i> RNAi, and Control or 5 µg/ml TM treatment. Known or predicted functions of these genes are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s008" target="_blank">Table S1</a>. Genes are grouped in (A) or (B) according to the extent of TM-induced activation, and plotted on different scales. All analyses of TM-regulated gene expression involved a 16 hr TM treatment, based upon a time-course experiment (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s001" target="_blank">Figure S1B</a>) and published work in <i>C. elegans </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701-Harding1" target="_blank">[15]</a>. Shorter time courses were chosen for other ER stress treatments (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen-1003701-g004" target="_blank">Figure 4</a>, legend). (C) Upregulation of SKN-1-regulated oxidative stress defense genes in response to TM. Error bars represent SEM, * p≤.05, ** p≤.01, *** p≤.001, relative to <i>pL4440</i> Control. All qRT-PCR p-values were calculated as one or two-sided t-test as appropriate with n≥3. (D) Activation of the <i>gcs-1</i>::<i>GFP</i> transgene in the intestine, with GFP expression scored as High, Medium, or Low. *** p<.0001 chi² method. See Experimental Procedures for scoring method. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s001" target="_blank">Figure S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s008" target="_blank">Table S1</a>.</p

UPR factors required for ER stress-induced SKN-1 activation.

<p>(A) ER stress-induced activation of <i>skn-1</i> and its target genes requires core UPR factors. RNA levels were assayed by qRT-PCR after RNAi against core UPR genes or in core UPR factor mutants (indicated by ^M) after TM treatment. (B-E) IRE-1 is required for ER stress-induced SKN-1 accumulation and activity at SKN-1 target genes <i>gst-4</i> and <i>pcp-2</i>. Presence of SKN-1 and transcription markers was assayed by ChIP as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen-1003701-g002" target="_blank">Figure 2</a>, and <i>ire-1</i> was knocked down by RNAi. (F–H) Endogenous XBP-1 (F), ATF-6 (G), and SKN-1 (H) bind within the <i>skn-1</i> gene locus in response to TM-induced ER stress, with binding assayed by ChIP. Multiple start sites are noted within the <i>skn-1</i> locus. Error bars represent SEM, and * p≤.05, ** p≤.01, *** p≤.001 by student's t-test, relative to <i>pL4440</i> Control unless otherwise indicated. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s005" target="_blank">Figure S5</a>.</p

TFIIH-Associated Cdk7 Kinase Functions in Phosphorylation of C-Terminal Domain Ser7 Residues, Promoter-Proximal Pausing, and Termination by RNA Polymerase II▿ †

The function of human TFIIH-associated Cdk7 in RNA polymerase II (Pol II) transcription and C-terminal domain (CTD) phosphorylation was investigated in analogue-sensitive Cdk7as/as mutant cells where the kinase can be inhibited without disrupting TFIIH. We show that both Cdk7 and Cdk9/PTEFb contribute to phosphorylation of Pol II CTD Ser5 residues on transcribed genes. Cdk7 is also a major kinase of CTD Ser7 on Pol II at the c-fos and U snRNA genes. Furthermore, TFIIH and recombinant Cdk7-CycH-Mat1 as well as recombinant Cdk9-CycT1 phosphorylated CTD Ser7 and Ser5 residues in vitro. Inhibition of Cdk7 in vivo suppressed the amount of Pol II accumulated at 5′ ends on several genes including c-myc, p21, and glyceraldehyde-3-phosphate dehydrogenase genes, indicating reduced promoter-proximal pausing or polymerase “leaking” into the gene. Consistent with a 5′ pausing defect, Cdk7 inhibition reduced recruitment of the negative elongation factor NELF at start sites. A role of Cdk7 in regulating elongation is further suggested by enhanced histone H4 acetylation and diminished histone H4 trimethylation on lysine 36—two marks of elongation—within genes when the kinase was inhibited. Consistent with a new role for TFIIH at 3′ ends, it was detected within genes and 3′-flanking regions, and Cdk7 inhibition delayed pausing and transcription termination