5 research outputs found
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
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
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 chi<sup>2</sup> 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 <sup>M</sup>) 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