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, 1 (3), e37

Genetic Interactions Due to Constitutive and Inducible Gene Regulation Mediated by the Unfolded Protein Response in C. Elegans

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Genetic Interactions Due to Constitutive and Inducible Gene Regulation Mediated by the Unfolded Protein Response in C. Elegans

Xiaohua Shen et al. PLoS Genet.

Abstract

The unfolded protein response (UPR) is an adaptive signaling pathway utilized to sense and alleviate the stress of protein folding in the endoplasmic reticulum (ER). In mammals, the UPR is mediated through three proximal sensors PERK/PEK, IRE1, and ATF6. PERK/PEK is a protein kinase that phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 to inhibit protein synthesis. Activation of IRE1 induces splicing of XBP1 mRNA to produce a potent transcription factor. ATF6 is a transmembrane transcription factor that is activated by cleavage upon ER stress. We show that in Caenorhabditis elegans, deletion of either ire-1 or xbp-1 is synthetically lethal with deletion of either atf-6 or pek-1, both producing a developmental arrest at larval stage 2. Therefore, in C. elegans, atf-6 acts synergistically with pek-1 to complement the developmental requirement for ire-1 and xbp-1. Microarray analysis identified inducible UPR (i-UPR) genes, as well as numerous constitutive UPR (c-UPR) genes that require the ER stress transducers during normal development. Although ire-1 and xbp-1 together regulate transcription of most i-UPR genes, they are each required for expression of nonoverlapping sets of c-UPR genes, suggesting that they have distinct functions. Intriguingly, C. elegans atf-6 regulates few i-UPR genes following ER stress, but is required for the expression of many c-UPR genes, indicating its importance during development and homeostasis. In contrast, pek-1 is required for induction of approximately 23% of i-UPR genes but is dispensable for the c-UPR. As pek-1 and atf-6 mainly act through sets of nonoverlapping targets that are different from ire-1 and xbp-1 targets, at least two coordinated responses are required to alleviate ER stress by distinct mechanisms. Finally, our array study identified the liver-specific transcription factor CREBh as a novel UPR gene conserved during metazoan evolution.

Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The Sequence Alignment of C. elegans, Human, and Murine ATF6 Homologs
The sequence alignment of C. elegans (ce), human (hs), and mouse (ms) shows five conserved regions in worm ATF-6 including a serine-rich domain, a bZIP domain, a transmembrane domain, and two C-terminal homology regions (HR-I and HR-II). Blue indicates residues that are conservative across species, green indicates blocks of similar residues, yellow indicates identical residues, and grey indicates weak similarity.
Figure 2
Figure 2. C. elegans atf-6 and pek-1 Display Partially Redundant Roles in Complementing ire-1/xbp-1 for Larval Survival and Development
(A) Characterization of C. elegans atf-6 and its ok551 deletion allele. The atf-6 gene structure is depicted in boxes and lines, representing exons and introns, respectively. The atf-6(ok551) allele lacks 1,900 bp of genomic sequence, and has the potential to encode a protein without the leucine zipper portion of the bZIP domain, the transmembrane domain, and ER lumenal domain. The atf-6(ok551) deletion allele can be detected by PCR, using the primers indicated by arrows. (B) Genetic interactions of atf-6, ire-1, and pek-1. Animals with the genotype ire-1(v33); atf-6(ok551) arrested as young larvae, showing that loss of both ire-1 and atf-6 is lethal. The ire-1(v33)/ mnC1; atf-6(ok551)/ pek-1(ok275) animals (P0) segregated healthy F1 progeny with the genotype ire-1(v33); atf-6(ok551)/ pek-1(ok275), which in turn produced dead F2 animals with exactly the same genotype, suggesting that ATF-6 and PEK-1 function synergistically to cope with endogenous ER stress during development. (C) Nomarski micrograph of a 3-d-old atf-6(RNAi); ire-1(v33) animal. The germline of this animal did not develop past the L2 larval stage. (D) Comparisons of intestinal degeneration in various double mutants: (i) ire-1(v33); pek-1(ok275), (ii) xbp-1(RNAi); pek-1(ok275), (iii) ire-1(v33); atf-6(RNAi), and (iv) atf-6(RNAi); pek-1(ok275). Normaski micrographs show a portion of the intestine. Mutants in (i)–(iii) arrested at the L2 larval stage and showed intestinal degeneration. The mutants in (iv) had an intestinal morphology similar to the wild-type. Yellow arrows indicate vacuoles in intestinal cells. Red arrowheads indicate light-reflective aggregates appearing in some mutants ([ii]and [iii]).
Figure 3
Figure 3. Transcriptional Targets of ire-1, xbp-1, pek-1, and atf-6
(A) Venn diagram showing the sets of i-UPR genes regulated by ire-1, xbp-1, pek-1, and atf-6. (B) Venn diagram showing the sets of c-UPR genes regulated by ire-1, xbp-1, pek-1, and atf-6.
Figure 4
Figure 4. Complex UPR Transcriptional Regulation of Genes with Known Functions in C. elegans
(A) The i-UPR pathway. Many conditions such as exogenous drug treatment, nutrient deprivation, viral infection, or protein overexpression block or overwhelm protein-folding reactions in the ER and result in ER stress. In this study, we used tunicamycin to block protein folding so as to activate the UPR. Following ER stress, ire-1 and xbp-1 act in a linear pathway that dominates the transcriptional response (total of 139 target genes with known functions), inducing genes that reshape the secretory pathway, adjust the metabolic profile, up-regulate functions involved in calcium homeostasis and anti-oxidative stress, and regulate other genes that might affect cell fate. Interestingly, ten genes require either ire-1 or xbp-1, but not both, for their induction upon ER stress. About 21 genes require only pek-1 for maximal induction, and eight genes regulated by ire-1/xbp-1 also share regulation by pek-1. Finally, atf-6 does not play a significant role in the i-UPR pathway (depicted by broken arrow). In addition to two genes that were also regulated by ire-1/xbp-1, the only gene that depends solely on atf-6 for its induction is cht-1, which encodes a chitinase orthologous to human chitotriosidase. (B) The c-UPR pathway. During development, active protein synthesis and secretion require the UPR signaling molecules ire-1, xbp-1, and atf-6 to maintain the expression of c-UPR genes, defined by the fact that they are not up-regulated by tunicamycin but are dependent on ER stress transducers for expression. Among the genes with known functions, there are only 12 that overlap between the set of 45 genes regulated by ire-1 and the set of 160 genes regulated by xbp-1. In addition, atf-6 is required by nine genes that are regulated by ire-1 and 35 that are genes regulated by xbp-1. Moreover, the expression of 19 genes is solely dependent on atf-6, suggesting an important role of atf-6 in the c-UPR pathway. By contrast, pek-1 is largely dispensable for regulation of the c-UPR as only nine genes require pek-1 in addition to their requirements for ire-1 to maintain basal expression.
Figure 5
Figure 5. CREBh Is a Novel UPR-Responsive Gene
(A) Microarray and quantitative RT-PCR analyses show that the expression of C. elegans (ce) CREBh requires ire-1, xbp-1, and atf-6. “Tuni” indicates tunicamycin treatment, as described in the Materials and Methods section. (B) ER stress induced by dithiothreitol in HepG2 cells activates CREBh transcription. HepG2 cells were treated with dithiothreitol and harvested at various time points from 0 h to 8 h. The relative expression of CREBh and spliced xbp-1 transcripts (Xbp1s) was analyzed by quantitative RT-PCR and normalized to GADPH. The induction pattern of CREBh resembles that of spliced xbp-1 transcripts.

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