1 Supporting information Global assessment of the integrated stress response in CF patient-derived airway and intestinal tissues Giovana B. Bampi, Robert Rauscher, Sebastian Kirchner, Kathryn E. Oliver, Marcel J.C. Bijvelds, Leonardo A. Santos, Johannes Wagner, Raymond A. Frizzell, Hugo R. de Jonge, Eric J. Sorscher, Zoya Ignatova Supporting text S1.1 Primary airway epithelia from three CF patients display ATF6-driven activation of the integrated stress response. The integrated unfolded protein response (UPR) describes coordinated relationships between three parallel but distinct cellular pathways, each of which initiates at different times through three classical endoplasmic reticulum (ER)-resident transmembrane protein sensors: (1) inositol-requiring protein 1 (IRE1); (2) protein kinase RNA (PRK)-like ER kinase (PERK); and (3) activating transcription factor (ATF)-6 [1]. In response to external stimuli, IRE1 and PERK undergo oligomerization and autophosphorylation, whereas ATF6 is translocated to the Golgi and then activated via proteolytic cleavage [2,3]. Following two sequential endonucleolytic cleavages, IRE1 activates the XBP1 (X-box binding protein 1) transcription factor by excision of a 26-nt fragment from XBP1 mRNA. PERK globally shuts down translation by inhibiting phosphorylation of eukaryotic initiation factor 2α (eIF2α), and in parallel, ATF4. Each transcription factor of the three signalling cascades (ATF4, ATF6 and XBP1) leads to expression of a subset of genes that regulate ER stress and restore cellular proteostasis. Stress-triggered IRE1 oligomerization stimulates the nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) pathway and maintains inflammatory signaling over a much longer time scale [4] (Suppl. Fig. 2A). To assess effect(s) of CF pathology on inter-related branches of the integrated stress response, we extracted expression levels of ATF4-, ATF6- and XBP1-activated gene targets in the freshly derived primary human bronchial epithelia (HBE) from CF lung transplants (#192, F508del/F508del; #209, F508del/F508del; #222, F508del/G542X) and a non-CF patient (#923, polymyositis). These transcript levels were compared to baseline values obtained from CF bronchial epithelia (CFBE) stably transduced to express wild-type CFTR. It should be noted that exogenous expression of F508del-CFTR in CFBE does not elicit ER stress, and most genes activated by these key transcription factors (ATF4, ATF6, XBP1 and NFkB) are silent in the cell model (Suppl. Fig. 2). F508del-triggered misfolding of CFTR also does not induce UPR, hence CFBE cells provide a robust negative control for comparing ER stress-mediated changes in gene expression. Among all four individuals (CF and non-CF), ATF4-targeted transcripts were significantly upregulated, with the polymyositis patient exhibiting the highest numbers of ATF4-activated genes (Suppl. Fig. 3A). Stimulation of ATF6-, XBP1- and NFkB-induced gene targets was more heterogeneous among CF patients, with no changes detected in the non-CF individual (Suppl. Fig. 3B-D).
Transcript
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Supporting information
Global assessment of the integrated stress response in CF patient-derived airway and
intestinal tissues
Giovana B. Bampi, Robert Rauscher, Sebastian Kirchner, Kathryn E. Oliver, Marcel J.C. Bijvelds, Leonardo A. Santos, Johannes Wagner, Raymond A. Frizzell, Hugo R. de Jonge, Eric
J. Sorscher, Zoya Ignatova
Supporting text
S1.1 Primary airway epithelia from three CF patients display ATF6-driven activation of the integrated stress response.
The integrated unfolded protein response (UPR) describes coordinated relationships between three parallel but distinct cellular pathways, each of which initiates at different times through three classical endoplasmic reticulum (ER)-resident transmembrane protein sensors: (1) inositol-requiring protein 1 (IRE1); (2) protein kinase RNA (PRK)-like ER kinase (PERK); and (3) activating transcription factor (ATF)-6 [1]. In response to external stimuli, IRE1 and PERK undergo oligomerization and autophosphorylation, whereas ATF6 is translocated to the Golgi and then activated via proteolytic cleavage [2,3]. Following two sequential endonucleolytic cleavages, IRE1 activates the XBP1 (X-box binding protein 1) transcription factor by excision of a 26-nt fragment from XBP1 mRNA. PERK globally shuts down translation by inhibiting phosphorylation of eukaryotic initiation factor 2α (eIF2α), and in parallel, ATF4. Each transcription factor of the three signalling cascades (ATF4, ATF6 and XBP1) leads to expression of a subset of genes that regulate ER stress and restore cellular proteostasis. Stress-triggered IRE1 oligomerization stimulates the nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) pathway and maintains inflammatory signaling over a much longer time scale [4] (Suppl. Fig. 2A).
To assess effect(s) of CF pathology on inter-related branches of the integrated stress response, we extracted expression levels of ATF4-, ATF6- and XBP1-activated gene targets in the freshly derived primary human bronchial epithelia (HBE) from CF lung transplants (#192, F508del/F508del; #209, F508del/F508del; #222, F508del/G542X) and a non-CF patient (#923, polymyositis). These transcript levels were compared to baseline values obtained from CF bronchial epithelia (CFBE) stably transduced to express wild-type CFTR. It should be noted that exogenous expression of F508del-CFTR in CFBE does not elicit ER stress, and most genes activated by these key transcription factors (ATF4, ATF6, XBP1 and NFkB) are silent in the cell model (Suppl. Fig. 2). F508del-triggered misfolding of CFTR also does not induce UPR, hence CFBE cells provide a robust negative control for comparing ER stress-mediated changes in gene expression.
Among all four individuals (CF and non-CF), ATF4-targeted transcripts were significantly upregulated, with the polymyositis patient exhibiting the highest numbers of ATF4-activated genes (Suppl. Fig. 3A). Stimulation of ATF6-, XBP1- and NFkB-induced gene targets was more heterogeneous among CF patients, with no changes detected in the non-CF individual (Suppl. Fig. 3B-D).
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S1.2 UPR is activated in ileal organoids from F508del homozygous patients. Organoids are three-dimensional, multicellular structures that maintain functional
expression of individual (personalized) genomes [5,6] and recapitulate features of tissue origin [7]. CFTR expression is tightly regulated during development and within different tissue types [8,9]. Levels of CFTR transcript vary between different cellular origins, indicating that tissue-specific regulatory features govern CFTR biogenesis. Intestinal organoids are a suitable model to address this question, because region-specific gene expression profiles are conserved in vitro [10]. Undifferentiated, stem cell-enriched intestinal organoids can be passaged and maintained in culture for many generations without procuring genetic aberration or loss of epithelial polarity. Tissue integrity is dependent upon Wnt signaling and consists mostly of resident proliferating stem cells, which can be directed towards a differentiated, goblet cell-enriched state by withdrawal of niche factors [11]. From this perspective, we next sought to investigate the extent to which a molecular stress signatures differ between distinct gastrointestinal tissues, e.g. intestinal organoids derived from the distal small intestine and colon of an F508del-CFTR homozygous patient. Furthermore, comparing ER-stress and inflammatory responses between undifferentiated and differentiated colonic organoids could provide insight regarding an acute versus chronic effect of F508del expression.
Ileal organoids displayed the strongest activation of targets downstream from ATF4, ATF6, XBP1 and NFkB (Suppl. Fig. 4A-D). We also found that UPR activation in undifferentiated colonic organoids was less pronounced than in the ileal samples (Suppl. Fig. 4A-D, ‘ileum’ versus ‘colon undiff’ columns). Differentiated goblet cell-enriched colonic organoids showed statistically significant upregulation of transcripts targeted by XBP1 and NFkB, whereas ATF4 and ATF6 activation was insignificant (Suppl. Fig. 4A-D). Together, our results suggest that F508del-triggered CFTR misfolding induces ER-stress and inflammatory signaling cascades in organoids from both the distal small intestine and colon, along with some tissue-specific patterning of UPR activation.
Our results did not provide evidence for UPR induction, nor pathways that stimulate tissue remodeling in CFBE stably expressing F508del-CFTR. In contrast, intestinal organoids endogenously expressing F508del display strong UPR activation, although CFTR mRNA levels in the organoids are two orders of magnitude lower than in CFBE (assessed from RNA-Seq data). Immortalization of CFBE cells likely increases robustness of a cancer-like phenotype, serving to confer stable growth and reproducibility under laboratory conditions. This setting may alter certain stress pathways [12], a finding that should be considered when interpreting results of innate immune activation in immortalized cell lines.
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Supporting figures
Figure S1. Tissue remodeling pathways in F508del-expressing CFBE cells. (A-C) Fold changes of downstream genes induced by the SRF (A), HIF1A (B) and CREB (C) transcription factors in CFBE stably transduced with F508del-CFTR, as compared to CFBE expressing wild-type CFTR. For each transcription factor, the total number (n) of downstream genes detected is given in the upper left corner; the total number of genes in each signaling pathway is shown in Fig. 1A. Different sizes of gene sets are detected for HIF1A, CREB and SRF (A-C) compared to the maximum total number of genes (Fig. 1A), which captures variation in sequencing detection levels for distinct cell lines. The number of transcripts upregulated more than 2-fold is italicized in the upper right corner. The vertical dashed line denotes the 2-fold change threshold. P-values for upregulated transcripts are defined with Wilcoxon rank sum test.
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Figure S2. ER-stress and inflammatory signatures of CFBE cells stably expressing F508del-CFTR. (A) Schematic of ER-stress induced signaling pathways. Key transcription factors are highlighted (yellow), and the total number (n) of downstream genes they activate is designated. (B-E) Fold changes of transcripts induced by ATF4 (B), ATF6 (C), XBP1 (D) and NFkB (E). XBP1u denotes unspliced XBP1. In each panel, mRNA levels obtained from F508del-expressing CFBE cells are compared to CFBE stably transduced with wild-type CFTR. For each transcription factor, the number of downstream genes detected is given in the upper left corner; the total number of genes in each signaling pathway is shown in panel A. The number of targets upregulated more than 2-fold is italicized in the upper right corner. The vertical dashed line denotes the 2-fold change threshold. P-values are defined with Wilcoxon rank sum test.
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Figure S3. ER-stress and inflammatory signatures of CF and non-CF airway epithelia from patient lung transplants. (A-C) Fold changes of transcripts targeted by ATF4 (A), ATF6 (B), XBP1 (C) and NFkB (D). In each panel, mRNA levels obtained from donors #209, #222, #192 (CF) and #923 (non-CF) are compared to CFBE cells stably transduced with wild-type CFTR. For each transcription factor, the number (n) of downstream genes detected is given in the upper left corner; the total number of genes in each signaling pathway is shown in Suppl. Fig. 2A. Different sizes of gene sets are noted within each patient (A-D) compared to the maximum total number of genes (Suppl. Fig. 2A), which captures variation in sequencing detection levels for distinct patient-derived materials. The number of targets upregulated more than 2-fold is italicized in the upper right corner. The vertical dashed line denotes the 2-fold change threshold. P-values are defined with Wilcoxon rank sum test.
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Figure S4. ER-stress and inflammatory signatures in CF patient-derived intestinal organoids. (A-D) Fold changes of transcripts targeted by ATF4 (A), ATF6 (B), XBP1 (C) and NFkB (D) in intestinal organoids collected from an F508del-CFTR homozygous patient. In each panel, mRNA levels obtained from CF organoids are compared to non-CF organoids (i.e. collected from a healthy donor). For each transcription factor, the number (n) of downstream genes
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detected is given in the upper left corner; the total number of genes in each signaling pathway is shown in Suppl. Fig. 2A. Different sizes of gene sets are noted for each transcription factor (A-D) compared to the maximum total number of genes (Suppl. Fig. 2A), which captures variation in sequencing detection levels for distinct intestinal sections. The number of targets upregulated more than 2-fold is italicized in the upper right corner. The vertical dashed line denotes the 2-fold change threshold. P-values are defined with Wilcoxon rank sum test.
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