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| Status |
Public on Apr 30, 2023 |
| Title |
XBP1s cutnrun |
| Organism |
Homo sapiens |
| Experiment type |
Other
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| Summary |
The unfolded protein response (UPR) has emerged as important signaling pathway mediating anti-viral defenses to Respiratory Syncytial Virus (RSV) infection. In this study, we examine how Inositol Requiring Enzyme (IREa X-Box Binding Protein spliced (XBP1s) arm of the Unfolded Protein Response (UPR) pathway controls the innate response integrating RNA-seq and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) analyses. XBP1s binds to ~4.2 K high-confidence genomic binding sites. Surprisingly these map to only a small subset of IL10/cytokine genes. We further find that that RSV infection enhances XBP1s occupancy on 786 genomic sites enriched in AP1/Fra-1, RELA and SP1 binding sites controlling a core subset of cytokine regulatory factors genes including IFN response factor 1 (IRF1), CSF2, NFKB1A and DUSP10. Selective IRF1 knockdown experiments demonstrates its requirement in control of type I and -III IFNs and IFN-stimulated genes (ISGs) indicating these genes are indirectly regulated through IRF1 transactivation. We conclude that RSV modulates the XBP1 binding complex to the IRF1 epromoter, providing novel insight into how the IRE1-XBP1s pathway potentiates airway mucosal anti-viral responses.
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| Overall design |
hSAECs were treated in triplicate replicates including FLAG-XBP1s transduced cells with or without RSV infection (MOI = 1.0, 24 h). Untransduced cells were negative control. After trypsinization, 4 x 106 cells were aliquoted and incubated on ice for 10 minutes in 1 ml of nuclear extraction buffer (20 mM HEPES, pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% glycerol, 1x cOmplete proteinase inhibitor, 1x protein phosphatase inhibitor cocktail and 0.5 mM spermidine), the released nuclei were pelleted at 600x g, 5 minutes at 4 oC. A basic wash buffer (WB) consisting of 20 mM HEPES, pH7.5, 150 mM NaCl, 0.05% Triton X-1000, 0.1% BSA, 1x cOmplete proteinase inhibitor, 1x protein phosphatase inhibitor cocktail and 0.5 mM spermidine was used throughout. Between incubation steps, the nuclei were pelleted at 600x g, 3 minutes at 4 oC. Prior to antibody binding, the isolated nuclei were incubated with nutation at 4 oC for 5 minutes in WB containing 2 mM EDTA, followed by nutation at 4 oC for 25 minutes in WB. The nuclei were resuspended in antibody buffer produced by diluting 5 mg of FLAG-M2 in 500 ml of Triton X-100-free WB, and incubated with nutation overnight at 4 oC. After washing three times in 500 ml of WB on ice (10 minutes each time), the nuclei were incubated at 4 oC for 1 h in 50 ml of WB containing 2.5 ml of EpiCypher pAG-MNase 20x stock (EpiCypher, NC). Washing as above, targeted chromatin cleavage was then conducted by incubating the nuclei on ice for 1 h in 150 ml of BSA-free WB containing 2 mM CaCl2. The cleavage was terminated by adding 150 ml of stop buffer (300 mM NaCl, 20 mM EDTA, 4 mM EGTA and 0.5 ng of E. coli Spike-in DNA (EpiCypher, NC) per 150 ml). The samples were further incubated with nutation at 4 oC for 1 h, centrifuged at 16,000x g for 5 min at 4 oC and the supernatant was collected. Phenol-chloroform DNA precipitation with 80 mg of glycogen was performed and the Cut&Run-enriched DNA was dissolved in 20 ml of 0.1x TE buffer. Following DNA quantitation by a Qubit fluorometer, Cut&Run DNA libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, MA) per the manufacturer’s instruction, with modification in SPRI bead clearance of adaptor ligation and library amplification reactions to retain small-sized DNA fragments (down to ~170 bp in post-library amplification size). The quality of the resultant DNA libraries was confirmed by Agilent TapeStation HS DNA assay (Agilend, CA), and paired-end Illumina NGS was carried out on NovaSeq 6000 with 5 million reads per sample.
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| Contributor(s) |
Brasier AR, Qiao D |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Mar 27, 2023 |
| Last update date |
May 02, 2023 |
| Contact name |
Allan Brasier |
| E-mail(s) |
abrasier@wisc.edu
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| Organization name |
university of Wissconsin
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| Department |
ICTR
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| Street address |
715 Highland
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| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53597 |
| Country |
USA |
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| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (9)
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| Relations |
| BioProject |
PRJNA949270 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE228328_RAW.tar |
584.7 Mb |
(http)(custom) |
TAR (of BED) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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