XBP1s cutnrun

Epithelial mesenchymal plasticity (EMP) is a complex cellular reprogramming event that plays a major role in tissue homeostasis to infections and injury. Recently we elucidated a mechanism wherein the unfolded protein response (UPR) triggers EMP through the inositol-requiring protein 1 (IRE1a)–X-box-binding protein 1 (XBP1) axis, enhancing glucose shunting to protein N glycosylation producing ER stress. To better the genomic targets for the IRE1-XBP1 pathway that activate compensatory metabolic changes in EMP, we systematically identified the genomic XBP1 targets using Cleavage Under Targets and Release Using Nuclease (CUT&RUN) of a FLAG-epitope tagged XBP1 in RSV infection. CUT&RUN identified 7,086 enriched binding sites mapped to 4,827 genes; of these XBP1 binds to 2,119 sites within 1 kb of the transcription start sites. Interestingly, XBP1 binds to 322 superenhancers associated with RHO GTPase signaling that were associated with largely inert gene expression. By contrast, XBP1 binds to proximal promoters inducing coordinate mRNA expression of hexosamine biosynthetic enzymes encoding sequential steps in UDP-GlcNAc biosynthesis through AGCTCA motifs. We demonstrate that IRE1-XBP1 signaling is necessary and sufficient to activate core HBP enzymatic pathway by recruiting transcriptional elongation-competent phospho-Ser2 CTD modified RNA Pol II (pSer-PolII). 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 10^6 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.