Threonine Phosphorylation of STAT1 Safeguards Gut Epithelial Integrity and Restricts Interferon-mediated Cytotoxicity [RNA-seq]

Uncontrolled inflammation drives tissue damage, highlighting the need for tightly regulated immune responses and tissue integrity, particularly in barrier tissues like the intestine. To maintain this exquisite balance, intestinal epithelial cells (IECs) employ molecular circuits that preserve tissue integrity following inflammation. STAT1 is traditionally viewed as a pro-inflammatory driver in the intestine, acting as a central signaling mediator downstream of interferons (IFN). Here, we identify threonine 748 (Thr748) phosphorylation of Stat1 as an evolutionarily conserved adaptation that reciprocally regulates IEC integrity and IFN responsiveness. Mice expressing a phospho-deficient T748A Stat1 mutant exhibit severe pathology similar to Stat1 deficient littermates, underscoring Thr748’s critical role in Stat1-driven protection following intestinal inflammation. Bone marrow transfer experiments further demonstrate that this protective effect is non-hematopoietic. Integrated genomic and transcriptomic analyses reveal that Thr748 phosphorylation modulates Stat1 DNA binding, directly activating the Itgb4 promoter and enhancing integrin β4 expression in IECs following inflamamtion. In vitro intestinal organoid models, combined with gain- and loss-of-function experiments, show that Stat1 promotes integrin β4 expression via Thr748 phosphorylation following damage, boosting epithelial resilience independently of IFN-induced Tyrosine 701 (Tyr701) phosphorylation. In contrast, IFN stimulation triggers Tyr701 phosphorylation of Stat1, upregulating Zbp1—a sensor of cytotoxic damage-associated nucleic acids—while suppressing integrin β4, leading to epithelial cytotoxicity, which is mitigated by Thr748 phosphorylation. Our findings uncover a modular architecture of Stat1 signaling that enables epithelial adaptation to damage, with Thr748 phosphorylation acting as a rheostat to preserve tissue integrity while restricting IFN-mediated cytotoxicity. To elucidate the protective epithelial transcriptional programs driven by the Thr748 phosphorylation of Stat1 following colitis-induced damage, we performed RNA sequencing (RNA-seq) on IECs isolated from Wt and Stat1T748A mice under both basal and DSS-treated conditions. A total of 2–5 × 10⁵ cells per condition were lysed in 0.5 mL of TRIzol Reagent (Thermo Fisher) following the indicated treatments. RNA-seq analysis was performed by the Genome Information Research Center (GIRC) at Osaka University. Briefly, total RNA was extracted, and cDNA libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit (Illumina, Cat. No. 20020594) according to the manufacturer’s instructions. Sequencing was conducted on an Illumina HiSeq 2500 platform using 75-base single-end reads. Base calling was performed with Illumina Casava v1.8.2 software. Sequenced reads were aligned to the mouse reference genome (mm10/GRCm38) using TopHat v2.0.13 with Bowtie2 v2.2.3 and SAMtools v0.1.19. Gene expression levels were quantified as fragments per kilobase of exon per million mapped reads (FPKM) using Cuffnorm v2.2.1. Further data processing and analysis were conducted on the Galaxy platform (https://usegalaxy.org/). Read quality was assessed using FastQC (v0.73), and adaptor sequences were trimmed prior to alignment with HISAT2 (v2.2.1) using default parameters. Gene counts were generated with featureCounts (v2.0.3), and differential expression analysis was performed using DESeq2 (v2.11.40.7). Visualization of results, including volcano plots and heatmaps, was performed using Volcano Plot (v0.0.5) and heatmap2 (v3.1.3), respectively. Gene ontology enrichment and pathway analysis were conducted using the PANTHER GO-Slim Biological Process database (v17.0, released 2022/02/22).