Threonine Phosphorylation of STAT1 Safeguards Gut Epithelial Integrity and Restricts Interferon-mediated Cytotoxicity [ChIP-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 ß4 expression in IECs following inflamamtion. In vitro intestinal organoid models, combined with gain- and loss-of-function experiments, show that Stat1 promotes integrin ß4 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 ß4, 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. Overall design: To  assess the impact of Thr748 phosphorylation on Stat1's transcriptional activity in intestinal epithelial cells (IECs), we performed chromatin immunoprecipitation sequencing (ChIP-seq) on IECs isolated from Wt and Stat1T748A mice following DSS treatment. ChIP assays were performed using the Pierce Magnetic ChIP Kit (Thermo Fisher) following the manufacturer's protocol. Briefly, IECs were isolated from DSS-treated Wt and Stat1T748A littermate mice and cross-linked with 3.7% formaldehyde for 10 minutes at room temperature. Cross-linking was quenched with 1.375 M glycine for 5 minutes at room temperature. Nuclei were isolated, and chromatin was enzymatically sheared using micrococcal nuclease for 17 minutes at 37 °C. Five percent of the sheared chromatin was reserved as input, and the remainder was subjected to immunoprecipitation using anti–STAT1 antibody (1:50 dilution). Immunocomplexes were reverse cross-linked overnight at 65 °C with Proteinase K, and DNA was purified using phenol/chloroform extraction. ChIP DNA libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems) according to the manufacturer's instructions. Paired-end sequencing (2 × 101 bp) was performed on an Illumina NovaSeq 6000 platform (Illumina Inc., San Diego, CA, USA). Adapter trimming was conducted with Cutadapt (v4.8), using the following adapter sequences: Read 1: 5'-AGATCGGAAGAGCACACGTCTGAACTCCAGTCA-3'; Read 2: 5'-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3'. Read quality was assessed using FastQC (default settings). Trimmed reads were aligned to the mouse reference genome (mm10) using Bowtie2 (v2.5.3) with the “very sensitive end-to-end” preset. BAM files were further processed using the Galaxy platform. Low-quality reads (MAPQ < 30), improperly paired reads, mitochondrial reads, and PCR duplicates were removed using 'Filter' (v2.4.2) and 'MarkDuplicates' (v3.1.1.0). Peak calling was performed using MACS2 callpeak (v2.2.9.1).