Essential role of single-stranded DNA-binding proteins Ssb1 and Ssb2 in maintaining intestinal epithelial homeostasis

Ssb1 and Ssb2 are single-stranded DNA-binding proteins with overlapping roles in genome stability. Both contain an OB-fold domain and interact with C9orf80 and IntS3, components of the Integrator complex which regulates snRNA processing, enhancer RNA biogenesis, and resolution of promoter-proximal RNA polymerase II (Pol II) pausing or early termination . We previously showed that combined loss of Ssb1 and Ssb2 leads to embryonic lethality or acute multi-organ failure in adult mice. Here, we show that intestine-specific deletion of both genes (DKO) causes severe radiomimetic damage to the intestinal proliferative crypt compartment, resulting in villous atrophy, marked malabsorption, and mortality, demonstrating that gut-specific mSSB loss is sufficient to drive systemic breakdown. An initial surge in intestinal progenitor cell proliferation preceded crypt depletion, suggesting transient regeneration followed by intestinal stem cell (ISC) exhaustion. DKO tissue failed to grow ex vivo , confirming a cell-autonomous requirement for mSsb2 in ISC maintenance. Mechanistically, mSSBs loss led to R-loop accumulation, p53 activation and interferon-stimulated gene expression, consistent with a failure to resolve transcription-associated genomic stress. Together, these findings uncover a critical, previously unrecognized role for Ssb1 and Ssb2 in regulating intestinal stem cell homeostasis and epithelial regeneration, with broad implications for gastrointestinal development, regeneration, and diseases such as cancer and inflammation.