Cooperative DNA binding is a key feature of transcriptional regulation. Here we examined the role of cooperativity in Notch signaling by CRISPR-mediated engineering of mice in which neither Notch1 nor Notch2 can homo-or heterodimerize, essential for cooperative binding to sequence-paired sites (SPS) located near many Notch-regulated genes. Although most known Notch-dependent phenotypes were unaffected in Notch1/2 dimerdeficient mice, a subset of tissues proved highly sensitive to loss of cooperativity. These phenotypes include heart development, compromised viability in combination with low gene dose, and the gut, developing ulcerative colitis in response to 1% dextran sulfate sodium (DSS). The most striking phenotypes-gender imbalance and splenic marginal zone B-cell lymphoma-emerged in combination with gene dose reduction or when challenged by chronic fur mite infestation. This study highlights the role of the environment in malignancy and colitis and is consistent with Notch-dependent anti-parasite immune responses being compromised in Notch dimer-deficient animals.

Intestinal exocrine secretory lineages, including goblet cells and Paneth cells, provide vital innate host defense to pathogens. However, how these cells are specified and maintained to ensure intestinal barrier function remains poorly defined. Here we show that endoplasmic reticulum membrane protein complex subunit 3 (Emc3) is essential for differentiation and function of exocrine secretory lineages. Deletion of Emc3 in intestinal epithelium decreases mucus production by goblet cells and Paneth cell population, along with gut microbial dysbiosis, which result in spontaneous inflammation and increased susceptibility to DSS-induced colitis. Moreover, Emc3 deletion impairs stem cell niche function of Paneth cells, thus resulting in intestinal organoid culture failure. Mechanistically, Emc3 deficiency leads to increased endoplasmic reticulum (ER) stress. Mitigating ER stress with tauroursodeoxycholate acid alleviates secretory dysfunction and restores organoid formation. Our study identifies a dominant role of Emc3 in maintaining intestinal mucosal homeostasis.