Discrete regulation of β-catenin-mediated transcription governs identity of intestinal epithelial stem cells

Abstract

The homeostasis of the gut epithelium relies upon continuous renewal and proliferation of crypt-resident intestinal epithelial stem cells (IESCs). Wnt/β-catenin signaling is required for IESC maintenance, however, it remains unclear how this pathway selectively governs the identity and proliferative decisions of IESCs. Here, we demonstrate that C-terminally-recruited transcriptional co-factors of β-catenin act as all-or-nothing regulators of Wnt-target gene expression. Blocking their interactions with β-catenin rapidly induces loss of IESCs and intestinal homeostasis. Conversely, N-terminally recruited co-factors fine-tune β-catenin's transcriptional output to ensure proper self-renewal and proliferative behaviour of IESCs. Impairment of N-terminal interactions triggers transient hyperproliferation of IESCs, eventually resulting in exhaustion of the self-renewing stem cell pool. IESC mis-differentiation, accompanied by intrinsic and extrinsic stress signalling results in a process resembling aberrant "villisation" of intestinal crypts. Our data suggest that IESC-specific Wnt/β-catenin output requires discrete regulation of transcription by transcriptional co-factors. Results Attenuation of N-vs. C-terminal β-catenin transcriptional outputs has contrasting effects on intestinal homeostasisWe previously generated transgenic β-catenin (Ctnnb1) alleles harboring mutations that prevent interactions with N-or C-terminal transcriptional co-factors (NTFs and CTFs, respectively) 7 . The D164A mutation abrogates interaction with NTFs, the ∆C truncation abrogates the interaction with the CTFs (Fig. 1a). To overcome embryonic lethality of these alleles, we used compound heterozygous mice carrying one mutant and one conditional βcatenin allele (Extended Data Fig. 1a). Similarly to constitutively hemizygous Ctnnb1 KO/wt mice, Ctnnb1 D164A/flox and Ctnnb1 ∆C/flox animals show no overt abnormalities, indicating haplosufficiency of β-catenin for the maintenance of intestinal homeostasis. Combination with the villin-CreER T2 driver 16 enables the inducible deletion of the conditional β-catenin allele (Ctnnb1 flox ) specifically in the intestinal epithelium, thus leaving the Wnt-transcriptional outputs solely under the control of mutant β-catenin (D164A or ∆C). While villin-CreER T2 ;Ctnnb1 wt/flox (control) mice are perfectly viable and indistinguishable from homozygous wild type animals, the presence of only mutant β-catenin is lethal: villin-CreER T2 ;Ctnnb1 ∆C/flox (∆C) animals exhibit atrophic crypts at 4d post Cre induction (pi) (Extended Data Fig. 1b). This is in accordance with our previous results in villin-CreER T2 ;Ctnnb1 dm/flox animals, which express double mutant (dm) β-catenin harboring both N-and C-terminal mutations 17 . Unlike dm and ∆C animals, villin-CreER T2 ;Ctnnb1 D164A/flox (D164A) animals only reach humane endpoint at 7d pi, as they suffer from severe colitis (Extended Data Fig. 1c). The different intestinal phenotypes that arise upon blocking C-or N-terminal transcriptional outputs of β-catenin are consist...