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 took advantage of knock-in mice harboring transgenic β-catenin alleles with mutations that specifically impair the recruitment of N- or C-terminal transcriptional co-factors. We show 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 unfolded protein response stress and immune infiltration, results in a process resembling aberrant "villisation" of intestinal crypts. Our data suggest that IESC-specific Wnt/β-catenin output requires selective modulation of gene expression by transcriptional co-factors.

• MeSH
• algoritmy MeSH
• beta-katenin chemie   metabolismus   MeSH
• buněčná diferenciace MeSH
• chromatin metabolismus   MeSH
• fenotyp MeSH
• genetická transkripce * MeSH
• homeostáza MeSH
• hyperplazie MeSH
• JNK mitogenem aktivované proteinkinasy metabolismus   MeSH
• kmenové buňky metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• mutace genetika   MeSH
• mutantní proteiny metabolismus   MeSH
• myši MeSH
• organoidy metabolismus   MeSH
• proliferace buněk MeSH
• restrukturace chromatinu MeSH
• sekvence nukleotidů MeSH
• signální transdukce MeSH
• střevní sliznice cytologie   MeSH
• transkripční faktory metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• beta-katenin MeSH
• chromatin MeSH
• JNK mitogenem aktivované proteinkinasy MeSH
• messenger RNA MeSH
• mutantní proteiny MeSH
• transkripční faktory MeSH

Production of small RNAs by ribonuclease III Dicer is a key step in microRNA and RNA interference pathways, which employ Dicer-produced small RNAs as sequence-specific silencing guides. Further studies and manipulations of microRNA and RNA interference pathways would benefit from identification of small-molecule modulators. Here, we report a study of a fluorescence-based in vitro Dicer cleavage assay, which was adapted for high-throughput screening. The kinetic assay can be performed under single-turnover conditions (35 nM substrate and 70 nM Dicer) in a small volume (5 µL), which makes it suitable for high-throughput screening in a 1536-well format. As a proof of principle, a small library of bioactive compounds was analyzed, demonstrating potential of the assay.

• Klíčová slova
• Dicer, high-throughput screening, siRNA,
• MeSH
• buněčné linie MeSH
• fluorescenční spektrometrie metody   MeSH
• knihovny malých molekul MeSH
• lidé MeSH
• objevování léků MeSH
• reprodukovatelnost výsledků MeSH
• ribonukleasa III antagonisté a inhibitory   genetika   metabolismus   MeSH
• rychlé screeningové testy * MeSH
• substrátová specifita MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• knihovny malých molekul MeSH
• ribonukleasa III MeSH