Studying effects of SOX9 induction and knockout on primary human intestinal stem cell monolayers

ABSTRACT: Background & Aims Little is known about mechanisms controlling cell cycle in intestinal cells, and while cell cycle arrest protects intestinal crypts against irradiation-induced damage, genetic regulators driving these changes are lacking. SOX9 expression is inverse to proliferation in crypt lineages, and SOX9 knockout increases proliferation, but our understanding of how SOX9 regulates cell cycle is lacking. Here, we test how SOX9 regulates cell cycle and whether this regulation confers cytoprotection in intestinal stem cells. Methods Healthy primary human intestinal stem cells were engineered to control SOX9 expression, visualize cell cycle, and alter known cell cycle regulators. Cells with altered levels of SOX9 were tested for stemness, differentiation, the ability to revert proliferation rates following SOX9 washout, and ability to survive 5-FU mediated injury. The downstream mechanism connected SOX9 to cell cycle was inferred transcriptionally and tested functionally using inducible CDKN2A (INK4A) and CCND2 cell lines. Results Inducing SOX9 expression lengthens total cell cycle largely by elongating G1 phase through the INK4A-Rb pathway. The effects of induced SOX9 repressing proliferation and stem cell activity are reversible. SOX9 induction confers protection against 5-FU toxicity, which is mimicked by INK4A overexpression or pharmacological cell cycle inhibition and repressed by CCND2 induction.. Conclusions SOX9 appears to be a master regulator of cell cycle within the intestinal crypt, acting through the INK4A-Rb pathway. Elongating G1 phase within healthy human intestinal cells is able to protect against chemotoxicity, making longer G1 phase a possible deciding factor on which crypt cells survive genotoxic injury to act as reserve stem cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE341884 | GEO | 2026/08/19

REPOSITORIES: GEO