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Background & Aims Self-renewal and multipotent differentiation are cardinal properties of intestinal stem cells (ISCs), mediated in part by WNT and NOTCH signaling. Although these pathways are well characterized, the molecular mechanisms that control the ‘stemness’ of ISCs are still not well defined. Here, we investigated the role of Kruppel-like factor 5 (KLF5) in regulating ISC functions. Methods We performed studies in adult Lgr5EGFP-IRES-creERT2;Rosa26LSLtdTomato (Lgr5Ctrl) and Lgr5EGFP-IRES-creERT2;Klf5fl/fl;Rosa26LSLtdTomato (Lgr5ΔKlf5) mice. Mice were injected with tamoxifen to activate Cre recombinase, which deletes Klf5 from the intestinal epithelium in Lgr5ΔKlf5 but not Lgr5Crtl mice. In experiments involving irradiation, mice were subjected to 12 Gy total body irradiation (TBI). Tissues were collected for immunofluorescence (IF) analysis and next generation sequencing. Oganoids were derived from fluoresecence activated cell sorted- (FACS-) single cells from tamoxifen-treated Lgr5ΔKlf5 or Lgr5Crtl mice and examined by immunofluorescence stain. Results Lgr5+ ISCs lacking KLF5 proliferate faster than control ISCs but fail to self-renew, resulting in a depleted ISC compartment. Transcriptome analysis revealed that Klf5-null Lgr5+ cells lose ISC identity and prematurely differentiate. Following irradiation injury, which depletes Lgr5+ ISCs, reserve Klf5-null progenitor cells fail to dedifferentiate and regenerate the epithelium. Absence of KLF5 inactivates numerous selected enhancer elements and direct transcriptional targets including canonical WNT- and NOTCH-responsive genes. Analysis of human intestinal tissues showed increased levels of KLF5 in the regenerating epithelium as compared to those of healthy controls. Conclusion We conclude that ISC self-renewal, lineage specification, and precursor dedifferentiation require KLF5, by its ability to regulate epigenetic and transcriptional activities of ISC-specific gene sets. These findings have the potential for modulating ISC functions by targeting KLF5 in the intestinal epithelium.

Tamoxifen is a synthetic non-steroidal Selective Estrogen Receptor Modulator used in the treatment of breast cancer and in treatment of male fertility. Earlier studies from our laboratory had demonstrated an increase in post-implantation embryo loss following tamoxifen treatment to adult male rats at a dose of 0.4mg/kg/day for 60 days. The post-implantation loss occurred at around 9-10 days of gestation suggesting that paternal factors involved in embryo development were affected by tamoxifen treatment. The present study was done to determine if any chromosomal aberrations occurred in the embryos sired by tamoxifen treated male rats. Chromosomal aberrations induced by tamoxifen treatment to adult male rats in the bone marrow (F(0) males) and in the embryos sired by these males (F(1) progeny) were determined. In addition, the reproductive performance of the F(1) progeny was assessed. A significant dose dependent reduction in mitotic activity in the bone marrow and embryonic cells was observed after tamoxifen treatment. In addition, tamoxifen also induced a significant dose dependent increase in the frequency of chromosomal aberrations, mainly gaps and breaks in bone marrow and embryonic cells. However, the embryos sired by the tamoxifen treated males had no effect on developmental milestones achieved and on their reproductive performance. The present study suggests that chromosomal aberrations observed in the embryos did not the affect their development until adulthood but could make the progeny of the tamoxifen treated males vulnerable to the development of adult onset diseases later in life.