Abstract
Neurogenin-3 (NEUROG3) is a helix-loop-helix (HLH) transcription factor involved in the production of endocrine cells in the intestine and pancreas of humans and mice. However, the human NEUROG3 loss-of-function phenotype differs subtly from that in mice, but the reason for this difference remains poorly understood. Because NEUROG3 expression precedes exit of the cell cycle and the expression of endocrine cell markers during differentiation, we investigated the effect of lentivirus-mediated overexpression of the human NEUROG3 gene on the cell cycle of BON4 cells and various human nonendocrine cell lines. NEUROG3 overexpression induced a reversible cell cycle exit, whereas expression of a neuronal lineage homolog, NEUROG1, had no such effect. In endocrine lineage cells, the cellular quiescence induced by short-term NEUROG3 expression required cyclin-dependent kinase inhibitor 1A (CDKN1A)/p21CIP1 expression. Expression of endocrine differentiation markers required sustained NEUROG3 expression in the quiescent, but not in the senescent, state. Inhibition of the phosphatase and tensin homolog (PTEN) pathway reversed quiescence by inducing cyclin-dependent kinase 2 (CDK2) and reducing p21CIP1 and NEUROG3 protein levels in BON4 cells and human enteroids. We discovered that NEUROG3 expression stimulates expression of CDKN2a/p16INK4a and BMI1 proto-oncogene polycomb ring finger (BMI1), with the latter limiting expression of the former, delaying the onset of CDKN2a/p16INK4a -driven cellular senescence. Furthermore, NEUROG3 bound to the promoters of both CDKN1a/p21CIP1 and BMI1 genes, and BMI1 attenuated NEUROG3 binding to the CDKN1a/p21CIP1 promoter. Our findings reveal how human NEUROG3 integrates inputs from multiple signaling pathways and thereby mediates cell cycle exit at the onset of differentiation.
Highlights
ARTICLE croThe cellular regulators phosphatase and tensin homolog (PTEN) and BMI1 help mediate NEUROGENIN-3–induced cell cycle arrest
Neurogenin-3 (NEUROG3) is a helix-loop-helix (HLH) transcription factor involved in the production of endocrine cells in the intestine and pancreas of humans and mice
Because NEUROG3 expression precedes exit of the cell cycle and the expression of endocrine cell markers during differentiation, we investigated the effect of lentivirus-mediated overexpression of the human NEUROG3 gene on the cell cycle of BON4 cells and various human nonendocrine cell lines
Summary
The cellular regulators PTEN and BMI1 help mediate NEUROGENIN-3–induced cell cycle arrest. Neurog expression seems to drive mouse pre-endocrine lineage cells from the cell cycle, initially inducing cellular quiescence. The role of BMI1 as a mediator of senescence and quiescence is further highlighted by the recent observation that the population of Bmi1ϩ cells in the mouse intestinal crypt is enriched with quiescent early GIE lineage cells [8], likely related to Neurog3-expressing cells known to be concentrated in the same crypt domain [2]. Similar quiescent cells, identified by mTert expression, respond to post-fasting refeeding by repressing Phosphatase and tensin homolog (Pten) expression, and re-entering the cell cycle [9] These observations suggest that in mice, Neurog3’s cell cycle effects occur in early GIE lineage cells and are mediated downstream by Bmi and Pten. The early NEUROG3-induced cellular quiescence is reversible by inhibition of PTEN, due to reduction in steady-state NEUROG3 levels in BON4 cells and human enteroids. We report that the onset of NEUROG3-induced senescence is slowed due to concomitant induction of BMI1 expression, which acts by attenuating p16INK4 expression
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