Abstract
E2F transcription factors are regulated by binding to the retinoblastoma (Rb) tumor suppressor family of proteins. Previously, we reported an E2FLQ mutation that disrupts the binding with Rb proteins without affecting the transcriptional activity of E2F. We also showed that mouse embryonic fibroblasts with an E2F3LQ mutation exhibit increased E2F activity and more rapid cell proliferation. In this report, we analyzed E2F3LQ mice to further characterize the in vivo consequences of Rb family-independent E2F3 activity. We found that homozygous E2F3LQ mice were viable and had no obvious developmental defects or tumor growth. Our results also indicated that E2F3LQ cells largely retain normal control of cell proliferation in vivo However, female E2F3LQ mice had partial nursing defects. Examination of the E2F3LQ mammary glands revealed increased caveolin-1 (CAV1) expression, reduced prolactin receptor/Stat5 signaling, and impaired pregnancy-induced cell proliferation and differentiation. Of note, ChIP experiments disclosed that E2F3 binds the CAV1 promoter. Furthermore, E2F3 overexpression induced CAV1 expression, and CRISPR/CAS9-mediated E2F3 knockout reduced CAV1 levels and also increased prolactin receptor-induced Stat5 signaling in mammary epithelial cells. Our results suggest that the Rb family-independent E2F3 LQ variant inhibits pregnancy-induced mammary gland cell proliferation and differentiation by up-regulating CAV1 expression and inhibiting Stat5 signaling.
Highlights
E2F transcription factors are regulated by binding to the retinoblastoma (Rb) tumor suppressor family of proteins
E2F3LQ/LQ females have small litter sizes, which is due to the fact that around 60% of the pups do not survive past parturition day 2 (Fig. 1A)
Normal weight at weaning was observed for pups from E2F3LQ/LQ mothers that were fostered by WT females, whereas a lower weight was observed for pups from WT females that were fostered by E2F3LQ/LQ females (Fig. S1)
Summary
The mouse Rb1R654W/ϩ mutation, which corresponds to the human RbR661W mutation identified in low-penetrance retinoblastomas, reduced the ability to bind activating E2F and promoted pituitary tumor development in a mouse model, similar to the Rbϩ/Ϫ mutation [12,13,14] These results suggest that inhibition of E2F activity is a critical function of Rb in normal and cancer development in both fly and mammalian systems. The Rb⌬G mutation, which disrupts the binding between Rb and the E2F transcription activation domain, does not cause spontaneous tumor development despite deregulated E2F target gene expression and accelerated G1/S transition after serum induction [15] These results could suggest that the free activating E2F proteins are not sufficient to induce tumor development. We describe the characterization of the effect of Rb family–independent E2F3LQ/LQ mutation on mammary gland remodeling during pregnancy
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