AffiliationUniv Arizona, Mol & Cellular Biol
MetadataShow full item record
PublisherHIGHER EDUCATION PRESS
CitationMathey-Prevot, B., Parker, B., Im, C. et al. Quantifying E2F1 protein dynamics in single cells. Quant Biol 8, 20–30 (2020). https://doi.org/10.1007/s40484-019-0193-6
Rights© Higher Education Press and Springer-Verlag GmbH Germany, part of Springer Nature 2020
Collection InformationThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at email@example.com.
AbstractBackground: E2F1 protein, a major effector of the Rb/E2F pathway plays a central role in regulating cell-fate decisions involved in proliferation, apoptosis, and differentiation. Its expression is highly dynamic and tightly modulated through a combination of transcriptional, translational and posttranslational controls. However, the mechanisms by which its expression and activity can promote different cellular outcomes remain to be fully elucidated. To better document E2F1 expression in live cells, we have engineered a series of fluorescent E2F1 protein reporters that quantitatively capture E2F1 protein dynamics. Methods: Reporter constructs, under the control of the mouse or human E2F1 proximal promoter, were designed to express an E2F1-Venus fusion protein incapable of binding DNA. In addition, constructs either included or excluded the 3 untranslated region (3UTR) of the E2F1 gene. These constructs were introduced into fibroblasts and epithelial cells, and expression of the fusion reporter protein was validated and quantified in single cells using live imaging. Results: In all cases, expression of the reporter protein effectively recapitulated the behavior of E2F1 under various conditions, including cell cycle progression and genotoxic stress. No or little fluorescent signal of the reporter was detected in G(0), but as the cycle progressed, expression of the reporter protein steadily increased in the nucleus, peaking a few hours before cell division, but declining to baseline 2-3 h prior to the onset of mitosis. The absence of the E2F1 3 ' UTR in the constructs led to considerably higher steady-state levels of the fusion protein, which although normally regulated, exhibited a slightly less complex dynamic profile during the cell cycle or genotoxic stress. Lastly, the presence or absence of Rb failed to impact the overall detection and levels of the reporter proteins. Conclusions: Our validated E2F1 protein reporters complement nicely other reporters of the Rb/E2F pathway and provide a unique tool to follow the complex dynamics of E2F1 expression in real time in single cells.
Note12 month embargo; published online: 6 March 2020
VersionFinal accepted manuscript
- Human herpesvirus 6 (HHV-6) alters E2F1/Rb pathways and utilizes the E2F1 transcription factor to express viral genes.
- Authors: Sharon E, Volchek L, Frenkel N
- Issue date: 2014 Jan 7
- Disruption of Rb/E2F pathway results in increased cyclooxygenase-2 expression and activity in prostate epithelial cells.
- Authors: Davis JN, McCabe MT, Hayward SW, Park JM, Day ML
- Issue date: 2005 May 1
- MyoD activity upregulates E2F1 and enhances transcription from the cyclin E promoter in differentiating myoblasts lacking a functional retinoblastoma protein.
- Authors: Tedesco D, Vesco C
- Issue date: 2001 Oct 1
- The role of the retinoblastoma/E2F1 tumor suppressor pathway in the lesion recognition step of nucleotide excision repair.
- Authors: Lin PS, McPherson LA, Chen AY, Sage J, Ford JM
- Issue date: 2009 Jul 4
- Regulation of Rb and E2F by signal transduction cascades: divergent effects of JNK1 and p38 kinases.
- Authors: Wang S, Nath N, Minden A, Chellappan S
- Issue date: 1999 Mar 15