Cell Rep. 2019 Jun 18;27(12):3547-3560.e5. doi: 10.1016/j.celrep.2019.05.004. Epub 2019 May 23.

Two Distinct E2F Transcriptional Modules Drive Cell Cycles and Differentiation.

Cuitiño MC¹, Pécot T¹, Sun D², Kladney R³, Okano-Uchida T¹, Shinde N⁴, Saeed R⁴, Perez-Castro AJ⁴, Webb A⁵, Liu T⁵, Bae SI³, Clijsters L⁶, Selner N¹, Coppola V³, Timmers C⁷, Ostrowski MC¹, Pagano M⁸, Leone G⁹.

Author information

1: Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA.
2: Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA; Department of Molecular Genetics, Ohio State University, Columbus, OH 43210, USA.
3: Department of Molecular Genetics, Ohio State University, Columbus, OH 43210, USA.
4: Department of Cancer Biology and Genetics, Ohio State University, Columbus, OH 43210, USA.
5: Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210, USA.
6: Department of Biochemistry and Molecular Pharmacology, Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016, USA.
7: Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA; Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210, USA.
8: Department of Biochemistry and Molecular Pharmacology, Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016, USA; Howard Hughes Medical Institute, New York University School of Medicine, New York, NY 10016, USA.
9: Department of Biochemistry and Molecular Biology, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425, USA. Electronic address: leoneg@musc.edu.

Abstract

Orchestrating cell-cycle-dependent mRNA oscillations is critical to cell proliferation in multicellular organisms. Even though our understanding of cell-cycle-regulated transcription has improved significantly over the last three decades, the mechanisms remain untested in vivo. Unbiased transcriptomic profiling of G₀, G₁-S, and S-G₂-M sorted cells from FUCCI mouse embryos suggested a central role for E2Fs in the control of cell-cycle-dependent gene expression. The analysis of gene expression and E2F-tagged knockin mice with tissue imaging and deep-learning tools suggested that post-transcriptional mechanisms universally coordinate the nuclear accumulation of E2F activators (E2F3A) and canonical (E2F4) and atypical (E2F8) repressors during the cell cycle in vivo. In summary, we mapped the spatiotemporal expression of sentinel E2F activators and canonical and atypical repressors at the single-cell level in vivo and propose that two distinct E2F modules relay the control of gene expression in cells actively cycling (E2F3A-8-4) and exiting the cycle (E2F3A-4) during mammalian development.

Published by Elsevier Inc.

KEYWORDS:

E2F; E2F-tagged knockin mice; FUCCI mouse embryos; RNA-seq; cell cycle; confocal microscopy; deep learning; image analysis; immunostaining; transcription

PMID:: 31130414
PMCID:: PMC6673649
DOI:: 10.1016/j.celrep.2019.05.004

Free PMC Article

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Figure 6.

Peak Expression of E2F3A in G₁-S, E2F8 in S, and E2F4 in G₁ and S-G₂

(A) Immunofluorescence of EdU (red) and pH3 (green) in the ventricular zone of the brain of E13.5 wild-type embryos (DAPI counterstain [blue]). Left, composite image; middle left, green and red channels; middle right, red and blue channels; right, green and blue channels.
(B) Schematic of the proposed distinct temporal patterns of EdU and pH3 displayed in (A).
(C) Immunofluorescence of EdU (red) and E2F3A (green) (top), and pH3 (red) and E2F3A (green) (bottom), on the intestine of E13.5 wild-type embryos (DAPI counterstain [blue]).
(D) Automated quantification of E2F3A and EdU (top) and E2F3A and pH3 (bottom) in the intestinal epithelium of E13.5 wild-type embryos. Data are presented as mean ± SD; n = 3 mice.
(E) Immunofluorescence of EdU (red) and E2F8 (green) (top), and pH3 (red) and E2F8 (green) (bottom), on the intestine of E2f8^myc/myc E13.5 embryos. E2F8 was detected using MYC antibody (DAPI counterstain [blue]).
(F) Automated quantification of E2F8 and EdU (top), and E2F8 and pH3 (bottom) in the intestinal epithelium of E2f8^myc/myc E13.5 embryos. Data are presented as mean ± SD; n = 3 mice.
(G) Immunofluorescence of EdU (red) and E2F4 (green) (top), and pH3 (red) and E2F4 (green) (bottom) on the intestine of E2f4^myc/myc E13.5 embryos. E2F4 was detected using MYC antibody (DAPI counterstain [blue]).
(H) Automated quantification of E2F4 and EdU (top) and E2F4 and pH3 (bottom) in the intestinal epithelium of E2f4^myc/myc E13.5 embryos. Data are presented as mean ± SD; n = 2 mice.
Images in (C), (E), and (G) are confocal micrographs. Composite and color-split images of the boxed area are shown on right panels; top left, green and red channels; top right, green channel; bottom left, red channel; bottom right, blue channel. Solid arrowhead, EdU diffuse (EdU-Di); open arrowhead, EdU punctate (EdU-Pu); arrow, pH3 punctate (pH3-Pu); asterisk, pH3 diffuse (pH3-Di). Scale bars, 20 μm. See also and .

Two Distinct E2F Transcriptional Modules Drive Cell Cycles and Differentiation

Cell Rep. 2019 Jun 18;27(12):3547-3560.e5.