Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jan;31(1):132-147.
doi: 10.1096/fj.201600631R. Epub 2016 Sep 30.

Role of FEN1 S187 phosphorylation in counteracting oxygen-induced stress and regulating postnatal heart development

Lina Zhou  1   2 , Huifang Dai  2 , Jian Wu  3 , Mian Zhou  2 , Hua Yuan  4 , Juan Du  2 , Lu Yang  5 , Xiwei Wu  5 , Hong Xu  6 , Yuejin Hua  6 , Jian Xu  3 , Li Zheng  7 , Binghui Shen  8
Affiliations

Role of FEN1 S187 phosphorylation in counteracting oxygen-induced stress and regulating postnatal heart development

Lina Zhou et al. FASEB J. 2017 Jan.

Abstract

Flap endonuclease 1 (FEN1) phosphorylation is proposed to regulate the action of FEN1 in DNA repair as well as Okazaki fragment maturation. However, the biologic significance of FEN1 phosphorylation in response to DNA damage remains unknown. Here, we report an in vivo role for FEN1 phosphorylation, using a mouse line carrying S187A FEN1, which abolishes FEN1 phosphorylation. Although S187A mouse embryonic fibroblast cells showed normal proliferation under low oxygen levels (2%), the mutant cells accumulated oxidative DNA damage, activated DNA damage checkpoints, and showed G1-phase arrest at atmospheric oxygen levels (21%). This suggests an essential role for FEN1 phosphorylation in repairing oxygen-induced DNA damage and maintaining proper cell cycle progression. Consistently, the mutant cardiomyocytes showed G1-phase arrest due to activation of the p53-mediated DNA damage response at the neonatal stage, which reduces the proliferation potential of the cardiomyocytes and impairs heart development. Nearly 50% of newborns with the S187A mutant died in the first week due to failure to undergo the peroxisome proliferator-activated receptor signaling-dependent switch from glycolysis to fatty acid oxidation. The adult mutant mice developed dilated hearts and showed significantly shorter life spans. Altogether, our results reveal an important role of FEN1 phosphorylation to counteract oxygen-induced stress in the heart during the fetal-to-neonatal transition.-Zhou, L., Dai, H., Wu, J., Zhou, M., Yuan, H., Du, J., Yang, L., Wu, X., Xu, H., Hua, Y., Xu, J., Zheng, L., Shen, B. Role of FEN1 S187 phosphorylation in counteracting oxygen-induced stress and regulating postnatal heart development.

Keywords: G1-phase arrest; dilated heart; flap endonuclease 1; p53 signaling.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Atmospheric O2 levels (21%) induce G1 cell cycle arrest and reduce the cell proliferation rate of S187A FEN1 but not WT FEN1 MEF cells. A, B) Cell cycle analysis of primary WT (A) and S187A (B) MEF cells (passage 1) exposed to 2% O2. C, D) Cell cycle analysis of primary WT (C) and S187A (D) MEF cells (passage 1) exposed to 21% O2. WT and S187A MEF cells were synchronized at the G1/S boundary in 2 or 21% O2 and released into S phase. The cells were harvested at the indicated time points, and the cell cycle stages were analyzed by flow cytometry. The values are the averages of 2 independent assays (A–D). E, F) Cell proliferation rate of WT and S187A MEF cells in response to 2% (E) or 21% (F) O2. Equal numbers of WT and S187A MEFs were seeded and cultured in DMEM (10% FBS) for 7 d. Cells were counted each day using a hemocytometer. The values are the means ± sem of 3 independent assays.
Figure 2.
Figure 2.
S187A MEF cells accumulate oxidative DNA damage and activate the p53-dependent cell cycle checkpoint at atmospheric O2 levels. A, B) S187A MEF cells accumulate oxidative DNA damage. WT and S187A MEF cells were cultured in 2 or 21% O2 for 24 h. The 8-oxoguanine DNA damage was detected by immunofluorescence staining. A) Representative images of 8-oxoguanine staining. B) Quantification. The values are the means ± sem of 5 independent assays. Scale bars, 20 μm. C, D) S187A MEF cells have more DNA damage foci than WT cells. γH2AX and 53BP1 DNA damage foci in WT and S187A MEF cells (21% O2) were detected by immunofluorescence staining. C) Representative images of γH2AX and 53BP1 staining. D) Quantification. The values are the means ± sem of 3 independent assays. Scale bars, 20 μm. E) S187A MEF cells show increased levels of nuclear γH2AX, 53BP1, p53, and p21 based on Western blot analysis of nuclear extracts from WT and S187A MEF cells (21% O2). F) The p53 protein level in control (siControl) and p53 knockdown (sip53) S187A MEFs based on Western blot analysis. The β-actin was used as an internal loading control. G) Cell proliferation rate of control and p53 knockdown S187A MEFs under 21% O2. Equal numbers of S187A MEFs treated with control siRNA or siRNA against mouse p53 were seeded and cultured in DMEM (10% FBS) for 3 d. Cells were counted each day using a hemocytometer. The values are the means ± sd of 4 independent assays.
Figure 3.
Figure 3.
S187A increases the mortality of newborn and adult mice and causes dilated cardiac hypertrophy. A) Kaplan-Meier survival curve of WT and S187A mice. A total 100 WT and 571 S187A mice were followed for 60 wk, and the ages of these mice at death were recorded. The significance (P < 0.001) was calculated by the log-rank test. B, C) Representative macroimages (B) and microimages (C) of WT and S187A hearts. Scale bars, 1 mm. D) Heart weight (HW) was measured for WT male mice (n = 12), WT female mice (n = 11), S187A male mice (n = 14), and S187A female mice (n = 15). The cutoff value was set as the mean + 2 sd of WT female or male mice. Any heart with HW/body weight (BW) ratio greater than the cutoff value was considered aberrantly enlarged. P values were calculated using a Student’s t test.
Figure 4.
Figure 4.
S187A impairs cardiomyocyte proliferation at the neonatal but not embryonic stage. A, B) Cell cycle analysis of heart cells from WT and S187A embryonic (E19.5) and newborn (P1) mice. A) Representative flow cytometry graphs from the heart cells. B) Cell cycle distribution. C, D) Tissue staining for proliferation markers. Heart tissue cryosections were stained for Ki-67 (proliferation marker, red) and troponin T cardiac isoform Ab-1 (cardiomyocyte marker, green). Nuclei were stained with DAPI (blue). C) Representative images of heart tissue staining. Scale bars, 100 μm. D) Quantification. The percentage of cardiomyocytes expressing proliferation markers was calculated by dividing the number of Ki-67/troponin T cardiac isoform Ab-1 double-positive cells (pink nuclear and green cytoplasm staining, proliferating cardiomyocytes) by the number of troponin T cardiac isoform AB-1-positive cells (blue nuclear and green cytoplasm staining, total cardiomyocytes). Values are the means ± sem of 5 hearts per group (B, D). E) Nuclear Cdc6 in WT and S187A hearts. Western blot analysis was conducted on nuclear extracts from WT and S187A embryonic and neonatal hearts. F) Total numbers of cardiomyocytes in WT and S187A hearts at the E19.5, P1, and P7 stages. The cells with rectangular shapes from cardiac cell suspensions were scored as cardiomyocytes. The values are the means ± sem of 10 independent assays.
Figure 5.
Figure 5.
RNA-Seq reveals altered pathways for DNA replication and energy production during the transition from the embryonic to postnatal stages. A) Schematic graph describing the experimental groups for gene expression profiling analysis by RNA-Seq. Hearts from three female embryos (E19.5) or newborns (P1) from the WT or S187A genetic background were used to investigate the transition from the embryonic to postnatal stages. B) PCA of gene expression profiles across experimental groups. C) Pathway analysis by IPA. The gene expression profiles of S187A E19.5 embryos vs. WT E19.5 embryos, WT P1 newborns vs. WT E19.5 embryos, and S187A P1 newborns vs. S187A E19.5 embryos were compared. Significantly changed pathways (P < 0.05 and false discovery rate <0.05) and corresponding affected genes are listed. Red: up-regulated pathways and genes; green: down-regulated pathways and genes.
Figure 6.
Figure 6.
Quantitative RT-PCR demonstrates that S187A alters newborn gene expression. Quantitative RT-PCR was conducted on genes from Fig. 5C in the following categories: myocardial fatty acid metabolism (A), glycolysis (B), cytokinesis (C), p53-mediated cell cycle arrest (D), inflammatory response (E), DNA replication (F), and DNA double-strand break response (G). The values are the means ± sem of assays from 3 hearts. The expression level of each gene was normalized with that of 18s rRNA. The normalized expression level of each gene in the WT E19.5 heart (control) was arbitrarily set as 1, and the relative gene expression level was calculated by comparison of the normalized level in each group to the control.
Figure 7.
Figure 7.
Down-regulation PPAR signaling is linked to S187A neonatal death. A) mRNA levels of PPAR-α and PPAR-γ target genes in WT (n = 5), S187A (moribund; n = 4), and S187A (live; n = 3) neonatal hearts (P1). The moribund state of S187A was predicted based on their inactivity and failure to suck milk. Such newborns would die within a couple of hours. Conversely, the live newborns were those that could actively suck milk. The mRNA levels were determined by RNA-Seq. The fold change was calculated by comparing the read value for each sample with the mean value of WT, which is arbitrarily set as 1. Values are means ± sem for each group. The P values were calculated by Students’ t test. The top PPAR-α and PPAR-γ target genes were defined by IPA. B) Quantitative PCR validates gene expression of ANGPTL4. Values are means ± sem of 3 independent analyses. C). mRNA levels of PPAR-α, PPAR-γ, PPARGC1a, and PPARGC1b. The mRNA levels were determined by RNA-Seq and the fold change, and P values were calculated as described in A. Values are means ± sem for each group. D) Model for the role of FEN1 phosphorylation in cell cycle-dependent oxidative DNA damage repair for maintaining the proliferation of postnatal cardiomyocytes. After birth, cardiomyocytes are exposed to increased levels of O2 and use fatty acid oxidation for energy production. These changes produce reaction oxidative species, which can damage DNA. Under normal physiologic conditions, oxidative DNA damage to G1-phase cells, which accounts for the majority of cells at any time, is readily repaired via FEN1-mediated long-patch base excision repair, allowing the cell cycle to progress. However, most FEN1 is degraded in the G2 phase, meaning that oxidative DNA damage to G2-phase cells may not be repaired. This oxidative damage will then activate ATM, leading to polyploidy and terminal differentiation of cardiomyocytes. The S187A FEN1 phosphorylation-deficient prevents DNA damage repair and leads to G1 cell cycle arrest. Therefore, FEN1 is critical for maintaining the proliferation potential of cardiomyocytes.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hanahan D., Weinberg R. A. (2011) Hallmarks of cancer: the next generation. Cell 144, 646–674 - PubMed
    1. Lombard D. B., Chua K. F., Mostoslavsky R., Franco S., Gostissa M., Alt F. W. (2005) DNA repair, genome stability, and aging. Cell 120, 497–512 - PubMed
    1. Aguilera A., Gómez-González B. (2008) Genome instability: a mechanistic view of its causes and consequences. Nat. Rev. Genet. 9, 204–217 - PubMed
    1. Branzei D., Foiani M. (2010) Maintaining genome stability at the replication fork. Nat. Rev. Mol. Cell Biol. 11, 208–219 - PubMed
    1. Lieber M. R. (1997) The FEN-1 family of structure-specific nucleases in eukaryotic DNA replication, recombination and repair. BioEssays 19, 233–240 - PubMed

Publication types

LinkOut - more resources

-