Poldip2 knockout results in perinatal lethality, reduced cellular growth and increased autophagy of mouse embryonic fibroblasts.

David I Brown,Rostam Zafari,Minyoung Lee,James S Long,Bernard Lassègue,Harold I Saavedra,Kathy K Griendling

doi:10.1371/journal.pone.0096657

Abstract

Polymerase-δ interacting protein 2 (Poldip2) is an understudied protein, originally described as a binding partner of polymerase delta and proliferating cell nuclear antigen (PCNA). Numerous roles for Poldip2 have been proposed, including mitochondrial elongation, DNA replication/repair and ROS production via Nox4. In this study, we have identified a novel role for Poldip2 in regulating the cell cycle. We used a Poldip2 gene-trap mouse and found that homozygous animals die around the time of birth. Poldip2−/− embryos are significantly smaller than wild type or heterozygous embryos. We found that Poldip2−/− mouse embryonic fibroblasts (MEFs) exhibit reduced growth as measured by population doubling and growth curves. This effect is not due to apoptosis or senescence; however, Poldip2−/− MEFs have higher levels of the autophagy marker LC3b. Measurement of DNA content by flow cytometry revealed an increase in the percentage of Poldip2−/− cells in the G1 and G2/M phases of the cell cycle, accompanied by a decrease in the percentage of S-phase cells. Increases in p53 S20 and Sirt1 were observed in passage 2 Poldip2−/− MEFs. In passage 4/5 MEFs, Cdk1 and CyclinA2 are downregulated in Poldip2−/− cells, and these changes are reversed by transfection with SV40 large T-antigen, suggesting that Poldip2 may target the E2F pathway. In contrast, p21CIP1 is increased in passage 4/5 Poldip2−/− MEFs and its expression is unaffected by SV40 transfection. Overall, these results reveal that Poldip2 is an essential protein in development, and underline its importance in cell viability and proliferation. Because it affects the cell cycle, Poldip2 is a potential novel target for treating proliferative conditions such as cancer, atherosclerosis and restenosis.

Highlights

Polymerase delta interacting protein 2 (Poldip2, PDIP38, Mitogenin 1) is a ubiquitously expressed, 368 amino acid protein consisting of an N-terminal mitochondrial localization sequence and two main highly conserved functional domains: a ApaG/F box A domain and a hemimethylated DNA binding domain called YccV
To clarify the role of Polymerase-d interacting protein 2 (Poldip2) in vivo, we used mice that have a gene trap construct inserted into the first intron of Poldip2, disrupting gene expression [11]
We uncovered a novel role of Poldip2 in cell cycle regulation (Figure 9)

Summary

Introduction

Polymerase delta interacting protein 2 (Poldip, PDIP38, Mitogenin 1) is a ubiquitously expressed, 368 amino acid protein consisting of an N-terminal mitochondrial localization sequence and two main highly conserved functional domains: a ApaG/F box A domain and a hemimethylated DNA binding domain called YccV. It was originally identified as a binding partner of polymerase-d and Proliferating Cell Nuclear Antigen (PCNA) [1]. Depletion of Poldip resulted in increased Polg foci in normal conditions and reduced cell survival after UV treatment. There has been no study directly testing the role of Poldip in regulating the proteins involved in cell cycle progression, nor has its role in apoptosis, senescence and autophagy been investigated

Methods

Results

Conclusion