POLD2 And FEN1 Contribute To Epigenetic And Genomic Stability

DNA replication is a fundamental process in cells. DNA replication factors play roles in transmitting both the genetic (DNA sequence) and epigenetic (DNA methylation and histone modification) information to the next generation precisely, safeguarding the integrity and accuracy of the genome. Interruption of DNA replication leads to replication stress, genomic instability and even cancer. The study on DNA replication in model plant Arabidopsis is little especially on epigenetic regulation because of the lack of mutant materials. Here, several components involving in the regulation of transcriptional gene silencing (TGS) were isolated from a transgenic system. These factors are histone deacetylase (HDA6), histone deubiquitinase (UBP26), DNA polymerase delta subunit 2 (POLD2), DNA replication factor C1 (RFC1) and flap endonuclease 1 (FEN1). Since the epigenetic roles of POLD2 and FEN1 are largely unknown, we chose these two proteins for this study.POLD2 is a nuclear-localized protein and expressed ubiquitously in Arabidopsis. Immunoprecipitation followed by mass spectrometry (IP-MS) demonstrated that POLD2 could be co-purified with POLD1, POLD3, POLD4 and REV3. Genetic analysis indicated that POLD2 has a synergistic role with Pola and ATR to control plant development. Furthermore, the pold2-1 mutant exhibits genome instability with a high frequency of homologous recombination (HR), hypersensitivity to DNA-damaging reagents, and shorter telomere length. Meanwhile, the expression level of cell cycle marker CYCB1;1 was higher in the pold2-1 mutant. Whole-genome bisulfite sequencing showed that POLD2 was not involved in the regulation of DNA methylation. Whole-genome ChIP-seq and RNA-seq analyses suggested that pold2-1 affects H3K27me3 and H3K4me3 modifications which are correlated to the gene expression levels.The expression of FEN1 was more abundant in the root and shoot meristems where DNA replication were more active. Transient expression of FEN1-GFP in tobacco cells indicated that FEN1-GFP is a nuclear protein and accumulates in nucleolus. fenl-1 mutant is hypersensitive to MMS and shows reduced telomere length. Genome-wide ChIP-seq and RNA-seq results demonstrate that FEN1 mutation leads to a decrease in H3K27me3 level and an increase in the expression of a subset of genes marked with H3K27me3.Above all, we functionally characterized two DNA replication factors, POLD2 and FEN1, in Arabidopsis. Dysfunction of these two genes resulted in genomic instability with hypersensitive to DNA-damaging reagent and shorter telomere length. Furthermore, the combination of genome-wide RNA-seq and ChIP-seq uncovered a role of DNA replication factors in maintaining H3K27me3 and silencing both endogenous and transgenic genes. These findings may shed light on the study about the relationship between genetic and epigenetic instability and DNA replication.