The catalytic subunit of Arabidopsis DNA polymerase α ensures stable maintenance of histone modification

Youbong Hyun 2 Hyein Yun 2 Kyunghyuk Park 2 Hyonhwa Ohr 2 Okchan Lee 2 Dong-Hwan Kim 1 Sibum Sung 1 Yeonhee Choi 0 2 0 Plant Genomics and Breeding Institute, Seoul National University , Seoul 151-747 , Korea 1 Section of Molecular Cell and Developmental Biology and Institute for Cellular and Molecular Biology, University of Texas at Austin , TX 78712 , USA 2 Department of Biological Sciences, Seoul National University , Seoul, 151-742 , Korea SUMMARY Mitotic inheritance of identical cellular memory is crucial for development in multicellular organisms. The cell type-specific epigenetic state should be correctly duplicated upon DNA replication to maintain cellular memory during tissue and organ development. Although a role of DNA replication machinery in maintenance of epigenetic memory has been proposed, technical limitations have prevented characterization of the process in detail. Here, we show that INCURVATA2 (ICU2), the catalytic subunit of DNA polymerase a in Arabidopsis, ensures the stable maintenance of repressive histone modifications. The missense mutant allele icu2-1 caused a defect in the mitotic maintenance of vernalization memory. Although neither the recruitment of CURLY LEAF (CLF), a SET-domain component of Polycomb Repressive Complex 2 (PRC2), nor the resultant deposition of the histone mark H3K27me3 required for vernalization-induced FLOWERING LOCUS C (FLC) repression were affected, icu2-1 mutants exhibited unstable maintenance of the H3K27me3 level at the FLC region, which resulted in mosaic FLC de-repression after vernalization. ICU2 maintains the repressive chromatin state at additional PRC2 targets as well as at heterochromatic retroelements. In icu2-1 mutants, the subsequent binding of LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1), a functional homolog of PRC1, at PRC2 targets was also reduced. We demonstrated that ICU2 facilitates histone assembly in dividing cells, suggesting a possible mechanism for ICU2-mediated epigenetic maintenance. INTRODUCTION During eukaryotic chromosome replication, both genetic and epigenetic information are accurately duplicated, which maintains identical cellular memory through mitoses (Margueron and Reinberg, 2010). DNA replication accompanies chromatin assembly, and the cooperation between these two processes has long been proposed to be important for the mitotic maintenance of epigenetic information (Alabert and Groth, 2012; Ransom et al., 2010; Smith and Whitehouse, 2012). DNA polymerase is responsible for initiating replication at both the origins and the lagging strand (Kunkel and Burgers, 2008). In Arabidopsis, the single copy gene INCURVATA2 (ICU2) encodes the catalytic subunit of DNA polymerase , and transfer DNA (T-DNA)-inserted knockout mutants of ICU2 showed a lethal phenotype during gametogenesis and embryogenesis (Barrero et al., 2007). A missense mutation in ICU2, icu2-1, was isolated by the resulting upward curling phenotype of the rosette leaves (Serrano-Cartagena et al., 2000). This mutant exhibits early flowering and homeotic transition of the floral organs, as observed in mutants of Polycomb Repressive Complex 2 (PRC2) (Barrero et al., 2007). Accordingly, certain PRC2 target genes are de-regulated in icu2-1 (Barrero et al., 2007). Another missense allele of ICU2, pol, was identified by suppressor mutant screening of the repressor of transcriptional silencing 1 (ros1) (Liu et al., 2010a), which has a defect in the DNA demethylase ROS1 (Gong et al., 2002). The pol mutant exhibited a similar phenotype to icu2-1, and showed de-repression of some heterochromatic elements in the Arabidopsis genome, although the DNA methylation levels in the centromeric repeats and transposons were not affected. ICU2 has a genetic relationship with CURLY LEAF (CLF), a SET-domain component of PRC2; LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1), a potential PRC1 component; and FASCIATA1 (FAS1), a chromatin assembly factor (Barrero et al., 2007). The vernalization response in Arabidopsis thaliana is an example of the mitotic inheritance of histone marks in plants (Kim et al., 2009). Vernalization is the acquisition of floral competence through exposure to prolonged cold in winter. In Arabidopsis, the two strong floral repressors FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) synergistically interact to cause late flowering (Shindo et al., 2005). FRI induces the expression of FLC, and the resulting high level of FLC inhibits floral induction. However, vernalization induces the downregulation of FLC, and this repression is mitotically remembered until reproduction, even after a return to warm conditions (Choi et al., 2009; Kim et al., 2009; Sheldon et al., 2008). As a result, plants exposed to long-term cold treatment undergo the transition to flowering. Covalent histone modifications at the FLC genomic region is a key process for maintaining FLC repression (Kim et al., 2009). During vernalization, FLC repression is initiated by VERNALIZATION INSENSITIVE 3 (VIN3) through the deacetylation of histone H3. Simultaneously, VERNALIZATION2 (VRN2)-PRC2 deposits H3K27me3 at the FLC region (De Lucia et al., 2008; Finnegan and Dennis, 2007; Sung and Amasino, 2004; Wood et al., 2006). The accumulation of H3K27me3 results in the stable repression of FLC. Accordingly, a loss-of-function vrn2 mutant normally shows a downregulation of FLC during vernalization. However, the mutant fails to accumulate H3K27me3, so FLC is de-repressed when the vrn2 plants return to warm conditions (Gendall et al., 2001). Remarkably, the H3K27me3 mark is inherited and propagated to nearby histones in the FLC region in subsequent warm growing conditions by an unknown mechanism (De Lucia et al., 2008; Finnegan and Dennis, 2007). Therefore, stable inheritance of H3K27me3 and the concomitant FLC silencing are crucial for the acquisition of floral competence after vernalization. In this study, we presented new evidence that ICU2 is specifically involved in the maintenance of repressive histone marks during mitoses, but not in the mark deposition on histones, by analyzing the mitotic maintenance of vernalization memory in icu2-1 mutants. In addition, the role of ICU2 in silencing diverse chromatin loci and the functional relationship of ICU2 with PRC2 and LHP1 were also examined. Lastly, we identified a possible mechanism for ICU2-mediated epigenetic inheritance by analyzing DNA replication-dependent chromatin assembly in icu2-1 mutant plants. MATERIALS AND METHODS Plant materials, growing conditions, histochemical GUS staining and microscopy All plants used in this study originated from the Col-0 background except for the icu2-1 (En-2), pol (C24) and clf-2 (Ler) mutants. To generate icu2 1 FRI and clf-2 FRI, each mutant allele was introduced into FRI-Col through five backcrosses. The plants were grown in either long-day (16 hour light/8 hour dark) or short-day (8 hour light/16 hour dark) photoperiodic conditions under cool white fluorescent light (100 mole/m2/s) at 22C with (...truncated)