The catalytic subunit ofArabidopsisDNA polymerase α ensures stable maintenance of histone modification

Abstract

SUMMARYMitotic 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. KEY WORDS: DNA polymerase a, Epigenetic maintenance, Chromatin assemblyThe catalytic subunit of Arabidopsis DNA polymerase a ensures stable maintenance of histone modification RESEARCH ARTICLEEpigenetic maintenance by DNA pol a 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 microscopyAll plants used in this study originated from the Col-0 background except for the icu2-1 (En-2), polα (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) ...

The second subunit of DNA-polymerase delta is required for genomic stability and epigenetic regulation

“…It was previously proposed that DNA polymerase d may play a role in preventing replication stress or HR and in depositing active H3K4me3 marks (Iglesias et al, 2015). Meanwhile, some studies indicate that the catalytic subunit of DNA polymerase a interacts with (Barrero et al, 2007) or perturbs the binding of like-heterochromatin protein 1 (LHP1; Hyun et al, 2013), which is the main reader/effector for H3K27me3 in Arabidopsis (Barrero et al, 2007;Zhang et al, 2007). Another link between DNA replication and H3K27me3 is MSI1 (multicopy suppressor of ira1), a chromatin assembly factor in Arabidopsis, which interacts with PcG protein and LHP1 (Derkacheva et al, 2013).…”

Ctf4-related protein recruits LHP1-PRC2 to maintain H3K27me3 levels in dividing cells in Arabidopsis thaliana

“…Mutations in the three DNA-pols enhance the abnormal phenotype of PcG mutants or cause deregulated expression of PcG target genes (25)(26)(27)(28). Although evidence for a direct interaction of LHP1 with ICU2 is slightly conflicting (25,27), it is conceivable that these proteins, which engage in higher-order protein complexes, possess several interfaces to other proteins.…”

“…Although evidence for a direct interaction of LHP1 with ICU2 is slightly conflicting (25,27), it is conceivable that these proteins, which engage in higher-order protein complexes, possess several interfaces to other proteins. Complexes may also share subunits, as reported for the WD40 domain protein MSI1, which is an integral part of PRC2 but also of Chromatin assembly factor 1 (CAF1), the histone H3-H4 chaperone that assists nucleosome assembly during replication (45).…”

“…Similar genetic interaction was suggested by the observation that a temperature-conditional DNA-polδ mutant, gigantea suppressor (gis) 5, expresses increased levels of SEPPALATA (SEP) 3 and FLOWERING LOCUS T (FT), which are also up-regulated in lhp1 and clf mutants (28). Physical interaction between LHP1 and ESD7 or ICU2 was shown by in vitro pull-down (25,26), but the latter could not be confirmed in a later study (27). Nevertheless, the genetic data suggest that there exists a tight link between DNA-pols and an LHP1-associated PRC2.…”

“…Mutations in INCURVATA (ICU) 2 and EARLY IN SHORT DAYS (ESD) 7, respectively encoding homologs of the priming DNA-polα and the leading strand-specific DNA-pole, were shown to enhance both lhp1 and clf mutant phenotypes (25)(26)(27). Similar genetic interaction was suggested by the observation that a temperature-conditional DNA-polδ mutant, gigantea suppressor (gis) 5, expresses increased levels of SEPPALATA (SEP) 3 and FLOWERING LOCUS T (FT), which are also up-regulated in lhp1 and clf mutants (28).…”