Arabidopsis Zinc-Finger-Like Protein ASYMMETRIC LEAVES2 (AS2) and Two Nucleolar Proteins Maintain Gene Body DNA Methylation in the Leaf Polarity Gene ETTIN (ARF3)

Abstract

Arabidopsis ASYMMETRIC LEAVES2 (AS2) plays a critical role in leaf adaxial-abaxial partitioning by repressing expression of the abaxial-determining gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3). We previously reported that six CpG dinucleotides in its exon 6 are thoroughly methylated by METHYLTRASFERASE1, that CpG methylation levels are inversely correlated with ETT/ARF3 transcript levels and that methylation levels at three out of the six CpG dinucleotides are decreased in as2-1. All these imply that AS2 is involved in epigenetic repression of ETT/ARF3 by gene body DNA methylation. The mechanism of the epigenetic repression by AS2, however, is unknown. Here, we tested mutations of NUCLEOLIN1 (NUC1) and RNA HELICASE10 (RH10) encoding nucleolus-localized proteins for the methylation in exon 6 as these mutations enhance the level of ETT/ARF3 transcripts in as2-1. Methylation levels at three specific CpGs were decreased in rh10-1, and two of those three overlapped with those in as2-1. Methylation levels at two specific CpGs were decreased in nuc1-1, and one of those three overlapped with that in as2-1. No site was affected by both rh10-1 and nuc1-1. One specific CpG was unaffected by these mutations. These results imply that the way in which RH10, NUC1 and AS2 are involved in maintaining methylation at five CpGs in exon 6 might be through at least several independent pathways, which might interact with each other. Furthermore, we found that AS2 binds specifically the sequence containing CpGs in exon 1 of ETT/ARF3, and that the binding requires the zinc-finger-like motif in AS2 that is structurally similar to the zinc finger-CxxC domain in vertebrate DNA methyltransferase1.

The formation of perinucleolar bodies is important for normal leaf development and requires the zinc‐finger DNA‐binding motif in Arabidopsis ASYMMETRIC LEAVES2

“…Similarly, in Arabidopsis, it is proposed that two orthologs of nucleolin, NUC1 and NUC2, are also abundant nucleolar proteins (Durut et al , ) and might act as components of the putative SSUP‐like complex (Matsumura et al , ). In this context, it is worth noting that NUC1, as well as the AS2–AS1 complex, is involved in maintaining methylated CpG dinucleotides in exon 6 in the target ETT/ARF3 gene (Vial‐Pradel et al , ). Such methylation depends on the activity of MET1, which is an Arabidopsis homolog of the vertebrate Dnmt1 methyltransferase that mediates maintenance of CpG methylation during DNA replication (Long et al , ; Iwasaki et al , ; Vial‐Pradel et al , ).…”

“…This motif in a wheat member of the AS2/LOB family binds a zinc ion, which is required for binding to DNA (Chen et al , ), an observation that implies that members of this family can form zinc‐finger (ZF) structures. The ZF motif is essential for binding of AS2 to the CpG repeat in exon 1 of its target ETT/ARF3 gene (Vial‐Pradel et al , ). The AS2/LOB domain includes two additional conserved regions: an internal‐co‐nserved‐glycine (ICG) region and a leucine‐zipper‐like (LZL) region (Iwakawa et al , ; Matsumura et al , ).…”

SIMON: Simple methods for analyzing DNA methylation by targeted bisulfite next-generation sequencing

“…Very small changes of methylation rate often have significant biological significance (Vial-Pradel et al 2018). We conducted power analyses of the Fisher's exact test in order to estimate the necessary number of samples (DNA-fragments) to detect a difference of 10% methylation.…”

“…Each of these proteins seems to be differentially involved in the maintenance of several specific combinations of CpG sites. These results imply that the molecular events of CpG methylation are achieved by the differential actions of these proteins (Vial-Pradel et al 2018). To understand the complex molecular pathways involved in the maintenance of CpG methylation and regulation of gene expression, we need a method that offers single base resolution.…”

Roles of ASYMMETRIC LEAVES2 (AS2) and Nucleolar Proteins in the Adaxial–Abaxial Polarity Specification at the Perinucleolar Region in Arabidopsis

“…Transcript levels of several abaxial-determining genes ( KAN2 , YAB5 , ETT/ARF3 , and ARF4 ) are slightly upregulated in the as2-1 single mutant and each of the modifier single mutants and are markedly increased in the as2-1 and modifier double mutants (for example, as2-1 rh10-1 ). When the double mutations of ETT/ARF3 and ARF4 are introduced to double mutants with as2-1 and one of the modifier mutations, such as as2-1 nuc1-1 or as2-1 rh10-1 , the abaxialized filamentous leaves phenotype (e.g., as2 rh10 leaves in Figure 3 ) is restored to the expanded shapes [ 59 , 64 , 65 ]. These results show that the upregulation of these ARF genes in the double mutants is responsible for the disappearance of their adaxial specification in their filamentous leaves.…”