Identification of a DNA methylation-independent imprinting control region at the Arabidopsis MEDEA locus.

Genomic imprinting is exclusive to mammals and seed plants and refers to parent-of-origin-dependent, differential transcription. As previously shown in mammals, studies in Arabidopsis have implicated DNA methylation as an important hallmark of imprinting. The current model suggests that maternally expressed imprinted genes, such as MEDEA (MEA), are activated by the DNA glycosylase DEMETER (DME), which removes DNA methylation established by the DNA methyltransferase MET1. We report the systematic functional dissection of the MEA cis-regulatory region, resulting in the identification of a 200-bp fragment that is necessary and sufficient to mediate MEA activation and imprinted expression, thus containing the imprinting control region (ICR). Notably, imprinted MEA expression mediated by this ICR is independent of DME and MET1, consistent with the lack of any significant DNA methylation in this region. This is the first example of an ICR without differential DNA methylation, suggesting that factors other than DME and MET1 are required for imprinting at the MEA locus.

The Polycomb group protein MEDEA and the DNA methyltransferase MET1 interact to repress autonomous endosperm development in Arabidopsis.

In flowering plants, double fertilization of the female gametes, the egg and the central cell, initiates seed development to give rise to a diploid embryo and the triploid endosperm. In the absence of fertilization, the FERTILIZATION-INDEPENDENT SEED Polycomb Repressive Complex 2 (FIS-PRC2) represses this developmental process by histone methylation of certain target genes. The FERTILIZATION-INDEPENDENT SEED (FIS) class genes MEDEA (MEA) and FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) encode two of the core components of this complex. In addition, DNA methylation establishes and maintains the repression of gene activity, for instance via DNA METHYLTRANSFERASE1 (MET1), which maintains methylation of symmetric CpG residues. Here, we demonstrate that Arabidopsis MET1 interacts with MEA in vitro and in a yeast two-hybrid assay, similar to the previously identified interaction of the mammalian homologues DNMT1 and EZH2. MET1 and MEA share overlapping expression patterns in reproductive tissues before and after fertilization, a prerequisite for an interaction in vivo. Importantly, a much higher percentage of central cells initiate endosperm development in the absence of fertilization in mea-1/MEA; met1-3/MET1 as compared to mea-1/MEA mutant plants. In addition, DNA methylation at the PHERES1 and MEA loci, imprinted target genes of the FIS-PRC2, was affected in the mea-1 mutant compared with wild-type embryos. In conclusion, our data suggest a mechanistic link between two major epigenetic pathways involved in histone and DNA methylation in plants by physical interaction of MET1 with the FIS-PRC2 core component MEA. This concerted action is relevant for the repression of seed development in the absence of fertilization.

Amiloride uptake and toxicity in fission yeast are caused by the pyridoxine transporter encoded by bsu1+ (car1+).

Amiloride, a diuretic drug that acts by inhibition of various sodium transporters, is toxic to the fission yeast Schizosaccharomyces pombe. Previous work has established that amiloride sensitivity is caused by expression of car1+, which encodes a protein with similarity to plasma membrane drug/proton antiporters from the multidrug resistance family. Here we isolated car1+ by complementation of Saccharomyces cerevisiae mutants that are deficient in pyridoxine biosynthesis and uptake. Our data show that Car1p represents a new high-affinity, plasma membrane-localized import carrier for pyridoxine, pyridoxal, and pyridoxamine. We therefore propose the gene name bsu1+ (for vitamin B6 uptake) to replace car1+. Bsu1p displays an acidic pH optimum and is inhibited by various protonophores, demonstrating that the protein works as a proton symporter. The expression of bsu1+ is associated with amiloride sensitivity and pyridoxine uptake in both S. cerevisiae and S. pombe cells. Moreover, amiloride acts as a competitor of pyridoxine uptake, demonstrating that both compounds are substrates of Bsu1p. Taken together, our data show that S. pombe and S. cerevisiae possess unrelated plasma membrane pyridoxine transporters. The S. pombe protein may be structurally related to the unknown human pyridoxine transporter, which is also inhibited by amiloride.