Control of DEMETER DNA demethylase gene transcription in male and female gamete companion cells inArabidopsis thaliana

Abstract

The DEMETER (DME) DNA glycosylase initiates active DNA demethylation via the base-excision repair pathway and is vital for reproduction in Arabidopsis thaliana. DME-mediated DNA demethylation is preferentially targeted to small, AT-rich, and nucleosome-depleted euchromatic transposable elements, influencing expression of adjacent genes and leading to imprinting in the endosperm. In the female gametophyte, DME expression and subsequent genome-wide DNA demethylation are confined to the companion cell of the egg, the central cell. Here, we show that, in the male gametophyte, DME expression is limited to the companion cell of sperm, the vegetative cell, and to a narrow window of time: immediately after separation of the companion cell lineage from the germline. We define transcriptional regulatory elements of DME using reporter genes, showing that a small region, which surprisingly lies within the DME gene, controls its expression in male and female companion cells. DME expression from this minimal promoter is sufficient to rescue seed abortion and the aberrant DNA methylome associated with the null dme-2 mutation. Within this minimal promoter, we found short, conserved enhancer sequences necessary for the transcriptional activities of DME and combined predicted binding motifs with published transcription factor binding coordinates to produce a list of candidate upstream pathway members in the genetic circuitry controlling DNA demethylation in gamete companion cells. These data show how DNA demethylation is regulated to facilitate endosperm gene imprinting and potential transgenerational epigenetic regulation, without subjecting the germline to potentially deleterious transposable element demethylation.S exual reproduction is characterized by fertilization of an egg by a sperm cell, generating the embryo. Uniquely in angiosperms, a second sperm cell fertilizes the companion cell of the egg, the central cell, to generate the endosperm, which supports development of the embryo. During reproduction in angiosperm Arabidopsis thaliana, the DEMETER (DME) DNA glycosylase exhibits a striking expression pattern. Within the ovule, the female gametophyte is generated by mitosis of the haploid megaspore, forming a mature gametophyte of seven cells. During this process, the egg and central cell lineages are separated, and, at this point, DME expression and DNA demethylation is initiated solely in the central cell (1, 2). DME expression is switched off after fertilization (2). This precise pattern of expression initiated in the central cell, and not in the egg cell, is responsible for hypomethylation specifically in the maternal endosperm genome and not in the maternal embryo genome (3). DME expression in the central cell is essential for plant reproduction and genomic imprinting, whereby its absence results in loss of genomic imprinting, aberrant endosperm development, and early seed abortion (2, 4, 5).In the male gametophyte, indirect evidence suggests that DME is expressed during development of the mature three-cell pollen gra...

3-Hydroxybutyrate Is Active Compound in Flax that Upregulates Genes Involved in DNA Methylation

“…DME glycosylase enzyme mainly operates in the DNA regions enriched with AT and acts to remove 5-methylcytosine and nick the DNA backbone, followed by repair and replacement with cytosine. Recent data suggested that in Arabidopsis DNA demethylation mediated by DME facilitates endosperm gene imprinting and potential transgenerational epigenetic regulation of transposable element demethylation required for plant reproduction [41]. We propose that remodeled, nucleosome-depleted chromatin resulted from the combined action of DDM1 and MET3, becomes preferentially targeted to DME demethylation at the sites enriched with AT such as the promoter region and influences expression of adjacent genes.…”

The Catalytic Core of DEMETER Guides Active DNA Demethylation in Arabidopsis

“…polypeptides of 1,729 (DME.1) and 1,987 (DME.2) amino--acids in length (17,29). The amino acid positions denoted in this study correspond to DME.2, the predominant isoform expressed in floral tissues (19). First, we detailed the domain structure and predicted characteristics of the catalytic and non--catalytic regions of the DME protein.…”

“…The A, Glycosylase, and B regions of the C--terminal half of DME are conserved among the DME/ROS1 DNA glycosylase clade, and are absolutely required for DME 5mC excision activity in vitro, comprising the minimal catalytic core for direct excision of 5mC from DNA (11,17,18). DME acts specifically in the central cell of the female gametophyte and the vegetative nucleus of pollen (17,19,20). The pollen grain contains the vegetative nucleus and two sperm cells, and the vegetative nucleus contributes to the germination and growth of the pollen tube, which delivers the to sperm cells to the female gametophyte.…”

The FACT complex is required for DNA demethylation at heterochromatin during reproduction in Arabidopsis

“…The vegetative and central cells, adjacent to the sperm and egg cells, respectively, are so-called gamete companion cells. In Arabidopsis thaliana, active DNA demethylation by the DNA glycosylase DEMETER (DME) occurs specifically in gamete companion cells, whereby highly specific transcriptional regulation during gametogenesis ensures DME expression is confined to these cells (9)(10)(11). DME-mediated DNA demethylation occurs at thousands of discrete loci genome-wide, including regulatory regions for genes encoding components of the Polycomb Repressive Complex 2 (PRC2); FERTILIZATION INDEPENDENT SEED 2 (FIS2) and MEDEA (MEA) inducing their monoallelic expression, i.e.…”