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...

SUMMARYTATA-binding protein-associated factors (TAFs) are general transcription factors within the transcription factor IID (TFIID) complex, which recognizes the core promoter of genes. In addition to their biochemical function, it is known that several TAFs are involved in the regulation of developmental processes. In this study, we found that TAF15b affects flowering time, especially through the autonomous pathway (AP) in Arabidopsis. The mutant taf15b shows late flowering compared with the wild type plant during both long and short days, and vernalization accelerates the flowering time of taf15b. In addition, taf15b shows strong upregulation of FLOWERING LOCUS C (FLC), a flowering repressor in Arabidopsis, and the flc taf15b double mutant completely offsets the late flowering of taf15b, indicating that TAF15b is a typical AP gene. The taf15b mutant also shows increased transcript levels of COOLAIR, an antisense transcript of FLC. Consistently, chromatin immunoprecipitation (ChIP) analyses showed that the TAF15b protein is enriched around both sense and antisense transcription start sites of the FLC locus. In addition, co-immunoprecipitation showed that TAF15b interacts with RNA polymerase II (Pol II), while ChIP showed increased enrichment of the phosphorylated forms, both serine 2 (Ser2) and Ser5, of the C-terminal domain of Pol II at the FLC locus, which is indicative of transcriptional elongation. Finally, taf15b showed higher enrichment of the active histone marker, H3K4me3, on FLC chromatin. Taken together, our results suggest that TAF15b affects flowering time through transcriptional repression of FLC in Arabidopsis.

The autonomous pathway promotes flowering by repressing a major flowering repressor, FLOWERING LOCUS C (FLC). Approximately 30 genes are involved in this pathway, and several of them are related to RNA processing; however, the molecular basis of the transcriptional regulation of FLC is yet to be understood. Recently, we discovered a new autonomous pathway gene, TATA-binding protein-associated factor 15b (TAF15b), which has a RNA recognition motif (RRM) and represses the level of FLC transcripts. TAF15b regulates the expression of FLC by directly interacting with RNA polymerase II (Pol II) at the transcription start sites on both the sense and antisense strands of the FLC locus. In addition to the transcriptional regulation in the nucleus, TAF15b accumulates in processing bodies (p-bodies), which are cytoplasmic RNA granules involved in translational repression, during heat stress. Here we discuss the implications of our findings and suggest a dual role of TAF15b in both transcriptional and translational regulation.