FACT complex is required for DNA demethylation at heterochromatin during reproduction in <i>Arabidopsis</i>

Frost, Jennifer M.,Kim, M. Yvonne,Park, Guen Tae,Hsieh, Ping-Hung,Nakamura, Miyuki,Lin, Samuel J. H.,Yoo, Hyunjin,Choi, Jaemyung,Ikeda, Yoko,Kinoshita, Tetsu,Choi, Yeonhee,Zilberman, Daniel,Fischer, R National Academy of Sciences 2018 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.115 No.20

<▼1><P><B>Significance</B></P><P>The chromatin remodeling activities of the FACT (facilitates chromatin transactions) complex are required for many cellular functions, including transcription, DNA replication, and repair. Here, we demonstrate that the two FACT subunits, SSRP1 and SPT16, are also required for genome-wide DNA demethylation and regulation of gene imprinting during <I>Arabidopsis</I> reproduction. Without FACT, <I>Arabidopsis</I> seeds undergo abnormal development and exhibit aberrant DNA hypermethylation, including at imprinting control region loci. We show that FACT associates with the DEMETER (DME) DNA demethylase, facilitating DNA demethylation at over half of DME’s targets, specifically those which reside in heterochromatin. These results provide insight into upstream events in the DNA demethylation pathway and reveal the importance of chromatin remodeling for DNA demethylation during <I>Arabidopsis</I> reproduction.</P></▼1><▼2><P>The DEMETER (DME) DNA glycosylase catalyzes genome-wide DNA demethylation and is required for endosperm genomic imprinting and embryo viability. Targets of DME-mediated DNA demethylation reside in small, euchromatic, AT-rich transposons and at the boundaries of large transposons, but how DME interacts with these diverse chromatin states is unknown. The STRUCTURE SPECIFIC RECOGNITION PROTEIN 1 (SSRP1) subunit of the chromatin remodeler FACT (facilitates chromatin transactions), was previously shown to be involved in the DME-dependent regulation of genomic imprinting in <I>Arabidopsis</I> endosperm. Therefore, to investigate the interaction between DME and chromatin, we focused on the activity of the two FACT subunits, SSRP1 and SUPPRESSOR of TY16 (SPT16), during reproduction in <I>Arabidopsis</I>. We found that FACT colocalizes with nuclear DME in vivo, and that DME has two classes of target sites, the first being euchromatic and accessible to DME, but the second, representing over half of DME targets, requiring the action of FACT for DME-mediated DNA demethylation genome-wide. Our results show that the FACT-dependent DME targets are GC-rich heterochromatin domains with high nucleosome occupancy enriched with H3K9me2 and H3K27me1. Further, we demonstrate that heterochromatin-associated linker histone H1 specifically mediates the requirement for FACT at a subset of DME-target loci. Overall, our results demonstrate that FACT is required for DME targeting by facilitating its access to heterochromatin.</P></▼2>

Optimized Methods for the Isolation of Arabidopsis Female Central Cells and Their Nuclei

Park, Kyunghyuk,Frost, Jennifer M.,Adair, Adam James,Kim, Dong Min,Yun, Hyein,Brooks, Janie S.,Fischer, Robert L.,Choi, Yeonhee Korean Society for Molecular and Cellular Biology 2016 Molecules and cells Vol.39 No.10

The Arabidopsis female gametophyte contains seven cells with eight haploid nuclei buried within layers of sporophytic tissue. Following double fertilization, the egg and central cells of the gametophyte develop into the embryo and endosperm of the seed, respectively. The epigenetic status of the central cell has long presented an enigma due both to its inaccessibility, and the fascinating epigenome of the endosperm, thought to have been inherited from the central cell following activity of the DEMETER demethylase enzyme, prior to fertilization. Here, we present for the first time, a method to isolate pure populations of Arabidopsis central cell nuclei. Utilizing a protocol designed to isolate leaf mesophyll protoplasts, we systematically optimized each step in order to efficiently separate central cells from the female gametophyte. We use initial manual pistil dissection followed by the derivation of central cell protoplasts, during which process the central cell emerges from the micropylar pole of the embryo sac. Then, we use a modified version of the Isolation of Nuclei TAgged in specific Cell Types (INTACT) protocol to purify central cell nuclei, resulting in a purity of 75-90% and a yield sufficient to undertake downstream molecular analyses. We find that the process is highly dependent on the health of the original plant tissue used, and the efficiency of protoplasting solution infiltration into the gametophyte. By isolating pure central cell populations, we have enabled elucidation of the physiology of this rare cell type, which in the future will provide novel insights into Arabidopsis reproduction.

Gametophytic Abortion in Heterozygotes but Not in Homozygotes: Implied Chromosome Rearrangement during T-DNA Insertion at the ASF1 Locus in Arabidopsis

Min, Yunsook,Frost, Jennifer M.,Choi, Yeonhee Korean Society for Molecular and Cellular Biology 2020 Molecules and cells Vol.43 No.5

T-DNA insertional mutations in Arabidopsis genes have conferred huge benefits to the research community, greatly facilitating gene function analyses. However, the insertion process can cause chromosomal rearrangements. Here, we show an example of a likely rearrangement following T-DNA insertion in the Anti-Silencing Function 1B (ASF1B) gene locus on Arabidopsis chromosome 5, so that the phenotype was not relevant to the gene of interest, ASF1B. ASF1 is a histone H3/H4 chaperone involved in chromatin remodeling in the sporophyte and during reproduction. Plants that were homozygous for mutant alleles asf1a or asf1b were developmentally normal. However, following self-fertilization of double heterozygotes (ASF1A/asf1a ASF1B/asf1b, hereafter AaBb), defects were visible in both male and female gametes. Half of the AaBb and aaBb ovules displayed arrested embryo sacs with functional megaspore identity. Similarly, half of the AaBb and aaBb pollen grains showed centromere defects, resulting in pollen abortion at the bi-cellular stage of the male gametophyte. However, inheritance of the mutant allele in a given gamete did not solely determine the abortion phenotype. Introducing functional ASF1B failed to rescue the AaBb- and aaBb-mediated abortion, suggesting that heterozygosity in the ASF1B gene causes gametophytic defects, rather than the loss of ASF1. The presence of reproductive defects in heterozygous mutants but not in homozygotes, and the characteristic all-or-nothing pollen viability within tetrads, were both indicative of commonly-observed T-DNA-mediated translocation activity for this allele. Our observations reinforce the importance of complementation tests in assigning gene function using reverse genetics.

Nucleoporin MOS7/Nup88 is required for mitosis in gametogenesis and seed development in <i>Arabidopsis</i>

Park, Guen Tae,Frost, Jennifer M.,Park, Jin-Sup,Kim, Tae Ho,Lee, Jong Seob,Oh, Sung Aeong,Twell, David,Brooks, Janie Sue,Fischer, Robert L.,Choi, Yeonhee National Academy of Sciences 2014 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.111 No.51

<P><B>Significance</B></P><P>During plant reproduction, meiosis generates haploid spores that undergo mitoses, forming gametophytes, in male and female parts of the flower. Haploid cells within gametophytes differentiate into sperm and eggs, which form the next generation. We discovered that the <I>MOS7</I> (Modifier of Snc1,7) gene plays a critical role in microtubule organization and dynamics during mitosis in <I>Arabidopsis</I> sexual reproduction. MOS7 absence results in impaired gametogenesis, failure of ovule and pollen development, and seed abortion. Although mitoses during gamete formation are unique to plants, the regulation of mitosis itself is highly conserved between plants and vertebrates. Notably, we find that MOS7 protein primarily produced prior to meiosis is inherited by gametophytes, and, only if there is sufficient MOS7 protein, will mitosis correctly occur during gametogenesis.</P><P>Angiosperm reproduction is characterized by alternate diploid sporophytic and haploid gametophytic generations. Gametogenesis shares similarities with that of animals except for the formation of the gametophyte, whereby haploid cells undergo several rounds of postmeiotic mitosis to form gametes and the accessory cells required for successful reproduction. The mechanisms regulating gametophyte development in angiosperms are incompletely understood. Here, we show that the nucleoporin Nup88-homolog MOS7 (Modifier of Snc1,7) plays a crucial role in mitosis during both male and female gametophyte formation in <I>Arabidopsis thaliana</I>. Using a mutagenesis screen, we identify the <I>mos7-5</I> mutant allele, which causes ovule and pollen abortion in <I>MOS7/mos7-5</I> heterozygous plants, and preglobular stage embryonic lethality in homozygous <I>mos7-5</I> seeds. During interphase, we show that MOS7 is localized to the nuclear membrane but, like many nucleoporins, is associated with the spindle apparatus during mitosis. We detect interactions between MOS7 and several nucleoporins known to control spindle dynamics, and find that in pollen from <I>MOS7</I>/<I>mos7-5</I> heterozygotes, abortion is accompanied by a failure of spindle formation, cell fate specification, and phragmoplast activity. Most intriguingly, we show that following gamete formation by <I>MOS7/mos7-5</I> heterozygous spores, inheritance of either the <I>MOS7</I> or the <I>mos7-5</I> allele by a given gamete does not correlate with its respective survival or abortion. Instead, we suggest a model whereby MOS7, which is highly expressed in the Pollen- and Megaspore Mother Cells, enacts a dosage-limiting effect on the gametes to enable their progression through subsequent mitoses.</P>

Optimized Methods for the Isolation of Arabidopsis Female Central Cells and Their Nuclei

Yeonhee Choi,Kyunghyuk Park,Jennifer M. Frost,Adam James Adair,Dong Min Kim,Hyein Yun,Janie S. Brooks,Robert L. Fischer 한국분자세포생물학회 2016 Molecules and cells Vol.39 No.10

The Arabidopsis female gametophyte contains seven cells with eight haploid nuclei buried within layers of sporophytic tissue. Following double fertilization, the egg and central cells of the gametophyte develop into the embryo and endosperm of the seed, respectively. The epigenetic status of the central cell has long presented an enigma due both to its inaccessibility, and the fascinating epigenome of the endosperm, thought to have been inherited from the central cell following activity of the DEMETER demethylase enzyme, prior to fertilization. Here, we present for the first time, a method to isolate pure populations of Arabidopsis central cell nuclei. Utilizing a protocol designed to isolate leaf mesophyll protoplasts, we systematically optimized each step in order to efficiently separate central cells from the female gametophyte. We use initial manual pistil dis-section followed by the derivation of central cell proto-plasts, during which process the central cell emerges from the micropylar pole of the embryo sac. Then, we use a modified version of the Isolation of Nuclei TAgged in specific Cell Types (INTACT) protocol to purify central cell nuclei, resulting in a purity of 75-90% and a yield sufficient to undertake downstream molecular analyses. We find that the process is highly dependent on the health of the original plant tissue used, and the efficiency of protoplasting solution infiltration into the gametophyte. By isolating pure central cell populations, we have enabled elucidation of the physiology of this rare cell type, which in the future will provide novel insights into Arabidopsis reproduction.

Control of DEMETER DNA demethylase gene transcription in male and female gamete companion cells in <i>Arabidopsis thaliana</i>

Park, Jin-Sup,Frost, Jennifer M.,Park, Kyunghyuk,Ohr, Hyonhwa,Park, Guen Tae,Kim, Seohyun,Eom, Hyunjoo,Lee, Ilha,Brooks, Janie S.,Fischer, Robert L.,Choi, Yeonhee National Academy of Sciences 2017 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.114 No.8

<P>The DEMETER (DME) DNA glycosylase initiates active DNA demethy-lation 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.</P>

The catalytic core of DEMETER guides active DNA demethylation in <i>Arabidopsis</i>

Zhang, Changqing,Hung, Yu-Hung,Rim, Hyun Jung,Zhang, Dapeng,Frost, Jennifer M.,Shin, Hosub,Jang, Hosung,Liu, Fang,Xiao, Wenyan,Iyer, Lakshminarayan M.,Aravind, L.,Zhang, Xiang-Qian,Fischer, Robert L. National Academy of Sciences 2019 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.116 No.35

<P><B>Significance</B></P><P>Flowering plants reproduce via a unique double-fertilization event, producing the zygote and the nutritive endosperm. The genome of the central cell, the precursor of the endosperm, undergoes extensive demethylation prior to fertilization. This epigenetic reconfiguration, directed by the DEMETER (DME) glycosylase at thousands of loci in <I>Arabidopsis</I>, differentiates the epigenetic landscapes of parental genomes and establishes parent of origin-specific expression of many imprinted genes in endosperm essential for seed development. However, how DME is targeted to various locations remains unknown. Here we show that the multidomain DME is organized into 2 functional regions: the C-terminal region, which guides localization and catalysis, and the N-terminal region, which likely recruits chromatin remodelers to facilitate demethylation within heterochromatin.</P><P>The <I>Arabidopsis</I> DEMETER (DME) DNA glycosylase demethylates the maternal genome in the central cell prior to fertilization and is essential for seed viability. DME preferentially targets small transposons that flank coding genes, influencing their expression and initiating plant gene imprinting. DME also targets intergenic and heterochromatic regions, but how it is recruited to these differing chromatin landscapes is unknown. The C-terminal half of DME consists of 3 conserved regions required for catalysis in vitro. We show that this catalytic core guides active demethylation at endogenous targets, rescuing <I>dme</I> developmental and genomic hypermethylation phenotypes. However, without the N terminus, heterochromatin demethylation is significantly impeded, and abundant CG-methylated genic sequences are ectopically demethylated. Comparative analysis revealed that the conserved DME N-terminal domains are present only in flowering plants, whereas the domain architecture of DME-like proteins in nonvascular plants mainly resembles the catalytic core, suggesting that it might represent the ancestral form of the 5mC DNA glycosylase found in plant lineages. We propose a bipartite model for DME protein action and suggest that the DME N terminus was acquired late during land plant evolution to improve specificity and facilitate demethylation at heterochromatin targets.</P>