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.

Experimental development of the epigenomic library construction method to elucidate the epigenetic diversity and causal relationship between epigenome and transcriptome at a single-cell level

Park, Kyunghyuk,Jeon, Min Chul,Kim, Bokyung,Cha, Bukyoung,Kim, Jong-Il Korea Genome Organization 2022 Genomics & informatics Vol.20 No.1

The method of single-cell RNA sequencing has been rapidly developed, and numerous experiments have been conducted over the past decade. Their results allow us to recognize various subpopulations and rare cell states in tissues, tumors, and immune systems that are previously unidentified, and guide us to understand fundamental biological processes that determine cell identity based on single-cell gene expression profiles. However, it is still challenging to understand the principle of comprehensive gene regulation that determines the cell fate only with transcriptome, a consequential output of the gene expression program. To elucidate the mechanisms related to the origin and maintenance of comprehensive single-cell transcriptome, we require a corresponding single-cell epigenome, which is a differentiated information of each cell with an identical genome. This review deals with the current development of single-cell epigenomic library construction methods, including multi-omics tools with crucial factors and additional requirements in the future focusing on DNA methylation, chromatin accessibility, and histone post-translational modifications. The study of cellular differentiation and the disease occurrence at a single-cell level has taken the first step with single-cell transcriptome and is now taking the next step with single-cell epigenome.

DEMETER-mediated DNA Demethylation in Gamete Companion Cells and the Endosperm, and its Possible Role in Embryo Development in Arabidopsis

Kyunghyuk Park,Seunga Lee,Hyunjin Yoo,Yeonhee Choi 한국식물학회 2020 Journal of Plant Biology Vol.63 No.5

Seed development begins upon double fertilization, producing the embryo and endosperm, which are genetically identical, except for their ploidy level. DEMETER (DME), a member of the DNA glycosylase family, functions as a DNA demethylase via the base excision repair pathway. DME is specifically expressed prior to fertilization in two gamete companion cells, central cell of the female gametophyte and vegetative cell of the male gametophyte, but not in the heritable gamete cells or embryo. Mutations in the DME gene cause hypermethylation in the endosperm, leading to endosperm overproliferation and seed abortion after fertilization. DME-mediated DNA demethylation preferentially targets euchromatic transposable elements (TEs), resulting in TE activation and initiation of de novo methylation through RNA-directed DNA methylation, and provides FERTILIZATION-INDEPENDENT SEED 2 (FIS2)-Polycomb Repressive Complex 2-binding sites, resulting in histone modifications and genomic imprinting during reproduction. The global demethylation of TEs in gamete companion cells and active de novo methylation in the embryo suggest a new role of sexual companion cells in reinforcing the genome integrity of the heritable tissue. In this review, we provide an overview of demethylation in sexual companion cells and the endosperm, and discuss its evolutionary effect on the heritable gamete cells and embryo.

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>

DNA demethylation is initiated in the central cells of <i>Arabidopsis</i> and rice

Park, Kyunghyuk,Kim, M. Yvonne,Vickers, Martin,Park, Jin-Sup,Hyun, Youbong,Okamoto, Takashi,Zilberman, Daniel,Fischer, Robert L.,Feng, Xiaoqi,Choi, Yeonhee,Scholten, Stefan National Academy of Sciences 2016 Proceedings of the National Academy of Sciences Vol.113 No.52

<P>Cytosine methylation is a DNA modification with important regulatory functions in eukaryotes. In flowering plants, sexual reproduction is accompanied by extensive DNA demethylation, which is required for proper gene expression in the endosperm, a nutritive extraembryonic seed tissue. Endosperm arises from a fusion of a sperm cell carried in the pollen and a female central cell. Endosperm DNA demethylation is observed specifically on the chromosomes inherited from the central cell in Arabidopsis thaliana, rice, and maize, and requires the DEMETER DNA demethylase in Arabidopsis. DEMETER is expressed in the central cell before fertilization, suggesting that endosperm demethylation patterns are inherited from the central cell. Down-regulation of the MET1 DNA methyltransferase has also been proposed to contribute to central cell demethylation. However, with the exception of three maize genes, central cell DNA methylation has not been directly measured, leaving the origin and mechanism of endosperm demethylation uncertain. Here, we report genome-wide analysis of DNA methylation in the central cells of Arabidopsis and rice-species that diverged 150 million years agoas well as in rice egg cells. We find that DNA demethylation in both species is initiated in central cells, which requires DEMETER in Arabidopsis. However, we do not observe a global reduction of CG methylation that would be indicative of lowered MET1 activity; on the contrary, CG methylation efficiency is elevated in female gametes compared with nonsexual tissues. Our results demonstrate that locus-specific, active DNA demethylation in the central cell is the origin of maternal chromosome hypomethylation in the endosperm.</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.

An Optimized Method for the Construction of a DNA Methylome from Small Quantities of Tissue or Purified DNA from Arabidopsis Embryo

Yoo, Hyunjin,Park, Kyunghyuk,Lee, Jaehoon,Lee, Seunga,Choi, Yeonhee Korean Society for Molecular and Cellular Biology 2021 Molecules and cells Vol.44 No.8

DNA methylation is an important epigenetic mechanism affecting genome structure, gene regulation, and the silencing of transposable elements. Cell- and tissue-specific methylation patterns are critical for differentiation and development in eukaryotes. Dynamic spatiotemporal methylation data in these cells or tissues is, therefore, of great interest. However, the construction of bisulfite sequencing libraries can be challenging if the starting material is limited or the genome size is small, such as in Arabidopsis. Here, we describe detailed methods for the purification of Arabidopsis embryos at all stages, and the construction of comprehensive bisulfite libraries from small quantities of input. We constructed bisulfite libraries by releasing embryos from intact seeds, using a different approach for each developmental stage, and manually picking single-embryo with microcapillaries. From these libraries, reliable Arabidopsis methylome data were collected allowing, on average, 11-fold coverage of the genome using as few as five globular, heart, and torpedo embryos as raw input material without the need for DNA purification step. On the other hand, purified DNA from as few as eight bending torpedo embryos or a single mature embryo is sufficient for library construction when RNase A is treated before DNA extraction. This method can be broadly applied to cells from different tissues or cells from other model organisms. Methylome construction can be achieved using a minimal amount of input material using our method; thereby, it has the potential to increase our understanding of dynamic spatiotemporal methylation patterns in model organisms.

A single-cell atlas of in vitro multiculture systems uncovers the in vivo lineage trajectory and cell state in the human lung

Lee Woochan,Lee Seyoon,Yoon Jung-Ki,Lee Dakyung,Kim Yuri,Han Yeon Bi,Kim Rokhyun,Moon Sungjin,Park Young Jun,Park Kyunghyuk,Cha Bukyoung,Choi Jaeyong,Kim Juhyun,Ha Na-young,Kim Kwhanmien,Cho Sukki,Cho 생화학분자생물학회 2023 Experimental and molecular medicine Vol.55 No.-

We present an in-depth single-cell atlas of in vitro multiculture systems on human primary airway epithelium derived from normal and diseased lungs of 27 individual donors. Our large-scale single-cell profiling identified new cell states and differentiation trajectories of rare airway epithelial cell types in human distal lungs. By integrating single-cell datasets of human lung tissues, we discovered immune-primed subsets enriched in lungs and organoids derived from patients with chronic respiratory disease. To demonstrate the full potential of our platform, we further illustrate transcriptomic responses to various respiratory virus infections in vitro airway models. Our work constitutes a single-cell roadmap for the cellular and molecular characteristics of human primary lung cells in vitro and their relevance to human tissues in vivo.