The authors thank Ms. Jennifer M. Lommel and Tara N. Rognan for maintenance of Arabidopsis plants. This work was supported by start-up funds from Saint Louis University and National Institutes of Health grants 1R15GM086846-01 and 3R15GM086846-01S1 to W. Xiao.

I. Genetic Crossing
1. Emasculating the female parent
In order to distinguish the maternal and paternal alleles by using DNA sequence polymorphism, two different ecotypes, e.g. Columbia-0 (Col-0) and Ler, will be chosen as female and male parents. The plants should be young and healthy. One can emasculate the female parent by using a dissecting microscope, magnifying visor, or naked eye. Locate stage-12 flowers (Smyth et al., 1990) and remove any flowers or siliques above and below them by clipping the base of the pedicel with the scissors. Sterilize forceps by dipping the base of the tip gently into a beaker of 95% ethanol, which will remove any pollen grains on the forceps and kill the pollen as well. Gently pry apart the flower buds using forceps, and gently remove 4 sepals, 4 petals, and 6 stamens, leaving the pistil bare and intact. Try to avoid damage to the carpels during this process.
2. Picking the pollen donor and carrying out pollination
The best pollen donors are anthesed open flowers at the stage 14 with the petals extending at a 90 &deg; angle to the pistil (Smyth et al., 1990), in which a lot of pollen is shedding. Grab the flower at the base and just above the pedicel, which will cause the flower to spread open. Dust the stigma of the prepared pistil with the anther. After the pollination, the stigma will be covered with the yellow pollen, which can be easily observed under a microscope.
3. Labeling the cross
After pollinating all the emasculated pistils on a plant, we label the cross with a jewelry tag and information of female and male parents and date. Place a stake in the soil close to the plant, use a string to tie the stem of the inflorescence to the stake, and cover the pollinated pistils with a plastic bag.
II. Isolation of Endosperm Tissues in Arabidopsis
1. Preparing materials
We usually get necessary materials ready before harvesting the endosperm and embryo tissues: a liquid nitrogen tank, liquid nitrogen, a dissecting microscope, two new pairs of fine-tip forceps (5 INOX. FST by Dumont biology, Switzerland), microscope glass slides (3" X 1" X 1.0 mm), a pH 5.7 solution of 0.3 M sorbitol and 5 mM MES.
2. Collecting siliques
At 7- or 8-day-after-pollination (DAP), the seeds are ready to be harvested at the mid- to late torpedo stage of embryogenesis and dissected for endosperm and embryo. Sometimes, it might take 9-10 DAP if the emasculated flowers are too young.
3. Isolating endosperm and embryo
Since Arabidopsis seeds are tiny, we use a dissecting microscope to isolate endosperm and embryo. Put an 8-DAP silique under a microscope, use a pair of forceps to hold the silique pedicel and use the tip of another pair of forceps to slide open the silique in the margin where two carpels fuse. Use a pair of forceps to pick up one seed onto pH 5.7 solutions of 0.3 M sorbitol and 5 mM MES, make a small cut at the micropyle end to slide out embryo, squeeze the uncut end to push out endosperm and separate endosperm from seed coat (Kinoshita et al., 2004). Put the embryo and endosperm into separate microtubes in liquid nitrogen. Continue this until we accumulate embryo or endosperm from 10-15 siliques and then store the tube in a -80&deg;C freezer. In some cases, endosperm does not have to be separated from seed coat, i.e., one can isolate total RNA from the endosperm and seed coat mixture for gene imprinting analysis.
III. Bisulfite Sequencing
1. Required reagents
Cetyltrimethyl ammonium bromide (CTAB) for preparing genomic DNA, restriction enzymes, 3 M NaOH (Freshly made), 6.42 M urea/4 M sodium bisulfite (2 M sodium metabisulfite, Sigma-Aldrich, S9000, Na2S2O5, Molecular Weight: 190), 10 mM hydroquinone, a DNA purification kit (Promega Wizard DNA Clean-up System, Cat. # A7280), TE buffer, 6.3 M NaOH (freshly made), 10 M NH4OAc, 20 &mu;g/&mu;L tRNA, and 100% ethanol.
2. Details of the bisulfite treatment protocol
<ol>
	<li>Isolate genomic endosperm or embryo DNA using a CTAB procedure (Rogers and Bendich, 1988).</li>
	<li>Digest 100 ng - 2 &mu;g of genomic DNA in 20 &mu;L to 100 &mu;L total volume with restriction enzymes that cut outside the region to be analyzed. For the MEA promoter, we use XhoI, NdeI, and PstI or HindIII.</li>
	<li>Denature the restriction enzymes by boiling the DNA for five minutes and then quench on ice.</li>
	<li>Add 1/9 volume (2.2 &mu;L for 20 &mu;L digested DNA) of 3 M NaOH and incubate at 37 &deg;C for 15 minutes.</li>
	<li>Transfer the solution to a 250 &mu;L PCR tube.</li>
	<li>Dissolve 7.5 g of urea in 10 mL of sterile distilled water; Slowly add 7.6 g of sodium metabisulfite over 1-2 hours and heating usually helps dissolving; Adjust the pH to 5 with freshly made 10 M NaOH; Add sterile distilled water to a final volume to 20 mL. This is 6.24 M urea/4 M sodium bisulfite solution.</li>
	<li>Add 6.24 M urea/4 M sodium bisulfite solution to a final concentration of 5.36 M and 3.44 M, respectively (Paulin et al., 1998). For example, add 208 &mu;L of 6.42 M urea/4 M sodium bisulfite solution to the above 22.2 &mu;L of denatured genomic DNA (Xiao et al., 2003).</li>
	<li>Add 10 mM hydroquinone to the DNA at a final concentration of 0.5 mM (12 &mu;L for 20 &mu;L digestion).</li>
	<li>Conduct bisulfite treatment in a PCR machine: 30 cycles of 55 &deg;C for 15 minutes and 95 &deg;C for 30 seconds.</li>
	<li>Desalt the bisulfite treated DNA using the Wizard DNA Clean-Up System from Promega and follow up the protocol (Jacobsen et al., 2000).</li>
	<li>Measure the exact volume of TE recovered from the column after desalting and add 6.3 M NaOH to a final concentration of 0.3 M. Incubate at 37 &deg;C for 15 minutes.</li>
	<li>Add 10 M NH4OAc (pH 7.0) to a final concentration of 3 M, 2 &mu;L of 20 &mu;g/&mu;L tRNA, and 3 volumes of 100% ethanol and then mix. Centrifuge for 15 minutes at 14,000 rpm.</li>
	<li>Wash the pellet once with 70% ethanol, do a short centrifuge, and remove extra ethanol.</li>
	<li>Dry pellet in a speedvac for 5-10 minutes and resuspend in 25 - 100 &mu;L TE buffer depending on starting amount of DNA. The sodium bisulfite-treated DNA is now ready for PCR analysis.</li>
</ol>
3. PCR amplification
<ol>
	<li>Since unmethylated cytosines are converted to uracil, it is difficult to amplify a large fragment using the bisulfite-treated DNA as a template. Thus, we usually design primers to amplify a product no longer than 500 bp. To sequence the 4-kb MEA promoter, we designed many sets of primers and amplified 14 overlapping fragments to cover the entire region (Xiao et al., 2003). </li>
	<li>To sequence the top-strand, in designing a forward primer, I) choose a G (guanine)-rich region in order to have a higher annealing temperature without extra long nucleotides in the primers; II) change C (cytosine ) to Y (pyrimidine) at CG and CNG contexts and change the remaining C to T (thymine). In designing a reverse primer, I) choose a C-rich region; II) change G to R (purine) at CG and CNG contexts and change the remaining G to A (adenine). </li>
	<li>To sequence the bottom-strand, in designing a forward primer, I) choose a C-rich region; II) change G to R at CG and CNG contexts and change the remaining G to A. In designing a reverse primer, I) choose a G-rich region; II) change C to Y (pyrimidine) at CG and CNG contexts and change the remaining C to T. </li>
	<li>We usually use 1-2 &mu;L of the sodium bisulfite-treated DNA as a template for each PCR amplification (Xiao et al., 2003). The PCR product needs to be analyzed by gel electrophoresis to confirm the correct size of the fragment, then to be gel purified and cloned into the TOPO TA cloning vector pCR2.1 (Invitrogen) as an insert. A single colony is picked up and cultured; plasmid DNA extracted and sent for sequencing.</li>
</ol>
4. Sequence analysis
The principle of bisulfite sequencing is that unmethylated cytosine will be converted to uracil due to hydrolytic deamination by high concentration of sodium bisulfite at pH5.0 which will be amplified as thymine in PCR product; while 5-methyl cytosine will not be modified by sodium bisulfite and remain to be cytosine after PCR amplification (Clark et al., 1994; Frommer et al., 1992). After obtaining the sequencing result, we compare it with the strand-specific template that is used for PCR amplification. If a cytosine residue in the template reads as a thymine in the sequencing result, it indicates that the cytosine is not methylated. If a cytosine residue in the template remains a cytosine in the sequencing, it means that the cytosine is methylated.
IV. Representative Results
<img src="/files/ftp_upload/2327/2327fig1.jpg" alt="Figure 1 /" />Figure 1.

<ol>
	<li>Clark, S. J., Harrison, J., Paul, C. L., Frommer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+mapping+of+methylated+cytosines.">High sensitivity mapping of methylated cytosines.</a> Nucleic Acids Res. 22, 2990-2997 (1994).</li><li>Cokus, S. J., Feng, S., Zhang, X., Chen, Z., Merriman, B., Haudenschild, C. D., Sriharsa Pradhan, S., Nelson, S. F., Pellegrini, M., Jacobsen, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shotgun+bisulphite+sequencing+of+the+Arabidopsis+genome+reveals+DNA+methylation+patterning.">Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning.</a> Nature. 452, 215-219 (2008).</li><li>Frommer, M., McDonald, L. E., Millar, D. S., Collis, C. M., Watt, F., Grigg, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomic+sequencing+protocol+that+yields+a+positive+display+of+5-methylcytosine+residues+in+individual+DNA+strands.">A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.</a> Proc Natl Acad Sci USA. 89, 1827-1831 (1992).</li><li>Gehring, M., Huh, J. H., Hsieh, T. F., Penterman, J., Choi, Y., Harada, J. J., Goldberg, R. B., Fischer, R. L. D. E. M. E. T. E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+glycosylase+establishes+MEDEA+polycomb+gene+self-imprinting+by+allele-specific+demethylation.">DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation.</a> Cell. 124, 495-506 (2006).</li><li>Henderson, I. R., Chan, S. R., Cao, X., Johnson, L., Jacobsen, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+sodium+bisulfite+sequencing+in+plants.">Accurate sodium bisulfite sequencing in plants.</a> Epigenetics. 5, 47-49 (2010).</li><li>Hsieh, T. F., Ibarra, C. A., Silva, P., Zemach, A., Eshed-Williams, L., Fischer, R. L., Zilberman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+demethylation+of+Arabidopsis+endosperm.">Genome-wide demethylation of Arabidopsis endosperm.</a> Science. 324, 1451-1454 (2009).</li><li>Jacobsen, S. E., Sakai, H., Finnegan, E. J., Cao, X., Meyerowitz, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+hypermethylation+of+flower-specific+genes+in+Arabidopsis.">Ectopic hypermethylation of flower-specific genes in Arabidopsis.</a> Curr. Biol. 10, 179-186 (2000).</li><li>Kinoshita, T., Miura, A., Choi, Y., Kinoshita, Y., Cao, X., Jacobsen, S. E., Fischer, R. L., Kakutani, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-way+control+of+FWA+imprinting+in+Arabidopsis+endosperm+by+DNA+methylation.">One-way control of FWA imprinting in Arabidopsis endosperm by DNA methylation.</a> Science. 303, 521-523 (2004).</li><li>Lister, R., O'Malley, R. C., Tonti-Filippini, J., Gregory, B. D., Berry, C. C., Millar, A. H., Ecker, J. 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No conflicts of interest declared.

It is relatively easy to separate embryo from endosperm and seed coat, but it is tedious to separate endosperm from seed coat, especially for seed at early or mid-torpedo stage of embryogenesis. Since seed coat only contributes a very small amount of tissue, for some genes, e.g., MEA and FWA, we do not have to separate endosperm from seed coat. That means that we can isolate RNAs from a mixture of endosperm and seed coat tissues, check expression of the maternal and paternal alleles of a gene, compare w...

Supplies
<ul>
 <li>Dissecting Microscope</li>
 <li>Scissors</li>
 <li>Fine Tip Forceps</li>
 <li>Jewelry Tag</li>
 <li>Plant Stakes</li>
 <li>String or Twist-Ties</li>
 <li>4" X 2" X 8" Polyethylene Bags</li>
 <li>3" X 1" X 1.0 mm Microscope Slides</li>
 <li>Liquid Nitrogen</li>
 <li>Liquid Nitrogen Containers</li>
 <li>Heat Block</li>
 <li>PCR Tubes</li>
 <li>Thermocycler</li>
 <li>Microcentrifuge Tubes</li>
 <li>Microcentrifuge</li>
 <li>Gel Electrophoresis facility</li>
 <li>Arabidopsis thaliana Columbia-0 Plants</li>
 <li>Arabidopsis thaliana Landsberg erecta Plants</li>
</ul>
Reagents
<ul>
 <li>70% Ethanol</li>
 <li>95% Ethanol</li>
 <li>100% Ethanol</li>
 <li>0.3 M Sorbitol and 5 mM MES-pH 5.7</li>
 <li>Cetyltrimethyl Ammonium Bromide (CTAB)</li>
 <li>100% Ethanol</li>
 <li>Cholorform</li>
 <li>Restriction Enzymes</li>
 <li>3 M NaOH</li>
 <li>6.3 M NaOH</li>
 <li>6.24 M Urea/ 4 M Sodium Bisulfite</li>
 <li>Sterile distilled H2O</li>
 <li>10 mM Hydroquinone</li>
 <li>Wizard DNA Clean-Up System (Promega)</li>
 <li>10 M NH4OAc</li>
 <li>20 &mu;g/ &mu;L tRNA</li>
 <li>TE buffer</li>
 <li>The TOPO TA Cloning Kit (Invitrogen)</li>
</ul>

determination of dna methylation of imprinted genes in arabidopsis endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Watch this Scientific Journal Video about Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm at JoVE.com

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

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Research

JoVE Journal

Biology

17.4K Aufrufe.  Saint Louis University. Imprinting ist ein Phänomen in Pflanzen-und Säugetier Reproduktion. DNA-Methylierung spielt eine wichtige Rolle in Mechanismen der Prägung. Isolating Endosperm und Bestimmung Methylierungsstatus von geprägten Genen in Arabidopsis Kann schwierig sein. In diesem Protokoll, beschreiben wir, wie Endosperm isolieren und zu bestimmen Methylierung durch Bisulfit-Sequenzierung.

Serie: Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Genetics

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is a useful measure to provide an accurate analysis of the methylation status of gastrointestinal (GI) malignancies. Given the multiple potential translational uses of DNA methylation analysis, practicing clinicians and others new to DNA methylation studies need to be able to understand step by step how these genome-wide analyses are performed. The goal of this protocol is to provide a detailed description of how this method is used for the biomarker identification in GI malignancies. Importantly, we describe three critical steps that are needed to obtain accurate results during genome-wide analysis. Clearly and concisely written, these three methods are often lacking and not noticeable to those new to epigenetic studies. We used 48 samples of a GI malignancy (gastric cancer) to highlight practically how genome-wide DNA methylation analysis can be performed for GI malignancies.

Herein, we describe a procedure for genome-wide analysis of DNA methylation in gastrointestinal cancers. The procedure is of relevance to studies that investigate relationships between methylation patterns of genes and factors contributing to carcinogenesis in gastrointestinal cancers.

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is ...

Cancer Research

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and sequencing (MeRIP-seq) is a method that aims to enrich for methylated RNA and ultimately identify modified transcripts. Briefly, sonicated RNA is incubated with an antibody for 5-methylated cytosines and precipitated with the assistance of protein G beads. The enriched fragments are then sequenced and the potential methylation sites are mapped based on the distribution of the reads and peak detection. MeRIP can be applied to any organism, as it does not require any prior sequence or modifying enzyme knowledge. In addition, besides fragmentation, RNA is not subjected to any other chemical or temperature treatment. However, MeRIP-seq does not provide single-nucleotide prediction of the methylation site as other methods do, although the methylated area can be narrowed down to a few nucleotides. The use of different modification-specific antibodies allows MeRIP to be adjusted for the different base modifications present on RNA, expanding the possible applications of this method.

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

The methylated RNA immunoprecipitation assay is an antibody-based method used to enrich for methylated RNA fragments. Coupled with deep sequencing, it leads to the identification of transcripts carrying the m5C modification.

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and ...

Sample Preparation to Bioinformatics Analysis of DNA Methylation: Association Strategy for Obesity and Related Trait Studies

Obesity is directly connected to lifestyle and has been associated with DNA methylation changes that may cause alterations in the adipogenesis and lipid storage processes contributing to the development of the disease. We demonstrate a complete protocol from selection to epigenetic data analysis of patients with and without obesity. All steps from the protocol were tested and validated in a pilot study. 32 women participated in the study, in which 15 individuals were classified with obesity according to Body Mass Index (BMI) (45.1 &#177; 5.4 kg/m2); and 17 individuals were classified without obesity according to BMI (22.6 &#177; 1.8 kg/m2). In the group with obesity, 564 CpG sites related to fat mass were identified by linear regression analysis. The CpG sites were in the promoter regions. The differential analysis found 470 CpGs hypomethylated and 94 hypermethylated sites in individuals with obesity. The most hypomethylated enriched pathwayswere in the RUNX, WNT signaling, and response to hypoxia. The hypermethylated pathways were related to insulin secretion, glucagon signaling, and Ca2+. We conclude that the protocol effectively identified DNA methylation patterns and trait-related DNA methylation. These patterns could be associated with altered gene expression, affecting adipogenesis and lipid storage. Our results confirmed that an obesogenic lifestyle could promote epigenetic changes in human DNA.

The present study describes the workflow to manage DNA methylation data obtained by microarray technologies. The protocol demonstrates steps from sample preparation to data analysis. All procedures are described in detail, and the video shows the significant steps.

Obesity is directly connected to lifestyle and has been associated with DNA methylation changes that may cause alterations in the adipogenesis and lipid ...

Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical role in early embryonic development. Enzymatic modifications drive active methylation and demethylation of cytosine into 5-methylcytosine (5-mC) and subsequent oxidation of 5-mC into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. Epigenetic reprogramming is a critical period during in utero development, and maternal exposure to chemicals has the potential to reprogram the epigenome within offspring. This can potentially cause adverse outcomes such as immediate phenotypic consequences, long-term effects on adult disease susceptibility, and transgenerational effects of inherited epigenetic marks. Although bisulfite-based sequencing enables investigators to interrogate cytosine methylation at base-pair resolution, sequencing-based approaches are cost-prohibitive and, as such, preclude the ability to monitor cytosine methylation across developmental stages, multiple concentrations per chemical, and replicate embryos per treatment. Due to the ease of automated in vivo imaging, genetic manipulations, rapid ex utero development time, and husbandry during embryogenesis, zebrafish embryos continue to be used as a physiologically intact model for uncovering xenobiotic-mediated pathways that contribute to adverse outcomes during early embryonic development. Therefore, using commercially available 5-mC-specific antibodies, we describe a cost-effective strategy for rapid and efficient spatiotemporal monitoring of cytosine methylation within individual, intact zebrafish embryos by leveraging whole-mount immunohistochemistry, automated high-content imaging, and efficient data processing using programming language prior to statistical analysis. To current knowledge, this method is the first to successfully detect and quantify 5-mC levels in situ within zebrafish embryos during early development. The method enables the detection of DNA methylation within the cell mass and also has the ability to detect cytosine methylation of yolk-localized maternal mRNAs during the maternal-to-zygotic transition. Overall, this method will be useful for the rapid identification of chemicals that have the potential to disrupt cytosine methylation in situ during epigenetic reprogramming.

This paper describes a protocol for the rapid and efficient spatiotemporal monitoring of normal and aberrant cytosine methylation within intact zebrafish embryos.

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical ...

Developmental Biology

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...