Progress 10/01/08 to 09/30/13
Outputs
Target Audience: The primary targets of our research are other scientists. During the term of this AES project I gave 24 presentations at scientific meetings and Universities. Knowledge gained during this project was also incorporated into classes that I teach at UC Berkeley: Biology 1A (genetics and molecular biology) and PMB 160 (molecular genetics of plant growth and development). Technology generated during the AES project was reviewed by biotechnology companies for integration into their product lines. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two graduate students (Jessica Rodrigues and Christian Ibarra) and two postdoctoral fellows (Jennifer Frost and Juhyun Shin) received training in genomics, epigenetics, computational biology, genetics and molecular biology. How have the results been disseminated to communities of interest? During the past year I presentations at three symposia – the Keystone Symposium on Epigenetics/Chromatin Dynamics, the Plant and Animal Genome Conference (San Diego, CA), and the Keystone Symposium on Nuclear Events in Plant Gene Expression. I also gave seminars at the Gregor Mendel Institute of Molecular Plant Biology (Vienna, Austria) and the Nara Institute of Science and Technology (Japan). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Arabidopsis thaliana endosperm, a transient tissue that nourishes the embryo, exhibits extensive localized DNA demethylation on maternally inherited chromosomes. Demethylation mediates parent-of-origin–specific (imprinted) gene expression but is apparently unnecessary for the extensive accumulation of maternally biased small RNA (sRNA) molecules detected in seeds. Endosperm DNA in the distantly related monocots rice and maize is likewise locally hypomethylated, but whether this hypomethylation is generally parent-of-origin specific is unknown. Imprinted expression of sRNA also remains uninvestigated in monocot seeds. Here, we report high-coverage sequencing of the Kitaake rice cultivar that enabled us to show that localized hypomethylation in rice endosperm occurs solely on the maternal genome, preferring regions of high DNA accessibility. Maternally expressed imprinted genes are enriched for hypomethylation at putative promoter regions and transcriptional termini and paternally expressed genes at promoters and gene bodies, mirroring our recent results in A. thaliana. However, unlike in A. thaliana, rice endosperm sRNA populations are dominated by specific strong sRNA-producing loci, and imprinted 24-nt sRNAs are expressed from both parental genomes and correlate with hypomethylation. Overlaps between imprinted sRNA loci and imprinted genes expressed from opposite alleles suggest that sRNAs may regulate genomic imprinting. Whereas sRNAs in seedling tissues primarily originate from small class II (cut-and-paste) transposable elements, those in endosperm are more uniformly derived, including sequences from other transposon classes, as well as genic and intergenic regions. Our data indicate that the endosperm exhibits a unique pattern of sRNA expression and suggest that localized hypomethylation of maternal endosperm DNA is conserved in flowering plants.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Yu, A., Lepere, G., Jay, F., Bapaume, L., Wang, J., Wang, Y., Abraham, A.-L., Penterman, J., Fischer, R.L., Voinnet, O., Navarro, L. (2013) Dynamics of biological relevance of DNA demethylation in Arabidopsis antibacterial defense. Proc. Natl. Acad. Sci. USA 110:2389-2394.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Rodrigues, J.A., Ruan, R., Nishimura, T., Sharma, M., Sharma, R., Ronald, P.C., Fischer, R.L., Zilberman, D. (2013) Imprinted expression of genes and small RNA is associated with localized hypomethylation of the maternal genome in rice endosperm. Proc. Natl. Acad. Sci. USA 110:7934-7939.
- Type:
Book Chapters
Status:
Published
Year Published:
2013
Citation:
Ibarra, C.A., Frost, J.M., Shin, J., Hsieh, T.-F. Fischer (2013) Epigenetic Control of Seed Gene Imprinting. In Seed Genomics. Beecraft, P.W., ed., John Wiley & Sons, Inc., Ames, Iowa, Pp. 63-82.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Seeds provide a significant fraction of the nutritional requirements of humans and their domesticated animals. Important oils and starches are also produced and harvested from seeds. Genes that are imprinted in the endosperm of the seed are important regulators of seed size and quality. DNA demethylation, catalyzed by the DEMETER (DME) DNA glycosylase, is a key regulator of gene imprinting in the endosperm. We conducted and analyzed experiments to identify a fundamentally new function of DNA demethylation. That is, DNA demethylation in companion cells reinforces transposon methylation in plant gametes and likely contributes to stable silencing of transposable elements across generations. Understanding how DNA demethylation influences gamete and embryo DNA methylation will improve our ability to control seed size and produce higher quality seeds. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The Arabidopsis thaliana central cell, the companion cell of the egg, undergoes DNA demethylation prior to fertilization, but the targeting preferences, mechanism, and biological significance of this process remain unclear. Here, we show that active DNA demethylation mediated by the DEMETER DNA glycosylase accounts for all of the demethylation in the central cell, and preferentially targets small, AT-rich and nucleosome-depleted euchromatic transposable elements. The vegetative cell, the companion cell of sperm, also undergoes DEMETER-dependent demethylation of similar sequences, and lack of DEMETER in vegetative cells causes reduced small RNA-directed DNA methylation of transposons in sperm. Our results demonstrate that demethylation in companion cells reinforces transposon methylation in plant gametes and likely contributes to stable silencing of transposable elements across generations.
Publications
- Ibarra, C.A., Feng, X., Schoft, V.K., Hsieh, T.-F., Uzawa, R., Rodrigues, J.A., Zemach, A., Chumak, F., Machlicova, A., Nishimura, T., Rojas, D., Fischer, R.L., Tamaru, H., Zilberman, D. (2012) Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes. Science 337:1360-1364.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Seeds provide a significant fraction of the nutritional requirements of humans and their domesticated animals. Important oils and starches are also produced and harvested from seeds. Genes that are imprinted in the endosperm of the seed are important regulators of seed size and quality. DNA demethylation, catalyzed by the DEMETER (DME) DNA glycosylase, is a key regulator of gene imprinting in the endosperm. We conducted and analyzed experiments to identify new genes whose imprinting is regulated by the DME glycosylase, to discover the role of the DME DNA glycosylase in male reproductive structures, and to develop new software for measuring DNA methylation. Understanding how DNA demethylation controls gene imprinting and expression in the endosperm will improve our ability to control seed size and produce higher quality seeds. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Imprinted genes are expressed primarily from either the maternal or paternal allele, a phenomenon that occurs in flowering plants and mammals. Plant imprinted gene expression occurs in endosperm, a terminal nutritive tissue consumed by the embryo during seed development or after germination. Imprinted expression in Arabidopsis endosperm is orchestrated by differences in cytosine DNA methylation between the paternal and maternal genomes as well as by Polycomb group proteins. We used extensive sequencing of cDNA libraries to identify new paternally expressed and maternally expressed imprinted genes in A. thaliana endosperm that are regulated by the DNA-demethylating glycosylase DEMETER, the DNA methyltransferase MET1, and/or the core Polycomb group protein FIE. These genes encode transcription factors, proteins involved in hormone signaling, components of the ubiquitin protein degradation pathway, regulators of histone and DNA methylation, and small RNA pathway proteins. We did not detect any imprinted genes in the embryo. Our results show that imprinted gene expression is an extensive mechanistically complex phenomenon that likely affects multiple aspects of seed development. In double fertilization, the vegetative cell of the male gametophyte (pollen) germinates and forms a pollen tube that brings to the female gametophyte two sperm cells that fertilize the egg and central cell to form the embryo and endosperm, respectively. The 5-methylcytosine DNA glycosylase DEMETER (DME), expressed in the central cell, is required for maternal allele demethylation and gene imprinting in the endosperm. By contrast, little is known about the function of DME in the male gametophyte. We discovered that reduced transmission of the paternal mutant dme allele in certain ecotypes reflects, at least in part, defective pollen germination. DME RNA is detected in pollen, but not in isolated sperm cells, suggesting that DME is expressed in the vegetative cell. Bisulfite sequencing experiments show that imprinted genes and a repetitive element are hypomethylated in the vegetative cell genome compared with the sperm genome, which is a process that requires DME. Moreover, we show that MEA and FWA RNA are detectable in pollen, but not in isolated sperm cells, suggesting that their expression occurs primarily in the vegetative cell. These results suggest that DME is active and demethylates similar genes and transposons in the genomes of the vegetative and central cells in the male and female gametophytes, respectively. We developed MethylCoder, a new software program that generates per-base methylation data given a set of bisulfite-treated reads. It provides the option to use either of two existing short-read aligners, each with different strengths. It accounts for soft-masked alignments and overlapping paired-end reads. MethylCoder outputs data in text and binary formats in addition to the final alignment in SAM format, so that common high-throughput sequencing tools can be used on the resulting output. It is more flexible than existing software and competitive in terms of speed and memory use.
Publications
- Hsieh, T.-F., Shin, J., Uzawa, R., Silva, P., Cohen, S., Bauer, M.J., Hashimoto, M., Kirkbride, R., Harada, J.J., Zilberman, D., Fischer, R.L. (2011) Regulation of Imprinted Gene Expression in Arabidopsis Endosperm. Proc. Natl. Acad. Sci. USA 108:1755-1762.
- Function of the DEMETER DNA Glycosylase in the Arabidopsis thaliana Male Gametophyte (2011) Schoft, V.K., Chumak, N., Choi, Y., Hannon, M., Garcia-Aguilar, M., Machlicova, A., Slusarz, L. Mosiolek, M., Park, J.-S., Park, G.T., Fischer, R.L., Tamaru, H. (2011) Proc. Natl. Acad. Sci. USA 108:8042-8047.
- Jeong, C.W., Roh, H., Dang, T.V.T., Choi, Y.D., Fischer, R.L., Lee, J.S., Choi, Y. (2011) An E3 Ligase Complex Regulates SET-Domain Polycomb Group Protein activity in Arabidopsis thaliana (2011) Proc. Natl. Acad. Sci. USA 108:8036-8041.
- Pedersen, B.S., Hsieh, T.-F., Ibarra, C., Fischer, R.L. (2011) MethylCoder: Software pipeline for bisulfite-treated sequences. Bioinformatics. 27:2435-2436.
- Mosher, R.A., Tan, E.H., Shin, J., Fischer, R.L., Pikaard, C.S., Baulcombe, D.C. (2011) An Atypical Epigenetic Mechanism Affects Uniparental Expression of Pol IV-Dependent siRNAs. PLoS One 6:e25756.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: DNA glycosylases initiate the base excision repair pathway (BER) by excising damaged, mismatched, or otherwise modified bases. Animals and plants independently evolved active BER-dependent DNA demethylation mechanisms important for epigenetic reprogramming. One such DNA demethylation mechanism is uniquely initiated in plants by DEMETER (DME)-class DNA glycosylases. Arabidopsis DME family glycosylases contain a conserved helix-hairpin-helix domain present in both prokaryotic and eukaryotic DNA glycosylases as well as two domains A and B of unknown function that are unique to this family. Here, we employed a mutagenesis approach to screen for DME residues critical for DNA glycosylase activity. This analysis revealed that amino acids clustered in all three domains, but not in the intervening variable regions, are required for in vitro 5-methylcytosine excision activity. Amino acids in domain A were found to be required for nonspecific DNA binding, a prerequisite for 5-methylcytosine excision. In addition, mutational analysis confirmed the importance of the iron-sulfur cluster motif to base excision activity. Thus, the DME DNA glycosylase has a unique structure composed of three essential domains that all function in 5-methylcytosine excision. Most of the transcription factors (TFs) responsible for controlling seed development are not yet known. To identify TF genes expressed at specific stages of seed development, including those unique to seeds, we used Affymetrix GeneChips to profile Arabidopsis genes active in seeds from fertilization through maturation and at other times of the plant life cycle. Seed gene sets were compared with those expressed in prefertilization ovules, germinating seedlings, and leaves, roots, stems, and floral buds of the mature plant. Most genes active in seeds are shared by all stages of seed development, although significant quantitative changes in gene activity occur. Each stage of seed development has a small gene set that is either specific at the level of the GeneChip or up-regulated with respect to genes active at other stages, including those that encode TFs. We identified 289 seed-specific genes, including 48 that encode TFs. Seven of the seed-specific TF genes are known regulators of seed development and include the LEAFY COTYLEDON (LEC) genes LEC1, LEC1-LIKE, LEC2, and FUS3. The rest represent different classes of TFs with unknown roles in seed development. Promoter-beta-glucuronidase (GUS) fusion experiments and seed mRNA localization GeneChip datasets showed that the seed-specific TF genes are active in different compartments and tissues of the seed at unique times of development. Collectively, these seed-specific TF genes should facilitate the identification of regulatory networks that are important for programming seed development. PARTICIPANTS: Postdoctoral fellows (Tzung-Fu Hsieh and Matthew Bauer) and graduate students (Christian Ibarra and Juhyun Shin) worked on the project. They received training in molecular biology, genetics, plant reproduction, and seed biology. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
DNA demethylation, catalyzed by the DEMETER (DME) DNA glycosylase, is a key regulator of gene imprinting in the endosperm. Understanding its modular structure provides insights into how it demethylates the maternal genome of the endosperm, which is essential process for endosperm and seed viability. Seeds provide a significant fraction of the nutritional requirements of humans and their domesticated animals. Important oils and starches are also produced and harvested from seeds. Genes that are imprinted in the endosperm of the seed are important regulators of seed size and quality. Understanding the gene expression network provides additional tools that can be used to control seed development and yield.
Publications
- Le, B.H., Cheng, C., Bui, A.Q., Wagmaister, J.A., Henry, K.F., Pelletier, J., Kwong, L., Belmonte, M., Kirkbride, R., Horvath, S., Drews, G.N., Fischer, R.L., Okamuro, J.K., Harada, J.J., Goldberg, R.B. (2010) Global Analysis of Gene Activity During Arabidopsis Seed Development and Identification of Seed-Specific Transcription Factors. Proc. Natl. Acad. Sci. USA 107:8063-8070.
- Mok, Y.G., Uzawa, R., Lee, J., Weiner, G.M., Eichman, B.F., Fischer, R.L., Huh, J.H. (2010) Domain structure of the DEMETER 5-methylcytosine DNA glycosylase. Proc. Natl. Acad. Sci. USA 107:19225-19230.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Parent-of-origin-specific (imprinted) gene expression is regulated in Arabidopsis thaliana endosperm by cytosine demethylation of the maternal genome mediated by the DNA glycosylase DEMETER, but the extent of the methylation changes is not known. Here, we show that virtually the entire endosperm genome is demethylated, coupled with extensive local non-CG hypermethylation of small interfering RNA-targeted sequences. Mutation of DEMETER partially restores endosperm CG methylation to levels found in other tissues, indicating that CG demethylation is specific to maternal sequences. Endosperm demethylation is accompanied by CHH hypermethylation of embryo transposable elements. Our findings demonstrate extensive reconfiguration of the endosperm methylation landscape that likely reinforces transposon silencing in the embryo. Arabidopsis APETALA2 (AP2) controls seed mass maternally, with ap2 mutants producing larger seeds than wild type. Here, we show that AP2 influences development of the three major seed compartments: embryo, endosperm, and seed coat. AP2 appears to have a significant effect on endosperm development. ap2 mutant seeds undergo an extended period of rapid endosperm growth early in development relative to wild type. This early expanded growth period in ap2 seeds is associated with delayed endosperm cellularization and overgrowth of the endosperm central vacuole. The subsequent period of moderate endosperm growth is also extended in ap2 seeds largely due to persistent cell divisions at the endosperm periphery. The effect of AP2 on endosperm development is mediated by different mechanisms than parent-of-origin effects on seed size observed in interploidy crosses. Seed coat development is affected; integument cells of ap2 mutants are more elongated than wild type. We conclude that endosperm overgrowth and/or integument cell elongation create a larger postfertilization embryo sac into which the ap2 embryo can grow. Morphological development of the embryo is initially delayed in ap2 compared with wild-type seeds, but ap2 embryos become larger than wild type after the bent-cotyledon stage of development. ap2 embryos are able to fill the enlarged postfertilization embryo sac, because they undergo extended periods of cell proliferation and seed filling. We discuss potential mechanisms by which maternally acting AP2 influences development of the zygotic embryo and endosperm to repress seed size. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Seeds provide a significant fraction of the nutritional requirements of humans and their domesticated animals. Important oils and starches are also produced and harvested from seeds. Genes that are imprinted in the endosperm of the seed are important regulators of seed size and quality. DNA demethylation, catalyzed by the DEMETER (DME) DNA glycosylase, is a key regulator of gene imprinting in the endosperm. The AP2 transcription factor is another key regulator of seed development. Understanding how DNA demethylation controls gene imprinting in the endosperm, and how AP2 regulates gene transcription, will improve our ability to control seed size and produce higher quality seeds.
Publications
- Hsieh, T.-F., Ibarra, C.A., Silva, P., Zemach, A., Eshed-Williams, L., Fischer, R.L., Zilberman, D. (2009) Genome-wide Demethylation of Arabidopsis Endosperm. Science. 324:1451-1454.
- Ohto, M., Floyd, S., Fischer, R.L., Goldberg, R.B., Harada, J.J. (2009) Effects of APETALA2 on Embryo, Endosperm, and Seed Coat Development Determine Seed Size in Arabidopsis. Sexual Plant Reproduction. 22:277-289.
- Fischer, R.L. and Mizukami, Y. (2009) Methods for altering organ mass in plants. U.S. Patent Number 7,595,434.
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Progress 10/01/08 to 12/31/08
Outputs
OUTPUTS: Seeds provide a significant fraction of the nutritional requirements of humans and their domesticated animals. Important oils and starches are also produced and harvested from seeds. Genes that are imprinted in the endosperm of the seed are important regulators of seed size and quality. DNA demethylation, catalyzed by the DEMETER (DME) DNA glycosylase, is a key regulator of gene imprinting in the endosperm. Understanding how DNA demethylation controls gene imprinting in the endosperm will improve our ability to produce higher quality seeds. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Parent-of-origin-specific (imprinted) gene expression is regulated in Arabidopsis thaliana endosperm by cytosine demethylation of the maternal genome mediated by the DNA glycosylase DEMETER, but the extent of the methylation changes is not known. To determine the methylation landscape during Arabidopsis seed development, we isolated DNA from wild-type embryos, wild-type endosperm, and endosperm from seeds with a defective maternal allele of DME, and used the Illumina Genome Analyzer platform to quantify DNA methylation by high-throughput bisulfite sequencing (bisulfite treatment converts unmethylated cytosine to uracil, fig. S1). We aligned 2.4 billion bases for embryo, 2.1 billion bases for wild-type endosperm, and 1.9 billion bases for dme endosperm, which corresponds to 20-fold, 17-fold, and 16-fold coverage of the Arabidopsis nuclear genome, respectively - a sequencing depth comparable to that used for single-locus analysis. Here we show that virtually the entire endosperm genome is demethylated, coupled with extensive local non-CG hypermethylation of siRNA-targeted sequences. Mutation of DEMETER partially restores endosperm CG methylation to levels found in the embryo, indicating that CG demethylation is specific to maternal sequences. Our findings demonstrate extensive reconfiguration of the endosperm methylation landscape that effectively results in imprinting of the entire maternal genome.
Publications
- No publications reported this period
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