Progress 10/01/08 to 10/01/13
Outputs
Target Audience: Plant biologists studying epigenetics, gene regulation and seed development Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Undergraduate students, graduate students, and postdoctoral fellows involved in this project were trained in experimental genetic, chromatin biology and genomic techniques, computational analysis, and effective written and oral research presentation. How have the results been disseminated to communities of interest? Research has been disseminated through presentation at national and international conferences and formal publication in scientific journals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objectives 1 and 4: Nucleosome remodelers of the DDM1/Lsh family are required for DNA methylation of transposable elements, but the reason for this was unknown. How DDM1 interacts with other methylation pathways, such as small RNA-directed DNA methylation (RdDM), which is thought to mediate plant asymmetric methylation through DRM enzymes, was also unclear. We showed that most asymmetric methylation is facilitated by DDM1 and mediated by the methyltransferase CMT2 separately from RdDM. We found that heterochromatic sequences preferentially require DDM1 for DNA methylation, and that this preference depends on linker histone H1. RdDM is instead inhibited by heterochromatin and absolutely requires the nucleosome remodeler DRD1. Together, DDM1 and RdDM mediate nearly all transposon methylation, and collaborate to repress transposition and regulate the methylation and expression of genes. Our results indicate that DDM1 provides DNA methyltransferases access to H1-containing heterochromatin to allow stable silencing of transposable elements in cooperation with the RdDM pathway. Objective 3: The regulation of eukaryotic chromatin relies on interactions between many epigenetic factors, including histone modifications, DNA methylation, and the incorporation of histone variants. H2A.Z, one of the most conserved but enigmatic histone variants that is enriched at the transcriptional start sites of genes, has been implicated in a variety of chromosomal processes. We reported a genome-wide anticorrelation between H2A.Z and DNA methylation, an epigenetic hallmark of heterochromatin that has also been found in the bodies of active genes in plants and animals. We investigated the basis of this anticorrelation using a novel h2a.z loss-of-function line in Arabidopsis thaliana. Through genome-wide bisulfite sequencing, we demonstrated that loss of H2A.Z in Arabidopsis has only a minor effect on the level or profile of DNA methylation in genes, and we proposed that the global anticorrelation between DNA methylation and H2A.Z is primarily caused by the exclusion of H2A.Z from methylated DNA. RNA sequencing and genomic mapping of H2A.Z showed that H2A.Z enrichment across gene bodies, rather than at the TSS, is correlated with lower transcription levels and higher measures of gene responsiveness. Loss of H2A.Z causes misregulation of many genes that are disproportionately associated with response to environmental and developmental stimuli. We proposed that H2A.Z deposition in gene bodies promotes variability in levels and patterns of gene expression, and that a major function of genic DNA methylation is to exclude H2A.Z from constitutively expressed genes. Objective 2: This objective was accomplished by another lab and therefore was not pursued by us.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Assaf Zemach, M. Yvonne Kim, Ping-Hung Hsieh, Devin Coleman-Derr, Leor Eshed-Williams, Ka Thao, Stacey L. Harmer and Daniel Zilberman. The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin. Cell 2013, 153: 193-205.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Activities: Conducted experiments analyzing evolution of DNA methylation in eukaryotic organisms, experiments analyzing differential DNA methylation in rice and Arabidopsis seeds, experiments analyzing the interactions of histones H1 and H2A.Z with DNA methylation, and experiments analyzing the role of chromatin remodelers DDM1 and DRD1 in maintenance of DNA methylation and regulation of transposition. Mentored four postdoctoral fellows, four graduate students and nine undergraduate students. Taught one undergraduate course: "Introduction to the Science of living Organisms". Events: Presented the following seminars/lectures: lecture at the Keystone Symposium on Epigenomics, Keystone, Colorado; lecture at the Plant Reproduction for Food Conference, Melbourne, Australia; lecture at the Keystone Symposium on Nuclear Events in Plant Gene Expression and Signaling, Taos, New Mexico; seminar at University Milano-Bicocca, Milan, Italy; seminar at Seoul National University, Korea; seminar at Yonsei University, Seoul, Korea; lecture at the Annual Meeting of the Korean Society for Biochemistry and Molecular Biology, Seoul, Korea; lecture at the 77th Cold Spring Harbor Laboratory Symposium: The Biology of Plants, Long Island, New York; lecture at the International Conference on Arabidopsis Research, Vienna, Austria; lecture at the Beckman Young Investigator Symposium, Irvine, California; lecture at the Bay Area Chromatin Club Symposium, San Francisco, California; session organizer at the 10th International Congress on Plant Molecular Biology, Jeju, Korea; lecture at the Cold Spring Harbor Asia Conference on Plant Epigenetics, Stress and Evolution, Suzhou, China. Products: Deep sequencing of allele-specific DNA methylation and small RNA expression in rice embryo and endosperm. These datasets are publicly available at the NCBI Gene Expression Omnibus web site (www.ncbi.nlm.nih.gov/geo). PARTICIPANTS: Daniel Zilberman (PI): Designed and supervised all research activities. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The Arabidopsis thaliana central cell, the companion cell of the egg, undergoes DNA demethylation prior to fertilization. Active and passive mechanisms have been proposed to contribute to this process, and the targeting preferences, if any, and biological significance, of central cell demethylation remain unclear. We showed 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 transposable elements. We also showed that the vegetative cell, the companion cell of sperm, undergoes DME-dependent demethylation of similar sequences. We found that small RNA molecules can travel from the central cell to the egg, and that 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, thereby assuring stable silencing of transposable elements across generations. The regulation of eukaryotic chromatin relies on interactions between many epigenetic factors, including histone modifications, DNA methylation, and the incorporation of histone variants. H2A.Z, one of the most conserved but enigmatic histone variants that is enriched at the transcriptional start sites of genes, has been implicated in a variety of chromosomal processes. Recently, we reported a genome-wide anticorrelation between H2A.Z and DNA methylation, an epigenetic hallmark of heterochromatin that has also been found in the bodies of active genes in plants and animals. We investigated the basis of this anticorrelation using a novel h2a.z loss-of-function line in Arabidopsis thaliana. Through genome-wide bisulfite sequencing, we demonstrated that loss of H2A.Z in Arabidopsis has only a minor effect on the level or profile of DNA methylation in genes, and we proposed that the global anticorrelation between DNA methylation and H2A.Z is primarily caused by the exclusion of H2A.Z from methylated DNA. RNA sequencing and genomic mapping of H2A.Z show that H2A.Z enrichment across gene bodies, rather than at the TSS, is correlated with lower transcription levels and higher measures of gene responsiveness. Loss of H2A.Z causes misregulation of many genes that are disproportionately associated with response to environmental and developmental stimuli. We propose that H2A.Z deposition in gene bodies promotes variability in levels and patterns of gene expression, and that a major function of genic DNA methylation is to exclude H2A.Z from constitutively expressed genes.
Publications
- Devin Coleman-Derr and Daniel Zilberman. DNA Methylation, H2A.Z, and the regulation of constitutive expression. Cold Spring Harbor Symposia on Quantitative Biology 2012, 77: doi: 10.1101/sqb.2012. 77.014944.
- Devin Coleman-Derr and Daniel Zilberman. Deposition of histone variant H2A.Z within gene bodies regulates responsive genes. PLoS Genetics 2012, 8(10): e1002988.
- Christian A. Ibarra, Xiaoqi Feng, Vera K. Schoft, Tzung-Fu Hsieh, Rie Uzawa, Jessica A. Rodrigues, Assaf Zemach, Nina Chumak, Adriana Machlicova, Toshiro Nishimura, Denisse Rojas, Robert L. Fischer, Hisashi Tamaru and Daniel Zilberman. Active DNA demethylation in plant companion cells reinforces transposon methylation in gametes. Science 2012, 337: 1360-1364.
- Sang Yeol Kim, Jungeun Lee, Leor Eshed-Williams, Daniel Zilberman and Z. Renee Sung. EMF1 and PRC2 cooperate to repress key regulators of Arabidopsis development. PLoS Genetics 2012, 8(3): e1002512.
- Jason T. Huff and Daniel Zilberman. Regulation of biological accuracy, precision, and memory by plant chromatin organization. Current Opinion in Genetics & Development 2012, 22: 132-138.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Activities: Conducted experiments analyzing regulation of imprinted gene expression in Arabidopsis seeds. Mentored four postdoctoral fellows, four graduate students and seven undergraduate students. Taught one undergraduate course: "Introduction to the Science of living Organisms". Events: Presented the following seminars/lectures: seminar at the University of Massachusetts, Amherst; lecture at the Epigenetics World Congress, Boston, Massachusetts; seminar at the Swedish University of Agricultural Sciences, Uppsala; lecture at the Epigenetics Gordon Research Conference, Easton, Massachusetts; seminar at the University of Zurich, Switzerland; lecture at the Botanikertagung Botanical Congress, Berlin, Germany; seminar at the University of California, Davis; two lectures at the Buenos Aires Plant Biology Lectures, Argentina. Products: Deep sequencing of allele-specific gene expression in Arabidopsis embryo and endosperm in wild-type plants and several mutant strains. These datasets are publicly available at the NCBI Gene Expression Omnibus web site (www.ncbi.nlm.nih.gov/geo). PARTICIPANTS: Daniel Zilberman (PI): Designed and supervised all research activities. Pedro Silva: Conducted computational analyses of allele-specific cDNA data. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Imprinted genes are expressed primarily or exclusively from either the maternal or paternal allele, a phenomenon that occurs in flowering plants and mammals. Flowering plant imprinted gene expression has been described primarily in endosperm, a terminal nutritive tissue consumed by the embryo during seed development or after germination. Imprinted expression in Arabidopsis thaliana endosperm is orchestrated by differences in cytosine DNA methylation between the paternal and maternal genomes, as well as by Polycomb group (PcG) proteins. Until recently, only 11 imprinted A. thaliana genes were known. We used extensive sequencing of cDNA libraries to identify nine new paternally expressed and 34 new 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 PcG 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 also identified maternally expressed genes that may be regulated by novel mechanisms or deposited from maternal tissues. We did not detect any imprinted genes in the embryo. Our results demonstrate that imprinted gene expression is an extensive, mechanistically complex phenomenon that likely affects multiple aspects of seed development.
Publications
- Tzung-Fu Hsieh, Juhyun Shin, Rie Uzawa, Pedro Silva, Stephanie Cohen, Matthew J. Bauer, Meryl Hashimoto, Ryan C. Kirkbride, John J. Harada, Daniel Zilberman, and Robert L. Fischer. (2011) "Regulation of imprinted gene expression in Arabidopsis endosperm." PNAS, 108: 1755-1762.
- Daniel Zilberman (2011) "Balancing parental contributions in plant embryonic gene activation." Developmental Cell, 20: 735-736.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Activities: Conducted experiments analyzing evolution of DNA methylation in eukaryotic organisms and experiments analyzing gene regulation by differential DNA methylation in rice seeds. Mentored two postdoctoral fellows, three graduate students and five undergraduate students. Taught two undergraduate courses: "Introduction to Evolutionary Ecology" and "Introduction to the Science of living Organisms". Events: Presented the following seminars/lectures: seminar at the University of Oregon, Eugene; seminar at the National Institute of Genetics, Mishima, Japan; lecture at the International Conference on Arabidopsis Research, Yokohama, Japan; lecture at the FASEB Conference on Biological Methylation, Carefree, Arizona; lecture at the GETA Conference on Epigenomics and Human Health, Oakland, California; lecture at the University of Minnesota Symposium on Next-Generation Sequencing, St. Paul, Minnesota; seminar at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing. Products: High coverage, single base pair-resolution whole genome maps of DNA methylation for seventeen eukaryotic organisms, and for five rice tissues (embryo, endosperm, seedling root, seedling leaf, adult leaf). These datasets are publicly available at the NCBI Gene Expression Omnibus web site (www.ncbi.nlm.nih.gov/geo). PARTICIPANTS: Daniel Zilberman (PI): Designed and supervised all research activities. Pedro Silva: Conducted computational analyses of DNA methylation data. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Eukaryotic cytosine methylation represses transcription, but also occurs in the bodies of active genes, and the extent of methylation biology conservation is unclear. We quantified DNA methylation in seventeen eukaryotic genomes, and found that gene body methylation is conserved between plants and animals, whereas selective methylation of transposons is not. We show that methylation of plant transposons in the CHG context extends to green algae, and present evidence for RNA-directed DNA methylation of fungal genes. Exclusion of histone H2A.Z from methylated DNA is conserved between plants and animals. Our data demonstrate that extant DNA methylation systems are mosaics of conserved and derived features, and indicate that gene body methylation is an ancient property of eukaryotic genomes. Plant DNA methylation is catalyzed by three families of enzymes, each with a preferred sequence context: CG, CHG (H = A, C or T) and CHH, with CHH methylation targeted by the RNA interference (RNAi) pathway. Arabidopsis thaliana endosperm, a placenta-like tissue that nourishes the embryo, is globally hypomethylated in the CG context while retaining high non-CG methylation. Global methylation dynamics in seeds of cereal crops that provide the bulk of human nutrition remain unknown. We showed that rice endosperm DNA is hypomethylated in all sequence contexts. Non-CG methylation is reduced evenly across the genome, while CG hypomethylation is localized. CHH methylation of small transposable elements is increased in embryos, suggesting that endosperm demethylation enhances transposon silencing. Genes preferentially expressed in endosperm, including those coding for major storage proteins and starch synthesizing enzymes, are frequently hypomethylated in endosperm, indicating that DNA methylation is a crucial regulator of rice endosperm biogenesis. Our data demonstrate that genome-wide reshaping of seed DNA methylation is conserved among angiosperms and has a profound effect on gene expression in cereal crops.
Publications
- Assaf Zemach, Ivy E. McDaniel, Pedro Silva and Daniel Zilberman. (2010) "Genome-wide evolutionary analysis of eukaryotic DNA methylation." Science, 328: 916-919.
- Assaf Zemach and Daniel Zilberman. (2010) "Evolution of eukaryotic DNA methylation and the pursuit of safer sex." Current Biology, 20: R780-R785.
- Assaf Zemach, M. Yvonne Kim, Pedro Silva, Jessica A. Rodrigues, Bradley Dotson, Matthew D. Brooks, and Daniel Zilberman. (2010) "Local DNA hypomethylation activates genes in rice endosperm." PNAS, 107: 18729-18734.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Activities: Experiments examining DNA methylation in the endosperm of Arabidopsis thaliana. Teaching: BIO 11, "Introduction to the Science of Living Organisms" and PMB 39D, "Life on Earth: Introduction to Evolutionary Ecology". PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Imprinted genes are expressed primarily or exclusively from either the maternal or paternal allele, a phenomenon that occurs in flowering plants and vertebrates. Flowering plant imprinted gene expression has been described primarily in endosperm, a terminal nutritive tissue consumed by the embryo during seed development or after germination. Imprinted expression in A. thaliana endosperm is orchestrated by differences in cytosine DNA methylation between the paternal and maternal genomes, as well as by Polycomb Group (PcG) proteins. Plant methylation is catalyzed by three families of enzymes, each with a preferred sequence context: CG, CHG (H = A, C or T) and CHH, with CHH methylation targeted by the RNA interference (RNAi) pathway. DNA hypomethylation of the maternal genome is mediated by the DNA glycosylase DEMETER, but the extent of the methylation changes was not known. We showed that virtually the entire endosperm genome is demethylated in the CG context, coupled with extensive local non-CG hypermethylation of siRNA-targeted sequences. Mutation of DEMETER partially restored endosperm CG methylation to levels found in other tissues, but led to large genome-wide decreases in CHG and CHH methylation. Endosperm demethylation was accompanied by CHH hypermethylation of embryo transposable elements, suggesting that demethylation and activation of transposons in the endosperm enhances transposon silencing in the embryo via the RNAi system. Our findings demonstrated extensive reconfiguration of the endosperm methylation landscape that likely reinforces transposon silencing in the embryo.
Publications
- Tzung-Fu Hsieh, Christian A. Ibarra, Pedro Silva, Assaf Zemach, Leor Eshed-Williams, Robert L. Fischer and Daniel Zilberman (2009) "Genome-wide demethylation of Arabidopsis endosperm." Science, 324: 1451-1454.
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