Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: I gave seminars at Vanderbilt University, a meeting on Translational Seed Biology Meeting at UC Davis, Texas A&M University, the Danforth Plant Science Center, at the Nara Institute of Science and Technology (Nara, Japan) and at the Advanced Industrial Science and Technology (Tsukuba Science Center, Japan). I taught classes in plant molecular biology (PMB160) and in genetics and molecular biology (Biology 1A). PARTICIPANTS: Robert Fischer (Principal Investigator), Jon Penterman (Postdoctoral Fellow), Jin Hue Huh (Postdoctoral Fellow), Tzung-Fu Hsieh (Postdoctoral Fellow), Matthew Bauer (Postdoctoral Fellow). All participants carried out experiments and helped write manuscripts on DNA demethylation and gene imprinting. TARGET AUDIENCES: Target audience includes biologists interested in epigenetic regulation of growth and development. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
DNA demethylation in Arabidopsis (Arabidopsis thaliana) is mediated by DNA glycosylases of the DEMETER family. Three DEMETER-LIKE (DML) proteins, REPRESSOR OF SILENCING1 (ROS1), DML2, and DML3, function to protect genes from potentially deleterious methylation. In Arabidopsis, much of the DNA methylation is directed by RNA interference (RNAi) pathways and used to defend the genome from transposable elements and their remnants, repetitive sequences. Here, we investigated the relationship between DML demethylation and RNAi-mediated DNA methylation. We found that genic regions demethylated by DML enzymes are enriched for small interfering RNAs and generally contain sequence repeats, transposons, or both. The most common class of small interfering RNAs was 24 nucleotides long, suggesting a role for an RNAi pathway that depends on RNA-DEPENDENT RNA POLYMERASE2 (RDR2). We show that ROS1 removes methylation that has multiple, independent origins, including de novo methylation directed by RDR2-dependent and -independent RNAi pathways. Interestingly, in rdr2 and drm2 mutant plants, we found that genes demethylated by ROS1 accumulate CG methylation, and we propose that this hypermethylation is due to the ROS1 down-regulation that occurs in these mutant backgrounds. Our observations support the hypothesis that DNA demethylation by DML enzymes is one mechanism by which Arabidopsis genes are protected from genome defense pathways. The Arabidopsis DEMETER (DME) DNA glycosylase is required for the maternal allele expression of imprinted Polycomb group (MEDEA and FIS2) and transcription factor (FWA) genes in the endosperm. Expression of DME in the central cell, not in pollen or stamen, establishes gene imprinting by hypomethylating maternal alleles. However, little is known about other genes regulated by DME. To identify putative DME target genes, we generated CaMV:DME plants which ectopically express DME in pollen and stamens. Comparison of mRNA profiles revealed 94 genes induced by ectopic DME expression in both stamen and pollen. Gene ontology analysis identified three molecular functions enriched in the DME-inducible RNA list: DNA or RNA binding, kinase activity, and transcription factor activity. Semi-quantitative RT-PCR verified the candidate genes identified by GeneChip analysis. The putative target genes identified in this study will provide insights into the regulatory mechanism of DME DNA glycosylase and the functions of DNA demethylation.
Publications
- Penterman, J., Uzawa, R., Fischer, R.L. (2007) Genetic Interactions between DNA demethylation and methylation in Arabidopsis thaliana. Plant Physiol. 145:1549-1557.
- Ohr, H., Bui, A.Q., Le, B.H., Fischer, R.L., Choi, Y. (2007) Identification of putative Arabidopsis DEMETER target genes by GeneChip analysis. Biochem. and Biophys. Res. Comm. 364:856-860.
- Huh, J.H., Bauer, M.J., Hsieh, T.-F., Fischer, R.L. (2007) Endosperm Gene Imprinting and Seed Development. Current Opinion Genetics and Development. 17:480-485.
- Penterman, J., Huh, J. H., Hsieh, T.-F., Fischer, R.L. (2007) Genomic Imprinting in Arabidopsis thaliana and Zea mays. In Plant Cell Monographs, Volume 8, Endosperm, Development and Molecular Biology. Olsen, O.A., ed., Springer-Verlag, Berlin, Pp. 219-239.
- Huh, J.H., Bauer, M.J., Hsieh, T.F., and Fischer, R.L. (2008) Cellular Programming of Plant Gene Imprinting. Cell. 132:735-744.
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Progress 09/01/05 to 08/31/08
Outputs
OUTPUTS: Seed, the end product of plant reproduction, represents the primary nutrient source for humans and domesticated animals. Seeds also produce many industrially important polymers such as oils and starches, as well as vaccines and pharmaceuticals. Genomic imprinting causes genes to be expressed according to their parental origin. Gene imprinting takes place in the endosperm of the seed and is essential for its viability. Imprinted genes also control seed size and likely represent a barrier to most forms of apomixis (parthenogenic seed formation) as well as wide species crosses. Information and tools from our experiments will improve our ability to control production of valuable nutrient and pharmaceutical commodities in endosperm and seed. The endosperm is an essential component of the seed. In dicot plants it supports the development of the embryo. In monocot plants it represents the major nutritional portion of the seed. Although a wealth of information about endosperm structure and biochemistry is available, little is known about the molecular mechanisms that control imprinting in the endosperm. Gene imprinting is observed exclusively in mammals and plants. In mammals, many of the imprinted genes are expressed in the extraembryonic membranes that transfer nutrients from the mother to the embryo. In plants, the endosperm performs a similar function and is the critical site of gene imprinting. Our experiments have led to genes, proteins, and the mechanism(s) that regulate endosperm imprinting. The molecular tools and information generated by these experiments will make it possible to control this important reproductive process and will allow us to derive novel strategies to generate better plant varieties and to increase food production. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We elucidated the mechanisms for plant gene imprinting, discovered fundamental differences in gene imprinting in plants and mammals, and showed how DNA is enzymatically demethylated. Gene imprinting, the differential expression of maternal and paternal derived alleles, is necessary for sexual plant and mammal reproduction. In mammals, imprinted genes influence fetal growth and human diseases are linked to mutations in imprinted genes. In plants, imprinted genes control the growth of seeds, which are the primary source of carbon, nitrogen, and energy for humans and domesticated animals. We discovered that double fertilization of egg and central cell to form embryo and endosperm, respectively, underlies the distinctive cellular programming of plant gene imprinting. In the central cell, a cascade of epigenetic regulation is initiated by DNA demethylation. Plants uniquely utilize the base excision DNA repair pathway to demethylate maternal alleles. The demethylation pathway is initiated by a DNA glycosylase, DME, which excises 5-methylcytosine and activates Polycomb group (MEA and FIS2) and transcription factor (FWA) maternal allele expression. In the endosperm, certain Polycomb group proteins methylate histones to maintain their imprinted status. Thus, the mechanisms for gene imprinting in plants and mammals are mirror images of each other. In mammals, active alleles are silenced by de novo DNA methylation, a process that must be reset every generation. In plants, silenced alleles are activated by DNA demethylation, a process that need never be reset because it occurs in the central cell, whose genome is not transmitted to the next generation.
Publications
- Fischer, R.L., Ohad, N., Kiyosue, T., Yadegari, R., Margossian, L., Harada, J., Goldberg, R.B. (2006) Nucleic Acids That Control Seed and Fruit Development in Plants. US Patent Number 7,029,917.
- Fischer, R.L., Ohad, N., Kiyosue, T., Yadegari, R., Margossian, L., Harada, J., Goldberg, R.B. (2006) Nucleic Acids That Control Seed and Fruit Development in Plants. US Patent Number 7,049,488.
- Fischer, R.L., Choi, Y., Hannon, M. (2006) Methods for Modulating Floral Organ Identity, Modulating Floral Organ Number, Increasing of Meristem Size, and Delaying Flowering Time. US Patent Number 7,109,394.
- Penterman, J., Zilberman, D., Huh, J.H., Ballinger, T., Henikoff, S., and Fischer, R.L. (2007) DNA Demethylation in the Arabidopsis Genome Proc. Natl. Acad. Sci. USA. 104:6752-6757.
- Penterman, J., Uzawa, R., Fischer, R.L. (2007) Genetic Interactions between DNA demethylation and methylation in Arabidopsis thaliana. Plant Physiol. 145:1549-1557.
- Gehring, M., Huh, J. H., Hsieh, T.-F., Penterman, J., Choi, Y., Harada, J.J., Goldberg, R.B., Fischer, R.L. (2006) DEMETER DNA glycosylase establishes MEDEA Polycomb gene self-imprinting by allele-specific demethylation. Cell 124:495-506.
- Xiao, W., Custard, K.D., Brown, R.C., Lemmon, B.E., Harada, J.J., Goldberg, R.B., Fischer, R.L. (2006) DNA Methylation Is Critical for Arabidopsis Embryogenesis and Seed Viability. Plant Cell 18:805-814.
- Xiao, W., Brown, R.C., Lemmon, B.E., Harada, J.J., Goldberg, R.B., Fischer, R.L. (2006) Regulation of Seed Size by Hypomethylation of Maternal and Paternal Genomes. Plant Physiology. 142:1160-1168.
- Ohr, H., Bui, A.Q., Le, B.H., Fischer, R.L., Choi, Y. (2007) Identification of putative Arabidopsis DEMETER target genes by GeneChip analysis. Biochem. and Biophys. Res. Comm. 364:856-860.
- Huh, J.H., Bauer, M.J., Hsieh, T.-F., Fischer, R.L. (2007) Endosperm Gene Imprinting and Seed Development. Current Opinion Genetics and Development. 17:480-485.
- Penterman, J., Huh, J. H., Hsieh, T.-F., Fischer, R.L. (2007) Genomic Imprinting in Arabidopsis thaliana and Zea mays. In Plant Cell Monographs, Volume 8, Endosperm: Development and Molecular Biology. Olsen, O.A., ed., Springer-Verlag, Berlin, Pp. 219-239.
- Fischer, R.L., Choi, Y., Hannon, M. (2008) Promoter for expression of a heterologous polynucleotide in a female gametophyte. US Patent Number 7,414,124.
- Huh, J.H., Bauer, M.J., Hsieh, T.F., and Fischer, R.L. (2008) Cellular Programming of Plant Gene Imprinting. Cell. 132:735-744.
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Progress 09/01/06 to 08/31/07
Outputs
DNA methylation is an epigenetic modification of cytosine that is important for silencing gene transcription and transposons, gene imprinting, development, and seed viability. DNA METHYLTRANSFERASE1 (MET1) is the primary maintenance DNA methyltransferase in Arabidopsis. Reciprocal crosses between antisense MET1 transgenic and wild type plants show that DNA hypomethylation has a parent-of-origin effect on seed size. However, due to the dominant nature of the antisense MET1 transgene, the parent with a hypomethylated genome, its gametophyte, and both the maternal and paternal genomes of the F1 seed become hypomethylated. Thus, the distinct role played by hypomethylation at each generation is not known. To address this issue, we examined F1 seed from reciprocal crosses using a loss-of-function recessive null allele, met1-6. Crosses between wild type and homozygous met1-6 parents show that hypomethylated maternal and paternal genomes result in significantly larger and
smaller F1 seeds, respectively. Our analysis of crosses between wild type and heterozygous MET1/met1-6 parents revealed that hypomethylation in the female or male gametophytic generation was sufficient to influence F1 seed size. A recessive mutation in another gene that dramatically reduces DNA methylation, DECREASE IN DNA METHYLATION1 (DDM1), also causes parent-of-origin effects on F1 seed size. By contrast, recessive mutations in genes that regulate a smaller subset of DNA methylation, CHROMOMETHYLASE3 (CMT3) and DOMAINS REARRANGED METHYLTRANSFERASES (DRM1/DRM2) had little effect on seed size. Collectively, these results show that maternal and paternal genomes play distinct roles in the regulation of seed size in Arabidopsis. Cytosine DNA methylation is considered to be a stable epigenetic mark, but active demethylation has been observed in both plants and animals. In Arabidopsis thaliana DNA glycosylases of the DEMETER (DME) family remove methylcytosines from DNA. Demethylation by
DME is necessary for genomic imprinting and demethylation by a related protein, REPRESSOR OF SILENCING1, prevents gene silencing in a transgenic background. However, the extent and function of demethylation by DEMETER-LIKE (DML) proteins in wild type plants is not known. Using genome tiling microarrays we mapped DNA methylation in mutant and wild type plants and identified 179 loci actively demethylated by DML enzymes. Mutations in DML genes lead to locus-specific DNA hypermethylation. Reintroducing wild type DML genes restores most loci to the normal pattern of methylation, although at some loci, hypermethylated epialleles persist. Over 80% of loci demethylated by DML enzymes are near or overlap genes. Genic demethylation by DML enzymes primarily occurs at the 5' and 3' ends, a pattern opposite to the overall distribution of wild type DNA methylation. Our results show that demethylation by DML DNA glycosylases edits the patterns of DNA methylation within the Arabidopsis genome to
protect genes from potentially deleterious methylation.
Impacts
Seed, the end product of plant reproduction, represents the primary nutrient source for humans and domesticated animals. Understanding the epigenetic mechanism of gene imprinting provides scientists with information to improve seed growth, development and viability.
Publications
- Xiao, W., Brown, R.C., Lemmon, B.E., Harada, J.J., Goldberg, R.B., Fischer, R.L. (2006) Regulation of Seed Size by Hypomethylation of Maternal and Paternal Genomes. Plant Physiology. 142:1160-1168.
- Penterman, J., Zilberman, D., Huh, J.H., Ballinger, T., Henikoff, S., and Fischer, R.L. (2007) DNA Demethylation in the Arabidopsis Genome Proc. Natl. Acad. Sci. USA. 104:6752-6757.
- Fischer, R.L., Choi, Y., Hannon, M. (2006) Methods for Modulating Floral Organ Identity, Modulating Floral Organ Number, Increasing of Meristem Size, and Delaying Flowering Time. US Patent Number 7,109,394.
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Progress 09/01/05 to 09/01/06
Outputs
Alleles of imprinted genes are expressed differently depending on whether they are inherited from the male or female parent. Imprinting regulates a number of genes essential for normal development in mammals and angiosperms. In mammals, imprinted genes contribute to the control of fetal growth and placental development. Diseases are linked to mutations in imprinted genes or aberrant regulation of their expression. The endosperm, one of the products of angiosperm double fertilization, is an important site of imprinting in plants. The endosperm has functions analogous to the placenta and supports seed growth and development. Failure to imprint certain genes results in embryo abortion and seed death. Using the model plant Arabidopsis, we discovered how gene imprinting is epigenetically regulated. Epigenetic information resides in chromatin, DNA and its associated proteins, which exists in an open conformation with the genes accessible to transcription factors or in a
compacted conformation that silences genes. Two interdependent processes regulate chromatin conformation: the chemical modification (methylation and acetylation) of histone proteins around which the DNA is wrapped, and the methylation of cytosine (5-methylcytosine). Both mechanisms contribute to the regulation of gene imprinting. A DNA glycosylase, called DEMETER (DME), excises 5-methylcytosine and creates an abasic site. Other enzymes (AP endonuclease, DNA polymerase, ligase) nick the DNA, insert cytosine, and seal the DNA. This pathway, reminiscent of the base-excision DNA repair pathway, demethylates DNA by replacing 5-methylcytosine with cytosine. DME specifically demethylates and activates expression of the maternal MEDEA (MEA) allele in the central cell of the female gametophyte. DME is not present in sperm cells and does not activate expression of the paternal MEA allele. The endosperm, generated from the fertilized central cell, inherits an active maternal MEA allele and a
silenced paternal MEA allele. The maternal MEA allele encodes a SET-domain Polycomb group protein that, in turn, methylates histones at the paternal MEA allele, causing the chromatin to be condensed, and ensuring that the paternal MEA allele is silenced. DNA methylation (5-methylcytosine) in mammalian genomes predominantly occurs at CpG dinucleotides, is maintained by DNA methyltransferase1 (Dnmt1), and is essential for embryo viability. The plant genome also has 5-methylcytosine at CpG dinucleotides, which is maintained by METHYLTRANSFERASE1 (MET1), a homolog of Dnmt1. In addition, plants have DNA methylation at CpNpG and CpNpN sites, maintained, in part, by the CHROMOMETHYLASE3 (CMT3) DNA methyltransferase. We found that Arabidopsis thaliana embryos with loss-of-function mutations in MET1 and CMT3 develop improperly, display altered planes and numbers of cell division, and have reduced viability. Genes that specify embryo cell identity are misexpressed, and auxin hormone gradients
are not properly formed in abnormal met1 embryos. Thus, DNA methylation is critical for the regulation of plant embryogenesis and for seed viability.
Impacts
Seed, the end product of plant reproduction, represents the primary nutrient source for humans and domesticated animals. Understanding the epigenetic mechanism of gene imprinting provides scientists with information to improve seed growth, development and viability.
Publications
- Gehring, M., Huh, J. H., Hsieh, T.-F., Penterman, J., Choi, Y., Harada, J.J., Goldberg, R.B., Fischer, R.L. (2006) DEMETER DNA glycosylase establishes MEDEA Polycomb gene self-imprinting by allele-specific demethylation. Cell 124:495-506.
- Xiao, W., Custard, K.D., Brown, R.C., Lemmon, B.E., Harada, J.J., Goldberg, R.B., Fischer, R.L. (2006) DNA Methylation Is Critical for Arabidopsis Embryogenesis and Seed Viability. Plant Cell 18:805-814.
- Fischer, R.L. and Mizukami, Y. (2006) Methods for Altering Organ Mass in Plants. US Patent Number 7,071,379.
- Fischer, R.L., Ohad, N., Kiyosue, T., Yadegari, R., Margossian, L., Harada, J., Goldberg, R.B. (2006) Nucleic Acids That Control Seed and Fruit Development in Plants. US Patent Number 7,029,917.
- Fischer, R.L., Ohad, N., Kiyosue, T., Yadegari, R., Margossian, L., Harada, J., Goldberg, R.B. (2006) Nucleic Acids That Control Seed and Fruit Development in Plants. US Patent Number 7,049,488.
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