Eric D. Richards - US grants

9306266 Richards Post-replicative methylation is the most common DNA modification present in eukaryotic cells. Although DNA methylation has been the subject of intense study, many questions remain about the function of this form of DNA modification in eukaryotes. The proposal outlines a genetic approach to the study of DNA methylation using the flowering plant Arabidopsis thaliana as a model. We have isolated A. thaliana mutants that contain reduced levels of cytosine methylation. Plans for further characterization of these hypomethylation mutants and strategies for isolating new methylation mutants are proposed. %%% It is anticipated that the A. thaliana mutants will provide a new system for studying eukaryotic DNA methylation. ***

Abstract

Although a general understanding that methylation of DNA serves to silence gene expression has been accepted, information about how that methylation is regulated and what other functions, if any, it serves is still forthcoming. A genetic approach to those questions has produced a collection of Arabidopsis thaliana mutants (ddm) that contain reduced cytosine methylation. ddm1 loss of function mutations lead to a rapid loss of methylation from most repetitive sequences in the genome, but only a slow loss of methylation from low copy sequences. The DDM1 gene has been recently cloned and found to encode a SW12/SNF2-like protein, implicating chromatin dynamics as an important process in DNA methylation. An experimental plan to test different models of DDM1 action has been created. Specific goals are to 1) test purified recombinant DDM1 and DDM1-associated complexes for DNA-dependent ATPase activity, nucleosome remodeling and chromatin assembly, 2) investigate whether DDM1 associates with cytosine methyltransferases or other chromatin modifying activities, 3) compare the ability of ddm1 and wild-type nuclear extracts to methylate chromatin substrates. A series of experiments to understand the consequences of DDM1 loss of function will be undertaken. Genetic analysis indicates that the ddm1-induced defects are due to the generation of stable variant alleles or epialleles at unlinked loci. The genomic region affected has been localized in one ddm1-induced dwarfing line (bal) to a 147 kb interval containing a leucine-rich repeat (LRR) "disease resistance" gene cluster. A change in gene expression in this cluster has been identified as a possible cause of the bal phenotype. Methylation and chromatin changes associated with bal variant (epi) alleles will be characterized. Transgenic experiments to determine if misexpression of the gene cluster is sufficient to cause the dwarfing phenotype will be done.

Although knowing the DNA sequence is necessary to an understanding of genetic control of life processes, it is not sufficient. The type of information gained from studies of this sort will have critical impact upon both our understanding and genetic engineering of both plants and animals.

This award provides funding to help junior U.S. scientists attend the Thirteenth International Conference on Arabidopsis Research to be held in Seville, Spain, June 28 to July 2, 2002. The aim is to provide an opportunity for these young researchers to exchange ideas with colleagues from around the world on all aspects of research concerning the model plant Arabidopsis. The conference will include sessions on genomics, metabolic regulation, responses to the environment (biotic and abiotic), hormones and signal transduction, genetic mechanisms and epigenetics, natural variation and evolution, cell biology, developmental phase transitions, embryogenesis and vegetative development, reproductive development, and light signaling and circadian regulation. Arabidopsis is an excellent model for crop plant development and physiology, and many of the findings made in Arabidopsis are readily applicable to crop plants. An important component of the meeting is that approximately thirty of those who submitted abstracts of their work will be invited to present that work in short talks. The conference will maximize participation of young scientists by inviting graduate students, postdoctoral fellows, and junior P.I.'s to present many of the talks.

Because this is the premier international forum for exchange of the latest results and approaches in Arabidopsis research, it is imperative that young scientists from the US have the opportunity to attend this conference. The funds from this award will provide travel fellowships for approximately thirty junior US scientists. First priority will be given to those junior scientists who have been selected by the meeting organizers to give oral presentations and to junior scientists from underrepresented groups. The fellowships, which will cover approximately half to two thirds of the expected travel and meeting costs, will be administered by the North American Arabidopsis Steering Committee, whose members are elected by electronic ballot by North American subscribers to the Arabidopsis electronic bulletin board (arab-gen@net.bio.net, http://www.bio.net/hypermail/ARABIDOPSIS/).

Genomes encode information in two ways, genetically and epigenetically. Genetic information is that contained in the nucleotide sequence of the genome, whereas epigenetic information is that contained elsewhere, typically in modifications to the nucleotides or to the chromatin. Epigenetic information in the form of differential DNA methylation and alternative chromatin packaging influences access to the underlying genetic information and thereby plays a central role in the modulation of gene expression. This project focuses on variation in epigenetic information and the extent to which this variation can affect inheritance from one generation to the next. The impetus for this project was the observation of the effects of altering cytosine methylation in Arabidopsis thaliana. In this plant, reduction in cytosine methylation leads to stable inherited variation in epigenetic information. This variation, in turn, generates abnormal phenotypes at the whole organism level. The project involves two major components. The first is the investigation of the molecular basis of the DNA hypomethylation-induced dwarfing defect, bal, caused by an epigenetic alteration that leads to elevated transcription of a pathogen resistance gene. The second objective is to determine the prevalence of epigenetic variation generated under more natural conditions (i.e. in the absence of mutations that destroy proper epigenetic modification). To establish a baseline for epigenetic variation in this model species, the investigator will study the occurrence of alternative epigenetic alleles among different Arabidopsis strains.

Understanding the influence of modifications to nucleotides in genes and of the packaging of chromosomes is essential to understanding inheritance. This project will help the scientific community in this endeavour.

Epigenetic information, in the form of differential cytosine methylation and alternative chromatin structures, plays a central role in the modulation of eukaryotic gene expression. In some cases, alternative epigenetic modifications can be inherited through meiosis, opening up the possibility that epigenetic variation plays a role in generating phenotypic and genomic diversity. This project is an investigation of two examples of epigenetic variation in the flowering plant Arabidopsis thaliana. The first epigenetic system to be investigated involves the complex BAL genomic locus, which contains paralogous pathogen defense genes and interspersed transposable elements. The defective bal epigenetic allele leads to overexpression of several genes from the locus and confers both developmental and pathogen defense phenotypes. The research will probe the molecular changes at the locus that cause this mis-regulation of gene expression, as well as the unusual metastable behavior of the bal allele. The second objective of the project is to characterize a newly discovered class of non-LTR retrotransposons, comprised of members that are epigenetically silenced in some strains of Arabidopsis but not others. The mechanistic basis and significance of this natural epigenetic variation will be investigated.

The slightly different forms of individual organisms in a population result from the interaction between the genetic potential of those individuals and the environment. Also important are epigenetic changes, which do not involve alterations in DNA sequence but rather the packaging of this genetic information. This project takes advantage of the experimental tools available in the small flowering plant Arabidopsis to study the role of epigenetic changes in inheritance and the control of morphological diversity among individuals and strains.

Intellectual Merit:
Flowering plants contain clusters of repeated genes, many of which are involved in recognition of pathogens. The structures of these genes are often highly variable and a combination of forces drives the diversification among repeated pathogen recognition genes. This project investigates a unique situation in the model flowering plant, Arabidopsis thaliana (mouse-ear cress), where the divergence between duplicated copies of a pathogen recognition gene called SNC1 is extremely rapid. The project focuses on this particular model to define the molecular mechanisms responsible for the frequent birth-and-death of pathogen resistance genes in plants.

Broader Impacts:
The project contains three different elements that broaden its impact. First, the research addresses fundamental questions regarding the ways that genetic change and rapid evolutionary processes occur. The results of the research will also provide insight into the forces that diversify a plant's arsenal of genes devoted to protection against agricultural pests. The second broader impact of the project will be the training and mentoring of scientists at the postdoctoral, graduate and undergraduate levels. Finally, the project directly engages high school students in the central New York region in an authentic collaborative research project, connecting these students, as well as their teachers, with scientists at the Boyce Thompson Institute.