Eric Selker, Ph.D. - US grants

DNA methylation is required for normal development of higher eukaryotes. Methylation is required for X-inactivation and genomic imprinting in mammals, and abnormal methylation is associated with cancer. In humans, mutation of a DNA methyltransferase (MTase) causes ICF syndrome and mutation of the methyI-DNA binding protein (MBP) MeCP2 causes Rett syndrome. DNA methylation is dispensable in the fungus Neurospora crassa, facilitating genetic studies. Isolation of Neurospora mutants defective in DNA methylation (dim) has led to insights into the control and function of methylation in eukaryotes. Most recently, identification of DIM-5 as a histone H3 MTase demonstrated that histone modifications control DNA methylation. The goal of the proposed research is to elucidate the mechanism of DNA methylation in eukaryotes by taking advantage of this outstanding model system. The work will be facilitated by valuable new resources and tools including: 1. the nearly complete sequence of the Neurospora genome, 2. a novel quelling-based system to recognize DNA methylation defects, 3. a mutagenic process, RIP (repeat-induced point mutation), for reverse-genetics, 4. antibodies specific for modified histones and 5. powerful new analytical methods (e.g., mass spectrometry and microarray analysis). Specific aims of the project are: 1. To identify and genetically characterize proteins that bind methylated and RIP-mutated DNA (MRBP-1, MBP-2, MBP-3); 2. To characterize the DIM-2 DNA MTase, which is responsible for all detected methylation in vegetative cells, and the potential DNA MTase RID, which is required for RIP in sexual cells; 3. To carry out mutational analyses of the N-terminal tails of histones H4, H3, H2B and H2A to assess their role in DNA methylation in vivo; 4. To determine the role of histone methylation in DNA methylation; 5. To elucidate the role of histone acetylation and phosphorylation in DNA methylation; 6. To test the possible role of chromatin remodeling in DNA methylation; 7. To identify additional components of the DNA methvlation machinerv.

The goal of the project is to elucidate mechanisms of genetic instability in Neurospora. Knowledge gained from the project should shed light on several general areas: recombination, mutation, and evolution. In addition, the project has practical significance for Neurospora geneticists and researchers working on related fungi (e.g. plant pathogens). During the grant period we will: 1) determine whether RIP is responsible for low- frequency inactivation of duplicate genes in vegetative tissue; 2) attempt to derepress RIP, by mutations, in vegetative cells for use in subsequent genetic and biochemical experiments; 3) isolate mutants deficient in RIP; 4) determine if an inverted linked duplication is subject to RIP, premeiotic reciprocal recombination, or both (to provide a test of the idea that high- frequency premeiotic deletion of sequences between linked duplications results from reciprocal recombination and to provide information for a potential approach to isolate RIP mutants); 5) determine if unlinked duplications are subject to recombination processes ("gene conversion" and reciprocal exchange) prior to meiosis; 6) identify the postulated DNA-cytosine deaminase involved in RIP; 7) investigate the resistance to RIP of rDNA, and the effect of RIP on the transposon Tad. Filamentous fungi have an unusual process, known as RIP, by which they inactive all but one copy of most genes present in more than one copy. This process was discovered by the Principal Investigator during a previous NSF award. The mechanism and significance of this process are unclear, and the PI hopes to learn more about them during this award. A better understanding of RIP will lead to more effective methods for genetic manipulation of industrially important fungi.

The goal of the research is to understand the function and control of DNA methylation in eukaryotes. We already know that methylation 1) can affect gene expression and 2) can cause mutations. The proposed collaboration will combine the biochemical expertise of my laboratory to dissect the mechanism and function of DNA methylation in Neurospora crassa, an organism well suited for both biochemical and genetic studies. Specific aims of the planned experiments are: 1. To define what constitutes a methylation "signal" in Neurospora. Discovery and characterization of RIP (repeat-induced point mutation) led to the conclusion that a sprinkling of G:C to A:T mutations can induced methylation. The proposed research will test a rapid in vivo assay for de novo methylation and apply it in experiments: a) to look for short sequences capable of preventing or inducing methylation of surrounding sequences, b) to determine how many G:C to A:T mutations are required to trigger methylation of a chromosomal region and which positions are critical, and c) to ascertain if various types of mutations (e.g., polarized transitions vs. transversions and frameshifts) are equally effective in triggering methylation. 2. To look for evidence of maintenance methylation in Neurospora. Evidence for cooperativity in methylation of opposite chains in Neurospora DNA will be sought and, if feasible, maintenance of methylation patterns will be directly assessed. 3. To determine whether methylated and unmethylated sequences of Neurospora are in different chromatin forms. A methyl-CpG binding protein available in the Bird laboratory will be employed to affinity-purify oligonucleosomes containing methylated sequences. This chromatin will be characterized with respect to acetylation state of histones H3 and H4 and presence of histone H1. 4. To determine whether Neurospora has a methyl-C binding protein. 5. To determine whether DNA methylation is associated with DNA replication in Neurospora.

9820195
Selker

This award will support a planning visit by Dr. Eric U. Selker of the University of Oregon, to go to Cordoba, Argentina to meet with Dr. Alberto Luis Rosa at the University of Cordoba. During this planning visit, they intend to develop a collaborative research proposal to address the question of structure and function of centromere sequences in neurospora.

DESCRIPTION (provided by applicant) This proposal requests support for a Gordon Research Conference on Epigenetic Regulation of Gene Expression to be held at Holderness School, New Hampshire, August 12-17, 2001. Epigenetic regulation is mediated by the creation and maintenance of heritable, but potentially reversible, changes in chromatin structure and/or DNA methylation which alters gene expression without altering DNA sequence. Epigenetic effects have been discovered in many organisms and they comprise some of the most intriguing and actively investigated phenomena with relevance to both basic and applied science. For example, epigenetic silencing of transgenes poses problems for the long term, commercial use of transgenic plants engineered to express novel phenotypes and can complicate human gene therapy trials. Moreover, loss of epigenetically imprinted events on the chromosome are shown to cause many human cancer types. In addition, mutations in genes encoding DNA methyltransferase and a methyl-DNA binding protein lead to diseases in humans, namely ICF syndrome and Rett Syndrome, respectively. Advances have been made towards understanding the molecular mechanisms that underlie epigenetic silencing in the last few years and this will be a major focus of this conference. Invited speakers are leading researchers working on fungal, plant and animal models who will cover topics such as control and function of DNA methylation, chromatin-based gene silencing, post-transcriptional gene silencing, imprinting, X-inactivation, prions, epigenetics and disease, genome defense systems, paramutation and position effects. The Epigenetics Gordon Conference provides a unique opportunity for researchers working on related phenomena in different organisms to come together and exchange recent results and ideas. It is in a cross-disciplinary environment such as this that intellectual leaps occur and innovative ideas flourish.

Eukaryotic genomes are more than just the sum of their genes. The chromosomal context of genes is important and certain chromosomal parts, including the centromeres, telomeres and rDNA regions, serve structural as well as coding roles in the cell. Several lines of evidence indicate that the chromatin structure of centromeric, telomeric and rDNA regions is more condensed than most chromosomal regions and that this "heterochromatic" chromatin causes gene silencing. The inactivated X chromosome in women represents an example of gene silencing by heterochromatin. Centromeric, telomeric, and rDNA provide excellent models to identify critical differences between alternative forms of chromatin. Parallels are emerging among organisms whose heterochromatic regions have been studied to date (principally yeasts and Drosophila) but significant differences have also been detected. The current project will broaden our understanding of the silencing that characterizes heterochromatic sequences by taking advantage of a model eukaryote, the filamentous fungus Neurospora crassa. The study builds on the well developed genetics of this organism and the recent availability of the nearly complete sequence of its genome. Efficient genetic approaches will be used to identify the important players in the cell that result in the formation, and normal function of, heterochromatin. Reporter genes will be placed in telomeric, centromeric and rDNA regions of Neurospora chromosomes by homologous recombination in a strain bearing a mutation in a silencer gene, nst-1. The resulting strains will be used as transformation hosts to test candidate genes for involvement in heterochromatin silencing. This part of the project takes advantage of a dominant post-transcriptional silencing process, quelling, to efficiently screen a large number of genes. Approximately 30 candidate genes identified by homology to genes of other organisms will be screened initially. In addition, novel silencer genes will be sought in a non-redundant set of sequenced cDNA clones. Genes showing evidence of involvement in silencing will be selectively disrupted using RIP (repeat-induced point mutation). Silenced reporter genes will also be used to select new silencing mutants generated by insertional mutagenesis. The mutated DNA will be isolated and sequenced to identify silencer genes. Finally, centromeric, telomeric and rDNA chromatin will be characterized in wildtype Neurospora and in silencing mutants. Both genetic and physical methods will be used to identify the nature of the defect(s) of the silencing mutants.

Knowledge gained from this project should both improve our understanding of specific mechanisms of gene silencing and shed light on mechanisms responsible for normal and abnormal chromosome behavior in a variety of eukaryotes. In addition to its scientific merit, this project will serve to train students and to advance the use of Neurospora as a practical model system for functional genomics.

DESCRIPTION (provided by applicant): Normal development of animals, plants and fungi rely heavily on chromatin-based signals. The proposed research focuses on a particularly central example, trimethylation of histone H3 Lysine 27 (H3K27me3). Work in flies, mammals and plants have implicated this mark in long-term repression of genes in development, as well as in X-inactivation, genomic imprinting and cancer. Details of how H3K27me3 functions and how it is controlled remain unknown, however. The recent discovery of regions of H3K27me3 in the genome of the simple eukaryote, Neurospora crassa, and identification of elements of the underlying methylation machinery in this organism provide an opportunity to apply the exceptional power of Neurospora molecular and genetic methods to explore the function and control of this histone mark. H3K27me3 has not been found in other simpler systems (e.g. yeasts). Specific aims of the project are: 1. To test for heritability of H3K27me3 and to determine the kinetics of de novo K27 methylation. We will test if methylation of H3K27 induced at an ectopic site is maintained after the inducing construct is removed. We will also use genetic and molecular methods to determine the kinetics of de novo H3K27 methylation. 2. To identify the components of the H3K27 methylation machinery. We will characterize the K27 methyltransferase complex and investigate what reads the H3K27me3 mark. Candidate binders (chromo domain proteins and the SET-7 complex) will be tested and proteomic and genetic (mutant hunt) methods will be used to identify unsuspected elements of the machinery. 3. To identify cis-acting sequences that control H3K27me3. To address the possibility that H3K27me3 in Neurospora is directed by sequences comparable to PREs of Drosophila, we will: a. use ChIP-Seq. to map binding sites of Neurospora PRC2 components; b. test deletions of native H3K27me3 regions for loss of H3K27me; c. test candidate control regions for the ability to direct H3K27 methylation at an ectopic site. 4. To define the role and mechanism of H3K27 methylation in gene repression. We will explore conditions that may control K27me3 genes and determine whether repression by K27 methylation results from a block in transcription initiation or elongation. 5. To test whether the SET-7 complex reads histone marks. We will explore the possibility that the SET-7 complex is sensitive to modifications in the N-terminus of H3 in vivo using our collection of mutants, and if the complex is found to bind H3K27me in vitro, we will follow up by testing it's binding to modified peptides. PUBLIC HEALTH RELEVANCE: Trimethylation of the lysine 27 residue of histone H3 plays a critical role in the epigenetic repression of genes during development, X-inactivation, genomic imprinting, and the aberrant inactivation of genes in cancer. Understanding how this epigenetic mark is regulated will lead to better insight of its role in tumorigenesis and ultimately to the development of therapeutic interventions. Neurospora crassa is the simplest model organism known to have the H3K27me3 mark and provides a system in which to make great strides in our understanding of the H3K27me3 mark.

Our broad aim is to elucidate how eukaryotic genomes are structured and how they work. We will focus on the role of heterochromatin. Normal development of animals, plants and fungi relies on chromatin features including methylation of DNA and histone H3K9 in constitutive heterochromatin, and methylation of histone H3K27 (H3K27me) in facultative heterochromatin. We have shown that the filamentous fungus Neurospora crassa is an extraordinarily favorable genetic/molecular system to elucidate the basic workings of both forms of heterochromatin. The project will involve genetic and molecular dissection of the interconnected roles of chromatin features implicated in heterochromatin including DNA signals, chromatin modifications, histone turnover, nucleosome organization, nuclear organization, and other factors. We have shown that a conserved protein complex (PRC2) is responsible for H3K27me in facultative heterochromatin and that this mark is repressive as in higher organisms. Importantly, H3K27me is not essential for viability of Neurospora, allowing for studies that would be difficult or impossible in higher organisms. A major objective of our study is to understand the mechanism of H3K27me-mediated transcriptional repression. We built a forward genetic scheme to identify genes required for H3K27me-mediated silencing and this has already identified interesting, unanticipated chromatin modifiers. We will both scale up our selection to identify more mutants and will characterize the factors already identified, testing if they act up- or down-stream of H3K27me and determining how they affect gene expression, nucleosome positioning, epigenetic modifications, and other features of chromatin. This will give insight into repression by H3K27me. In addition, we will focus on a tryptophan- inducible H3K27me-marked locus, kyn-1, for a controlled and in depth dissection of H3K27me regulation. We will also take several complementary approaches to elucidate what controls the genomic placement of this epigenetic mark. Our recent studies defined telomere-dependent (TD) and telomere-independent (TI) H3K27me and revealed that telomere repeats are capable of inducing H3K27me. We will investigate the underlying mechanism of TD H3K27me, for example by testing the possible roles of structural features (e.g. G- quadruplex DNA), telomere-associated proteins, and nuclear organization (e.g. placement at nuclear periphery). Use of gene knockouts and our LexA tethering system will allow us to test both necessity and sufficiency of candidate features. We will also experimentally dissect TI domains, which may identify ?PRE?-like elements and DNA binding factors involved in recruitment of H3K27me machinery. Finally, we will test how the broader chromatin environment controls H3K27me, following up our leads that suggest constitutive heterochromatin, H3K36me, H3K56ac, nucleosome turnover, and transcription all influence H3K27me distribution. We are optimistic that our findings on the control and function of heterochromatin in Neurospora will elucidate fundamental processes that also operate in higher eukaryotes.