Genetic Analysis of Silenced Chromatin in Neurospora

University Of Oregon Eugene, Eugene OR

Investigators

Abstract

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.