Dataset Information

Decoupling epigenetic and genetic effects through systematic analysis of gene position

ABSTRACT: Classic ‘position effect’ experiments repositioned genes to the telomere to demonstrate that the epigenetic landscape can dramatically alter gene expression. Here we show that systematic gene knockout collections provide an exceptional resource for interrogating position effects, not only at the telomere but at every single genetic locus. Because deleted genes are replaced by the same reporter gene, interrogation of this reporter provides a sensitive probe into many different chromatin environments while controlling for genetic context. Using this approach we find that, whereas replacement of yeast genes with the kanMX marker does not perturb the chromatin landscape, differences due to gene position account for more than 35% of marker gene activity. We observe chromatin influences different from those reported previously, including an antagonistic interaction between histone H3 lysine 36 trimethylation (H3K36me3) and the Rap1 transcriptional activation site in kanMX that is mediated through a Set2-Rpd3-dependent pathway. This interaction explains why some yeast genes have been resistant to deletion and allows successful generation of these deletion strains using a modified transformation procedure. These findings demonstrate that chromatin regulation is not governed by a uniform ‘histone code’, but by specific interactions between chromatin and genetic factors.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE42554 | GEO | 2012/11/28

SECONDARY ACCESSION(S): PRJNA182259

REPOSITORIES: GEO

ACCESS DATA

Similar Datasets

Project description:The mutation in the budding yeast gene PCNA, pol30-8, as well as deletion of DOT1 (dot1Δ), encoding the only histone H3 K79 methyltransferase in budding yeast, have been implicated in telomeric silencing. To further analyze these mutants, we used microarrays to study whether either pol30-8, dot1Δ or the double mutant leads to changes in gene expression levels when compared to isogenic wild-type strains. ABSTRACT: Telomere-associated position effect variegation (TPEV) in budding yeast has been used as a model for understanding epigenetic inheritance and gene silencing. A widely used assay to identify mutants with improper TPEV employs the URA3 gene at the telomere of chromosome VII-L that can be counter-selected with 5-fluoroorotic acid (5-FOA). 5-FOA resistance has been inferred to represent lack of transcription of URA3 and therefore to represent heterochromatin-induced gene silencing. For two genes implicated in telomere silencing, POL30 and DOT1, we show that the URA3 telomere reporter assay does not reflect their role in heterochromatin formation. Rather, an imbalance in ribonucleotide reductase (RNR), which is induced by 5-FOA, and the specific promoter of URA3 fused to ADH4 at telomere VII-L are jointly responsible for the variegated phenotype. We conclude that metabolic changes caused by the drug employed and certain mutants being studied are incompatible with the use of certain prototrophic markers for TPEV. 3 replicates each of the S. cerevisiae MATalpha wild-type, pol30-8, dot1Δ and pol30-8 dot1Δ strains carrying the epigenetic silencing reporter genes hmr::ADE2 and URA3-VIIL (in the W303 genetic background; the ade2-1 and ura3-1 alleles normally present in this background were deleted) were grown to mid-logarithmic phase and harvested for RNA extraction and hybridization on Affymetrix microarrays.

Project description:Genomic instability associated with DNA replication stress is linked to cancer and genetic pathologies in humans. If not properly regulated, replication stress, such as fork stalling and collapse, can be induced at natural replication impediments present throughout the genome. The fork protection complex (FPC) is thought to play a critical role in stabilizing stalled replication forks at several known replication barriers including eukaryotic rDNA genes and the fission yeast mating-type locus. However, little is known about the role of the FPC at other natural impediments including telomeres. Telomeres are considered to be difficult to replicate due to the presence of repetitive GT-rich sequences and telomere-binding proteins. However, the regulatory mechanism that ensures telomere replication is not fully understood. Here, we report the role of the fission yeast Swi1/Timeless, a subunit of the FPC, in telomere replication. Loss of Swi1 causes telomere shortening in a telomerase-independent manner. Our epistasis analyses suggest that heterochromatin and telomere-binding proteins are not major impediments for telomere replication in the absence of Swi1. Instead, repetitive DNA sequences impair telomere integrity in swi1Î mutant cells, leading to the loss of repeat DNA. In the absence of Swi1, telomere shortening is accompanied with an increased recruitment of Rad52 recombinase and more frequent amplification of telomere/subtelomeres, reminiscent of tumor cells that utilize the alternative lengthening of telomeres pathway (ALT) to maintain telomeres. These results suggest that Swi1 ensures telomere replication by suppressing recombination and repeat instability at telomeres. Our studies may also be relevant in understanding the potential role of the FPC in regulation of telomere stability in cancer cells. Genome-wide distributions of Rad52 in wild type and in swi1 deletion in fission yeast The'SP1173_WT ChIP-seq' is an input sample (non-tagged data).

Dataset Information

Decoupling epigenetic and genetic effects through systematic analysis of gene position

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure