Telomere Formation Systems in Budding and Fission Yeasts

Telomeres are specialized structures essential for genomic stability in eukaryotic cells. Inducible systems causing telomere shortening or telomere formation from short tracts of telomere repeats were developed in the late 1990s in Saccharomyces cerevisiae and have been adapted to investigate multiple aspects of telomere biology. In the formation system, an internal tract of telomere repeats is placed next to an inducible double-strand break. Inducing the break converts the telomere tract into a functional telomere whose fate can be followed kinetically and allows one to assay elongation, protein recruitment, and the DNA damage checkpoint activation. This work was extended to Schizosaccharomyces pombe, as it shares some features of telomeric chromatin with mammalian cells that are missing in S. cerevisiae. The S. pombe system has revealed novel aspects of telomeric chromatin formation and similarities with S. cerevisiae. This chapter will review these past discoveries in different yeast model organisms, and what they reveal about telomere physiology that may well be conserved in mammals

Mec1p associates with functionally compromised telomeres

In many organisms, telomere DNA consists of simple sequence repeat tracts that are required to protect the chromosome end. In the yeast Saccharomyces cerevisiae, tract maintenance requires two checkpoint kinases of the ATM family, Tel1p and Mec1p. Previous work has shown that Tel1p is recruited to functional telomeres with shorter repeat tracts to promote telomerase-mediated repeat addition, but the role of Mec1p is unknown. We found that Mec1p telomere association was detected as cells senesced when telomere function was compromised by extreme shortening due to either the loss of telomerase or the double-strand break binding protein Ku. Exonuclease I effects the removal of the 5' telomeric strand, and eliminating it prevented both senescence and Mec1p telomere association. Thus, in contrast to Tel1p, Mec1p associates with short, functionally compromised telomeres

The inhibition of checkpoint activation by telomeres does not involve exclusion of dimethylation of histone H4 lysine 20 (H4K20me2) [version 2; referees: 2 approved, 1 not approved]

DNA double-strand breaks (DSBs) activate the DNA damage checkpoint machinery to pause or halt the cell cycle.  Telomeres, the specific DNA-protein complexes at linear eukaryotic chromosome ends, are capped DSBs that do not activate DNA damage checkpoints.  This “checkpoint privileged” status of telomeres was previously investigated in the yeast Schizosaccharomyces pombelacking the major double-stranded telomere DNA binding protein Taz1. Telomeric DNA repeats in cells lacking Taz1 are 10 times longer than normal and contain single-stranded DNA regions. DNA damage checkpoint proteins associate with these damaged telomeres, but the DNA damage checkpoint is not activated. This severing of the DNA damage checkpoint signaling pathway was reported to stem from exclusion of histone H4 lysine 20 dimethylation (H4K20me2) from telomeric nucleosomes in both wild type cells and cells lacking Taz1.  However, experiments to identify the mechanism of this exclusion failed, prompting our re-evaluation of H4K20me2 levels at telomeric chromatin.  In this short report, we used an extensive series of controls to identify an antibody specific for the H4K20me2 modification and show that the level of this modification is the same at telomeres and internal loci in both wild type cells and those lacking Taz1.  Consequently, telomeres must block activation of the DNA Damage Response by another mechanism that remains to be determined

Identification of regulatory variants associated with genetic susceptibility to meningococcal disease.

Non-coding genetic variants play an important role in driving susceptibility to complex diseases but their characterization remains challenging. Here, we employed a novel approach to interrogate the genetic risk of such polymorphisms in a more systematic way by targeting specific regulatory regions relevant for the phenotype studied. We applied this method to meningococcal disease susceptibility, using the DNA binding pattern of RELA - a NF-kB subunit, master regulator of the response to infection - under bacterial stimuli in nasopharyngeal epithelial cells. We designed a custom panel to cover these RELA binding sites and used it for targeted sequencing in cases and controls. Variant calling and association analysis were performed followed by validation of candidate polymorphisms by genotyping in three independent cohorts. We identified two new polymorphisms, rs4823231 and rs11913168, showing signs of association with meningococcal disease susceptibility. In addition, using our genomic data as well as publicly available resources, we found evidences for these SNPs to have potential regulatory effects on ATXN10 and LIF genes respectively. The variants and related candidate genes are relevant for infectious diseases and may have important contribution for meningococcal disease pathology. Finally, we described a novel genetic association approach that could be applied to other phenotypes

Two paralogs involved in transcriptional silencing that antagonistically control yeast life span

AbstractIn the yeast Saccharomyces cerevisiae, one determinant of aging or life span is the accumulation of extrachromosomal copies of rDNA circles in old mother cells [1]. The production of rDNA circles depends upon intrachromosomal recombination within the rDNA tandem array, a process regulated by the protein Sir2 (Sir2p). Together with Sir1p, Sir3p, Sir4p and Orc1p, Sir2p is also involved in transcriptional silencing of genes at the silent mating-type cassettes, in the rDNA array, and at telomeres. Using a ‘triple silencer’ strain that can monitor an increase or decrease in gene expression at these three loci, we found that deletion of the ZDS1 gene caused an increase in silencing in the rDNA and at a silent mating-type cassette at the expense of telomere silencing. The zds1 deletion also resulted in an increase in life span and a decrease in Sir3p phosphorylation. In contrast, deletion of its paralog ZDS2 caused a decrease in rDNA silencing, a decrease in life span and an increase in Sir3p phosphorylation. As Zds2p, but not Zds1p, had strong two-hybrid interactions with Orc1p and the four Sir proteins, Zds1p might indirectly control Sir3p through a Sir3p kinase