Vincent Géli

How telomeres are replicated

The replication of the ends of linear chromosomes, or telomeres, poses unique problems, which must be solved to maintain genome integrity and to allow cell division to occur. Here, we describe and compare the timing and specific mechanisms that are required to initiate, control and coordinate synthesis of the leading and lagging strands at telomeres in yeasts, ciliates and mammals. Overall, it emerges that telomere replication relies on a strong synergy between the conventional replication machinery, telomere protection systems, DNA-damage-response pathways and chromosomal organization.

Histone H3 lysine 4 trimethylation marks meiotic recombination initiation sites

The function of histone modifications in initiating and regulating the chromosomal events of the meiotic prophase remains poorly understood. In Saccharomyces cerevisiae, we examined the genome‐wide localization of histone H3 lysine 4 trimethylation (H3K4me3) along meiosis and its relationship to gene expression and position of the programmed double‐strand breaks (DSBs) that initiate interhomologue recombination, essential to yield viable haploid gametes. We find that the level of H3K4me3 is constitutively higher close to DSB sites, independently of local gene expression levels. Without Set1, the H3K4 methylase, 84% of the DSB sites exhibit a severely reduced DSB frequency, the reduction being quantitatively correlated with the local level of H3K4me3 in wild‐type cells. Further, we show that this differential histone mark is already established in vegetative cells, being higher in DSB‐prone regions than in regions with no or little DSB. Taken together, our results demonstrate that H3K4me3 is a prominent and preexisting mark of active meiotic recombination initiation sites. Novel perspectives to dissect the various layers of the controls of meiotic DSB formation are discussed.

RPA regulates telomerase action by providing Est1p access to chromosome ends

Replication protein A (RPA) is a highly conserved single-stranded DNA–binding protein involved in DNA replication, recombination and repair. We show here that RPA is present at the telomeres of the budding yeast Saccharomyces cerevisiae, with a maximal association in S phase. A truncation of the N-terminal region of Rfa2p (associated with the rfa2Δ40 mutated allele) results in severe telomere shortening caused by a defect in the in vivo regulation of telomerase activity. Cells carrying rfa2Δ40 show impaired binding of the protein Est1p, which is required for telomerase action. In addition, normal telomere length can be restored by expressing a Cdc13-Est1p hybrid protein. These findings indicate that RPA activates telomerase by loading Est1p onto telomeres during S phase. We propose a model of in vivo telomerase action that involves synergistic action of RPA and Cdc13p at the G-rich 3′ overhang of telomeric DNA.

The DNA damage response at eroded telomeres and tethering to the nuclear pore complex

The ends of linear eukaryotic chromosomes are protected by telomeres, which serve to ensure proper chromosome replication and to prevent spurious recombination at chromosome ends. In this study, we show by single cell analysis that in the absence of telomerase, a single short telomere is sufficient to induce the recruitment of checkpoint and recombination proteins. Notably, a DNA damage response at eroded telomeres starts many generations before senescence and is characterized by the recruitment of Cdc13 (cell division cycle 13), replication protein A, DNA damage checkpoint proteins and the DNA repair protein Rad52 into a single focus. Moreover, we show that eroded telomeres, although remaining at the nuclear periphery, move to the nuclear pore complex. Our results link the DNA damage response at eroded telomeres to changes in subnuclear localization and suggest the existence of collapsed replication forks at eroded telomeres.

A two-step model for senescence triggered by a single critically short telomere

Telomeres protect chromosome ends from fusion and degradation. In the absence of a specific telomere elongation mechanism, their DNA shortens progressively with every round of replication, leading to replicative senescence. Here, we show that telomerase-deficient cells bearing a single, very short telomere senesce earlier, demonstrating that the length of the shortest telomere is a major determinant of the onset of senescence. We further show that Mec1p–ATR specifically recognizes the single, very short telomere causing the accelerated senescence. Strikingly, before entering senescence, cells divide for several generations despite complete erosion of their shortened telomeres. This pre-senescence growth requires RAD52 (radiation sensitive) and MMS1 (methyl methane sulfonate sensitive), and there is no evidence for major inter-telomeric recombination. We propose that, in the absence of telomerase, a very short telomere is first maintained in a pre-signalling state by a RAD52–MMS1-dependent pathway and then switches to a signalling state leading to senescence through a Mec1p-dependent checkpoint.

Protein interactions within the SET1 complex and their roles in the regulation of histone 3 lysine 4 methylation

Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc1 strongly interacts with itself. Inactivation of either Bre2 or Sdc1 has very similar effects. Neither is required for complex integrity, and their removal results in an increase of H3K4 mono- and dimethylation and a severe decrease of trimethylation at the 5′ end of active coding regions but a decrease of H3K4 dimethylation at the 3′ end of coding regions. Cells lacking Spp1 have a reduced amount of Set1 and retain a fraction of trimethylated H3K4, whereas cells lacking Shg1 show slightly elevated levels of both di- and trimethylation. Set1C associates with both serine 5- and serine 2-phosphorylated forms of polymerase II, indicating that the association persists to the 3′ end of transcribed genes. Taken together, our results suggest that Set1C subunits stimulate Set1 catalytic activity all along active genes.

Set1 is required for meiotic S-phase onset, double-strand break formation and middle gene expression

The Set1 protein of Saccharomyces cerevisiae is a histone methyltransferase (HMTase) acting on lysine 4 of histone H3. Inactivation of the SET1 gene in a diploid leads to a sporulation defect. We have studied various processes that take place during meiotic differentiation in set1Δ diploid cells. The absence of Set1 leads to a delay of meiotic S‐phase onset, which reflects a defect in DNA replication initiation. The timely induction of meiotic DNA replication does not require the Set1 HMTase activity, but depends on the SET domain. In addition, set1Δ displays a severe impairment of the DNA double‐strand break formation, which is not only the consequence of the replication delay. Transcriptional profiling experiments show that the induction of middle meiotic genes, but not of early meiotic genes, is affected by the loss of Set1. In contrast to meiotic replication, the transcriptional induction of the middle meiotic genes appears to depend on the methylation of H3‐K4. Our results unveil multiple roles of Set1 in meiotic differentiation and distinguish between HMTase‐dependent and ‐independent Set1 functions.

CST Meets Shelterin to Keep Telomeres in Check

Telomere protection in budding yeast requires the heterotrimer named CST (for Cdc13-Stn1-Ten1). Recent data show that CST components are conserved and required for telomere stability in a wide range of eukaryotes, even those utilizing the shelterin complex to protect their telomeres. A common function of these proteins might be to stimulate priming at the C-strand gap that remains after telomerase elongation, replication termination, and terminal processing. In light of the budding yeast situation, another conserved function of CST might well be the regulation of telomerase. The cohabitation at telomeres of CST and shelterin components highlights the complexity of telomere biology.

Interaction between Set1p and checkpoint protein Mec3p in DNA repair and telomere functions.

The yeast protein Set1p, inactivation of which alleviates telomeric position effect (TPE), contains a conserved SET domain present in chromosomal proteins involved in epigenetic control of transcription. Mec3p is required for efficient DNA–damage–dependent checkpoints at G1/S, intra–S and G2/M (refs 3, 4,5,6 and 7). We show here that the SET domain of Set1p interacts with Mec3p. Deletion of SET1 increases the viability of mec3Δ mutants after DNA damage (in a process that is mostly independent of Rad53p kinase, which has a central role in checkpoint control) but does not significantly affect cell–cycle progression. Deletion of MEC3 enhances TPE and attenuates the set1Δ–induced silencing defect. Furthermore, restoration of TPE in a set1Δ mutant by overexpression of the isolated SET domain requires Mec3p. Finally, deletion of MEC3 results in telomere elongation, whereas cells with deletions of both SET1 and MEC3 do not have elongated telomeres. Our findings indicate that interactions between SET1 and MEC3 have a role in DNA repair and telomere function.

Ubiquitylation of the COMPASS component Swd2 links H2B ubiquitylation to H3K4 trimethylation

Mono-ubiquitylation of histone H2B correlates with transcriptional activation and is required for di- and trimethylation at Lys 4 on the histone H3 tail (H3K4) by the SET1/COMPASS methyltransferase complex through a poorly characterized trans-tail pathway. Here we show that mono-ubiquitylation of histone H2B promotes ubiquitylation at Lys 68 and Lys 69 of Swd2, the essential component of SET1/COMPASS in Saccharomyces cerevisiae. We found that Rad6/Bre1 ubiquitylation enzymes responsible for H2B ubiquitylation also participate directly in Swd2 modification. Preventing Swd2 or H2B ubiquitylation did not affect Set1 stability, interaction of Swd2 with Set1 or the ability of Swd2 to interact with chromatin. However, we found that mutation of Lys 68 and Lys 69 of Swd2 markedly reduced trimethylation, and to a lesser extent dimethylation, of H3K4 at the 5′-end of transcribing genes without affecting monomethylation. This effect results from the ability of Swd2 ubiquitylation to control recruitment of Spp1, a COMPASS subunit necessary for trimethylation. Our results further indicate that Swd2 is a major H3-binding component of COMPASS. Swd2 thus represents a key factor that mediates crosstalk between H2B ubiquitylation and H3K4 trimethylation on chromatin.