Epigenetic rewriting at centromeric DNA repeats leads to increased chromatin accessibility and chromosomal instability.

BACKGROUND: Centromeric regions of human chromosomes contain large numbers of tandemly repeated α-satellite sequences. These sequences are covered with constitutive heterochromatin which is enriched in trimethylation of histone H3 on lysine 9 (H3K9me3). Although well studied using artificial chromosomes and global perturbations, the contribution of this epigenetic mark to chromatin structure and genome stability remains poorly known in a more natural context. RESULTS: Using transcriptional activator-like effectors (TALEs) fused to a histone lysine demethylase (KDM4B), we were able to reduce the level of H3K9me3 on the α-satellites repeats of human chromosome 7. We show that the removal of H3K9me3 affects chromatin structure by increasing the accessibility of DNA repeats to the TALE protein. Tethering TALE-demethylase to centromeric repeats impairs the recruitment of HP1α and proteins of Chromosomal Passenger Complex (CPC) on this specific centromere without affecting CENP-A loading. Finally, the epigenetic re-writing by the TALE-KDM4B affects specifically the stability of chromosome 7 upon mitosis, highlighting the importance of H3K9me3 in centromere integrity and chromosome stability, mediated by the recruitment of HP1α and the CPC. CONCLUSION: Our cellular model allows to demonstrate the direct role of pericentromeric H3K9me3 epigenetic mark on centromere integrity and function in a natural context and opens interesting possibilities for further studies regarding the role of the H3K9me3 mark.

Short loop length and high thermal stability determine genomic instability induced by G-quadruplex-forming minisatellites.

G-quadruplexes (G4) are polymorphic four-stranded structures formed by certain G-rich nucleic acids, with various biological roles. However, structural features dictating their formation and/or function in vivo are unknown. In S. cerevisiae, the pathological persistency of G4 within the CEB1 minisatellite induces its rearrangement during leading-strand replication. We now show that several other G4-forming sequences remain stable. Extensive mutagenesis of the CEB25 minisatellite motif reveals that only variants with very short (≤ 4 nt) G4 loops preferentially containing pyrimidine bases trigger genomic instability. Parallel biophysical analyses demonstrate that shortening loop length does not change the monomorphic G4 structure of CEB25 variants but drastically increases its thermal stability, in correlation with the in vivo instability. Finally, bioinformatics analyses reveal that the threat for genomic stability posed by G4 bearing short pyrimidine loops is conserved in C. elegans and humans. This work provides a framework explanation for the heterogeneous instability behavior of G4-forming sequences in vivo, highlights the importance of structure thermal stability, and questions the prevailing assumption that G4 structures with short or longer loops are as likely to form in vivo.

Stimulation of gross chromosomal rearrangements by the human CEB1 and CEB25 minisatellites in Saccharomyces cerevisiae depends on G-quadruplexes or Cdc13.

Genomes contain tandem repeats that are at risk of internal rearrangements and a threat to genome integrity. Here, we investigated the behavior of the human subtelomeric minisatellites HRAS1, CEB1, and CEB25 in Saccharomyces cerevisiae. In mitotically growing wild-type cells, these GC-rich tandem arrays stimulate the rate of gross chromosomal rearrangements (GCR) by 20, 1,620, and 276,000-fold, respectively. In the absence of the Pif1 helicase, known to inhibit GCR by telomere addition and to unwind G-quadruplexes, the GCR rate is further increased in the presence of CEB1, by 385-fold compared to the pif1Δ control strain. The behavior of CEB1 is strongly dependent on its capacity to form G-quadruplexes, since the treatment of WT cells with the Phen-DC(3) G-quadruplex ligand has a 52-fold stimulating effect while the mutation of the G-quadruplex-forming motif reduced the GCR rate 30-fold in WT and 100-fold in pif1Δ cells. The GCR events are telomere additions within CEB1. Differently, the extreme stimulation of CEB25 GCR depends on its affinity for Cdc13, which binds the TG-rich ssDNA telomere overhang. This property confers a biased orientation-dependent behavior to CEB25, while CEB1 and HRAS1 increase GCR similarly in either orientation. Furthermore, we analyzed the minisatellites' distribution in the human genome and discuss their potential role to trigger subtelomeric rearrangements.

Assessment of human Nter and Cter BRCA1 mutations using growth and localization assays in yeast.

A large number of missense mutations have been identified within the tumor suppressor gene BRCA1. Most of them, called "variants of unknown significance" (VUS), cannot be classified as pathogenic or neutral by genetic methods, which complicates their cancer risk assessment. Functional assays have been developed to circumvent this uncertainty. They aim to determine how VUS impact the BRCA1 protein structure or function, thereby giving an indication of their potential to cause cancer. So far, three relevant assays have been designed in yeast and used on large sets of variants. However, they are limited to variants mapped in restricted domains of BRCA1. One of them, the small colony phenotype (SCP) assay, monitors the BRCA1-dependent growth of yeast colonies that increases with pathogenic but not neutral mutations positioned in the Cter region. Here, we extend this assay to the Nter part of BRCA1. We also designed a new assay, called the "yeast localization phenotype (YLP) assay," based on the accumulation of BRCA1 in a single inclusion body in the yeast nucleus. This phenotype is altered by variants positioned both in the Nter and Cter regions. Together, these assays provide new perspectives for the functional assessment of BRCA1 mutations in yeast.

G-quadruplex-induced instability during leading-strand replication.

G-quadruplexes are four-stranded nucleic acid structures whose biological functions remain poorly understood. In the yeast S. cerevisiae, we report that G-quadruplexes form and, if not properly processed, pose a specific challenge to replication. We show that the G-quadruplex-prone CEB1 tandem array is tolerated when inserted near ARS305 replication origin in wild-type cells but is very frequently destabilized upon treatment with the potent Phen-DC(3) G-quadruplex ligand, or in the absence of the G-quadruplex-unwinding Pif1 helicase, only when the G-rich strand is the template of leading-strand replication. The orientation-dependent instability is associated with the formation of Rad51-Rad52-dependent X-shaped intermediates during replication detected by two-dimensional (2D) gels, and relies on the presence of intact G-quadruplex motifs in CEB1 and on the activity of ARS305. The asymmetrical behaviour of G-quadruplex prone sequences during replication has implications for their evolutionary dynamics within genomes, including the maintenance of G-rich telomeres.

Genetic instability triggered by G-quadruplex interacting Phen-DC compounds in Saccharomyces cerevisiae.

G-quadruplexes are nucleic acid secondary structures for which many biological roles have been proposed but whose existence in vivo has remained elusive. To assess their formation, highly specific G-quadruplex ligands are needed. Here, we tested Phen-DC(3) and Phen-DC(6), two recently released ligands of the bisquinolinium class. In vitro, both compounds exhibit high affinity for the G4 formed by the human minisatellite CEB1 and inhibit efficiently their unwinding by the yeast Pif1 helicase. In vivo, both compounds rapidly induced recombination-dependent rearrangements of CEB1 inserted in the Saccharomyces cerevisiae genome, but did not affect the stability of other tandem repeats lacking G-quadruplex forming sequences. The rearrangements yielded simple-deletion, double-deletion or complex reshuffling of the polymorphic motif units, mimicking the phenotype of the Pif1 inactivation. Treatment of Pif1-deficient cells with the Phen-DC compounds further increased CEB1 instability, revealing additional G4 formation per cell. In sharp contrast, the commonly used N-methyl-mesoporphyrin IX G-quadruplex ligand did not affect CEB1 stability. Altogether, these results demonstrate that the Phen-DC bisquinolinium compounds are potent molecular tools for probing the formation of G-quadruplexes in vivo, interfere with their processing and elucidate their biological roles.

The yeast Pif1 helicase prevents genomic instability caused by G-quadruplex-forming CEB1 sequences in vivo.

In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Delta cells. Hence, we conclude that CEB1 instability in pif1Delta cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences.

Yeast genes involved in cadmium tolerance: Identification of DNA replication as a target of cadmium toxicity.

Cadmium (Cd(2+)) is a ubiquitous environmental pollutant and human carcinogen. The molecular basis of its toxicity remains unclear. Here, to identify the landscape of genes and cell functions involved in cadmium resistance, we have screened the Saccharomyces cerevisiae deletion collection for mutants sensitive to cadmium exposure. Among the 4866 ORFs tested, we identified 73 genes whose inactivation confers increased sensitivity to Cd(2+). Most were previously unknown to play a role in cadmium tolerance and we observed little correlation between the cadmium sensitivity of a gene deletant and the variation in the transcriptional activity of that gene in response to cadmium. These genes encode proteins involved in various functions: intracellular transport, stress response and gene expression. Analysis of the sensitive phenotype of our "Cd(2+)-sensitive mutant collection" to arsenite, cobalt, mercury and H(2)O(2) revealed 17 genes specifically involved in cadmium-induced response. Among them we found RAD27 and subsequently DNA2 which encode for proteins involved in DNA repair and replication. Analysis of the Cd(2+)-sensitivity of RAD27 (rad27-G67S) and DNA2 (dna2-1) separation of function alleles revealed that their activities necessary for Okazaki fragment processing are essential in conditions of cadmium exposure. Consistently, we observed that wild-type cells exposed to cadmium display an enhanced frequency of forward mutations to canavanine resistance and minisatellite destabilisation. Taken together these results provide a global picture of the genetic requirement for cadmium tolerance in yeast and strongly suggest that DNA replication, through the step of Okazaki fragment processing, is a target of cadmium toxicity.

Complex minisatellite rearrangements generated in the total or partial absence of Rad27/hFEN1 activity occur in a single generation and are Rad51 and Rad52 dependent.

Genomes contain tandem repeat blocks that are at risk of expansion or contraction. The mechanisms of destabilization of the human minisatellite CEB1 (arrays of 36- to 43-bp repeats) were investigated in a previously developed model system, in which CEB1-0.6 (14 repeats) and CEB1-1.8 (42 repeats) alleles were inserted into the genome of Saccharomyces cerevisiae. As in human cells, CEB1 is stable in mitotically growing yeast cells but is frequently rearranged in the absence of the Rad27/hFEN1 protein involved in Okazaki fragments maturation. To gain insight into this mode of destabilization, the CEB1-1.8 and CEB1-0.6 human alleles and 47 rearrangements derived from a CEB1-1.8 progenitor in rad27Delta cells were sequenced. A high degree of polymorphism of CEB1 internal repeats was observed, attesting to a large variety of homology-driven rearrangements. Simple deletion, double deletion, and highly complex events were observed. Pedigree analysis showed that all rearrangements, even the most complex, occurred in a single generation and were inherited equally by mother and daughter cells. Finally, the rearrangement frequency was found to increase with array size, and partial complementation of the rad27Delta mutation by hFEN1 demonstrated that the production of novel CEB1 alleles is Rad52 and Rad51 dependent. Instability can be explained by an accumulation of unresolved flap structures during replication, leading to the formation of recombinogenic lesions and faulty repair, best understood by homology-dependent synthesis-strand displacement and annealing.

Instability of the human minisatellite CEB1 in rad27Delta and dna2-1 replication-deficient yeast cells.

Convergent studies in human and yeast model systems have shown that some minisatellite loci are relatively stable in somatic cells but not in the germline, and little is known about the mechanism(s) that can destabilize them. Unlike microsatellite sequences, mini satellites are not destabilized by mismatch repair mutations. We report here that the absence of Rad27 and Dna2 functions but not RNase H(35) or Exo1, which play an essential role in the processing of Okazaki fragments during replication, destabilize the human minisatellite CEB1 in mitotically growing Saccharomyces cerevisiae cells, up to 14% per generation in rad27Delta cells. Analysis using minisatellite variant repeat mapping by polymerase chain reaction of the internal structure of 17 variants reveals that the majority of rearrangements in rad27Delta cells are extremely complex contraction events that contain deletions, often accompanied by duplications of motif unit. Altogether, these results suggest that the improperly processed 5' flap structures that accumulate when replication is impaired can act as a potent stimulator of minisatellite destabilization and can provoke an unexpectedly broad range of mutagenic events. This replication-dependent phenomenon differs from the recombination-induced instability in yeast meiotic cells.