Genetics, epigenetics and back again: Lessons learned from neocentromeres.

The duplication and segregation of the genome during cell division is crucial to maintain cell identity, development of organisms and tissue maintenance. Centromeres are at the basis of accurate chromosome segregation as they define the site of assembly of the kinetochore, a large complex of proteins that attaches to spindle microtubules driving chromosome movement during cell division. Here we summarize nearly 40 years of research focussed on centromere specification and the role of local cis elements in creating a stable centromere. Initial discoveries in budding yeast in the 1980s opened up the field and revealed essential DNA sequence elements that define centromere position and function. Further work in humans discovered a centromeric DNA sequence-specific binding protein and centromeric α-satellite DNA was found to have the capacity to seed centromeres de novo. Despite the early indication of genetic elements as drivers of centromere specification, the discovery in the nineties of neocentromeres that form on unrelated DNA sequences, shifted the focus to epigenetic mechanisms. While specific sequence elements appeared non-essential, the histone H3 variant CENP-A was identified as a crucial component in centromere specification. Neocentromeres, occurring naturally or induced experimentally, have become an insightful tool to understand the mechanisms for centromere specification and will be the focus of this review. They have helped to define the strong epigenetic chromatin-based component underlying centromere inheritance but also provide new opportunities to understand the enigmatic, yet crucial role that DNA sequence elements play in centromere function and inheritance.

Histone depletion prevents telomere fusions in pre-senescent cells.

Upon telomerase inactivation, telomeres gradually shorten with each cell division until cells enter replicative senescence. In Saccharomyces cerevisiae, the kinases Mec1/ATR and Tel1/ATM protect the genome during pre-senescence by preventing telomere-telomere fusions (T-TFs) and the subsequent genetic instability associated with fusion-bridge-breakage cycles. Here we report that T-TFs in mec1Δ tel1Δ cells can be suppressed by reducing the pool of available histones. This protection associates neither with changes in bulk telomere length nor with major changes in the structure of subtelomeric chromatin. We show that the absence of Mec1 and Tel1 strongly augments double-strand break (DSB) repair by non-homologous end joining (NHEJ), which might contribute to the high frequency of T-TFs in mec1Δ tel1Δ cells. However, histone depletion does not prevent telomere fusions by inhibiting NHEJ, which is actually increased in histone-depleted cells. Rather, histone depletion protects telomeres from fusions by homologous recombination (HR), even though HR is proficient in maintaining the proliferative state of pre-senescent mec1Δ tel1Δ cells. Therefore, HR during pre-senescence not only helps stalled replication forks but also prevents T-TFs by a mechanism that, in contrast to the previous one, is promoted by a reduction in the histone pool and can occur in the absence of Rad51. Our results further suggest that the Mec1-dependent depletion of histones that occurs during pre-senescence in cells without telomerase (tlc1Δ) prevents T-TFs by favoring the processing of unprotected telomeres by Rad51-independent HR.

Defective histone supply causes condensin-dependent chromatin alterations, SAC activation and chromosome decatenation impairment.

The structural organization of chromosomes is essential for their correct function and dynamics during the cell cycle. The assembly of DNA into chromatin provides the substrate for topoisomerases and condensins, which introduce the different levels of superhelical torsion required for DNA metabolism. In particular, Top2 and condensin are directly involved in both the resolution of precatenanes that form during replication and the formation of the intramolecular loop that detects tension at the centromeric chromatin during chromosome biorientation. Here we show that histone depletion activates the spindle assembly checkpoint (SAC) and impairs sister chromatid decatenation, leading to chromosome mis-segregation and lethality in the absence of the SAC. We demonstrate that histone depletion impairs chromosome biorientation and activates the Aurora-dependent pathway, which detects tension problems at the kinetochore. Interestingly, SAC activation is suppressed by the absence of Top2 and Smc2, an essential component of condensin. Indeed, smc2-8 suppresses catenanes accumulation, mitotic arrest and growth defects induced by histone depletion at semi-permissive temperature. Remarkably, SAC activation by histone depletion is associated with condensin-mediated alterations of the centromeric chromatin. Therefore, our results reveal the importance of a precise interplay between histone supply and condensin/Top2 for pericentric chromatin structure, precatenanes resolution and centromere biorientation.

Transcription and FACT facilitate the restoration of replication-coupled chromatin assembly defects.

Genome duplication occurs through the coordinated action of DNA replication and nucleosome assembly at replication forks. Defective nucleosome assembly causes DNA lesions by fork breakage that need to be repaired. In addition, it causes a loss of chromatin integrity. These chromatin alterations can be restored, even though the mechanisms are unknown. Here, we show that the process of chromatin restoration can deal with highly severe chromatin defects induced by the absence of the chaperones CAF1 and Rtt106 or a strong reduction in the pool of available histones, and that this process can be followed by analyzing the topoisomer distribution of the 2µ plasmid. Using this assay, we demonstrate that chromatin restoration is slow and independent of checkpoint activation, whereas it requires the action of transcription and the FACT complex. Therefore, cells are able to "repair" not only DNA lesions but also chromatin alterations associated with defective nucleosome assembly.

Effects of Sesquiterpene Lactones on Primary Cilia Formation (Ciliogenesis).

Sesquiterpene lactones (SLs), plant-derived metabolites with broad spectra of biological effects, including anti-tumor and anti-inflammatory, hold promise for drug development. Primary cilia, organelles extending from cell surfaces, are crucial for sensing and transducing extracellular signals essential for cell differentiation and proliferation. Their life cycle is linked to the cell cycle, as cilia assemble in non-dividing cells of G0/G1 phases and disassemble before entering mitosis. Abnormalities in both primary cilia (non-motile cilia) and motile cilia structure or function are associated with developmental disorders (ciliopathies), heart disease, and cancer. However, the impact of SLs on primary cilia remains unknown. This study evaluated the effects of selected SLs (grosheimin, costunolide, and three cyclocostunolides) on primary cilia biogenesis and stability in human retinal pigment epithelial (RPE) cells. Confocal fluorescence microscopy was employed to analyze the effects on primary cilia formation (ciliogenesis), primary cilia length, and stability. The effects on cell proliferation were evaluated by flow cytometry. All SLs disrupted primary cilia formation in the early stages of ciliogenesis, irrespective of starvation conditions or cytochalasin-D treatment, with no effect on cilia length or cell cycle progression. Interestingly, grosheimin stabilized and promoted primary cilia formation under cilia homeostasis and elongation treatment conditions. Thus, SLs have potential as novel drugs for ciliopathies and tumor treatment.

Induction of spontaneous human neocentromere formation and long-term maturation.

Human centromeres form primarily on α-satellite DNA but sporadically arise de novo at naive ectopic loci, creating neocentromeres. Centromere inheritance is driven primarily by chromatin containing the histone H3 variant CENP-A. Here, we report a chromosome engineering system for neocentromere formation in human cells and characterize the first experimentally induced human neocentromere at a naive locus. The spontaneously formed neocentromere spans a gene-poor 100-kb domain enriched in histone H3 lysine 9 trimethylated (H3K9me3). Long-read sequencing revealed this neocentromere was formed by purely epigenetic means and assembly of a functional kinetochore correlated with CENP-A seeding, eviction of H3K9me3 and local accumulation of mitotic cohesin and RNA polymerase II. At formation, the young neocentromere showed markedly reduced chromosomal passenger complex (CPC) occupancy and poor sister chromatin cohesion. However, long-term tracking revealed increased CPC assembly and low-level transcription providing evidence for centromere maturation over time.

Actin and Nuclear Envelope Components Influence Ectopic Recombination in the Absence of Swr1.

The accuracy of most DNA processes depends on chromatin integrity and dynamics. Our analyses in the yeast Saccharomyces cerevisiae show that an absence of Swr1 (the catalytic and scaffold subunit of the chromatin-remodeling complex SWR) leads to the formation of long-duration Rad52, but not RPA, foci and to an increase in intramolecular recombination. These phenotypes are further increased by MMS, zeocin, and ionizing radiation, but not by double-strand breaks, HU, or transcription/replication collisions, suggesting that they are associated with specific DNA lesions. Importantly, these phenotypes can be specifically suppressed by mutations in: (1) chromatin-anchorage internal nuclear membrane components (mps3∆75-150 and src1∆); (2) actin and actin regulators (act1-157, act1-159, crn1∆, and cdc42-6); or (3) the SWR subunit Swc5 and the SWR substrate Htz1 However, they are not suppressed by global disruption of actin filaments or by the absence of Csm4 (a component of the external nuclear membrane that forms a bridging complex with Mps3, thus connecting the actin cytoskeleton with chromatin). Moreover, swr1∆-induced Rad52 foci and intramolecular recombination are not associated with tethering recombinogenic DNA lesions to the nuclear periphery. In conclusion, the absence of Swr1 impairs efficient recombinational repair of specific DNA lesions by mechanisms that are influenced by SWR subunits, including actin, and nuclear envelope components. We suggest that these recombinational phenotypes might be associated with a pathological effect on homologous recombination of actin-containing complexes.

Crosstalk between chromatin structure, cohesin activity and transcription.

BACKGROUND: A complex interplay between chromatin and topological machineries is critical for genome architecture and function. However, little is known about these reciprocal interactions, even for cohesin, despite its multiple roles in DNA metabolism. RESULTS: We have used genome-wide analyses to address how cohesins and chromatin structure impact each other in yeast. Cohesin inactivation in scc1-73 mutants during the S and G2 phases causes specific changes in chromatin structure that preferentially take place at promoters; these changes include a significant increase in the occupancy of the - 1 and + 1 nucleosomes. In addition, cohesins play a major role in transcription regulation that is associated with specific promoter chromatin architecture. In scc1-73 cells, downregulated genes are enriched in promoters with short or no nucleosome-free region (NFR) and a fragile "nucleosome - 1/RSC complex" particle. These results, together with a preferential increase in the occupancy of nucleosome - 1 of these genes, suggest that cohesins promote transcription activation by helping RSC to form the NFR. In sharp contrast, the scc1-73 upregulated genes are enriched in promoters with an "open" chromatin structure and are mostly at cohesin-enriched regions, suggesting that a local accumulation of cohesins might help to inhibit transcription. On the other hand, a dramatic loss of chromatin integrity by histone depletion during DNA replication has a moderate effect on the accumulation and distribution of cohesin peaks along the genome. CONCLUSIONS: Our analyses of the interplay between chromatin integrity and cohesin activity suggest that cohesins play a major role in transcription regulation, which is associated with specific chromatin architecture and cohesin-mediated nucleosome alterations of the regulated promoters. In contrast, chromatin integrity plays only a minor role in the binding and distribution of cohesins.