Heterochromatin: Guardian of the Genome.

Constitutive heterochromatin is a major component of the eukaryotic nucleus and is essential for the maintenance of genome stability. Highly concentrated at pericentromeric and telomeric domains, heterochromatin is riddled with repetitive sequences and has evolved specific ways to compartmentalize, silence, and repair repeats. The delicate balance between heterochromatin epigenetic maintenance and cellular processes such as mitosis and DNA repair and replication reveals a highly dynamic and plastic chromatin domain that can be perturbed by multiple mechanisms, with far-reaching consequences for genome integrity. Indeed, heterochromatin dysfunction provokes genetic turmoil by inducing aberrant repeat repair, chromosome segregation errors, transposon activation, and replication stress and is strongly implicated in aging and tumorigenesis. Here, we summarize the general principles of heterochromatin structure and function, discuss the importance of its maintenance for genome integrity, and propose that more comprehensive analyses of heterochromatin roles in tumorigenesis will be integral to future innovations in cancer treatment.

Timely double-strand break repair and pathway choice in pericentromeric heterochromatin depend on the histone demethylase dKDM4A.

Repair of DNA double-strand breaks (DSBs) must be orchestrated properly within diverse chromatin domains in order to maintain genetic stability. Euchromatin and heterochromatin domains display major differences in histone modifications, biophysical properties, and spatiotemporal dynamics of DSB repair. However, it is unclear whether differential histone-modifying activities are required for DSB repair in these distinct domains. We showed previously that the Drosophila melanogaster KDM4A (dKDM4A) histone demethylase is required for heterochromatic DSB mobility. Here we used locus-specific DSB induction in Drosophila animal tissues and cultured cells to more deeply interrogate the impact of dKDM4A on chromatin changes, temporal progression, and pathway utilization during DSB repair. We found that dKDM4A promotes the demethylation of heterochromatin-associated histone marks at DSBs in heterochromatin but not euchromatin. Most importantly, we demonstrate that dKDM4A is required to complete DSB repair in a timely manner and regulate the relative utilization of homologous recombination (HR) and nonhomologous end-joining (NHEJ) repair pathways but exclusively for heterochromatic DSBs. We conclude that the temporal kinetics and pathway utilization during heterochromatic DSB repair depend on dKDM4A-dependent demethylation of heterochromatic histone marks. Thus, distinct pre-existing chromatin states require specialized epigenetic alterations to ensure proper DSB repair.

A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin.

Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context.Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.

The impact of chromatin on double-strand break repair: Imaging tools and discoveries.

Eukaryotic nuclei are constantly being exposed to factors that break or chemically modify the DNA. Accurate repair of this DNA damage is crucial to prevent DNA mutations and maintain optimal cell function. To overcome the detrimental effects of DNA damage, a multitude of repair pathways has evolved. These pathways need to function properly within the different chromatin domains present in the nucleus. Each of these domains exhibit distinct molecular- and bio-physical characteristics that can influence the response to DNA damage. In particular, chromatin domains highly enriched for repetitive DNA sequences, such as nucleoli, centromeres and pericentromeric heterochromatin require tailored repair mechanisms to safeguard genome stability. Work from the past decades has led to the development of innovative imaging tools as well as inducible DNA damage techniques to gain new insights into the impact of these repetitive chromatin domains on the DNA repair process. Here we summarize these tools with a particular focus on Double-Strand Break (DSB) repair, and discuss the insights gained into our understanding of the influence of chromatin domains on DSB -dynamics and -repair pathway choice.

Elevating the frequency of chromosome mis-segregation as a strategy to kill tumor cells.

The mitotic checkpoint has evolved to prevent chromosome mis-segregations by delaying mitosis when unattached chromosomes are present. Inducing severe chromosome segregation errors by ablating the mitotic checkpoint causes cell death. Here we have analyzed the consequences of gradual increases in chromosome segregation errors on the viability of tumor cells and normal human fibroblasts. Partial reduction of essential mitotic checkpoint components in four tumor cell lines caused mild chromosome mis-segregations, but no lethality. These cells were, however, remarkably more sensitive to low doses of taxol, which enhanced the amount and severity of chromosome segregation errors. Sensitization to taxol was achieved by reducing levels of Mps1 or BubR1, proteins having dual roles in checkpoint activation and chromosome alignment, but not by reducing Mad2, functioning solely in the mitotic checkpoint. Moreover, we find that untransformed human fibroblasts with reduced Mps1 levels could not be sensitized to sublethal doses of taxol. Thus, targeting the mitotic checkpoint and chromosome alignment simultaneously may selectively kill tumor cells by enhancing chromosome mis-segregations.

The Sound of Silence: How Silenced Chromatin Orchestrates the Repair of Double-Strand Breaks.

The eukaryotic nucleus is continuously being exposed to endogenous and exogenous sources that cause DNA breaks, whose faithful repair requires the activity of dedicated nuclear machineries. DNA is packaged into a variety of chromatin domains, each characterized by specific molecular properties that regulate gene expression and help maintain nuclear structure. These different chromatin environments each demand a tailored response to DNA damage. Silenced chromatin domains in particular present a major challenge to the cell's DNA repair machinery due to their specific biophysical properties and distinct, often repetitive, DNA content. To this end, we here discuss the interplay between silenced chromatin domains and DNA damage repair, specifically double-strand breaks, and how these processes help maintain genome stability.

Degree and site of chromosomal instability define its oncogenic potential.

Most human cancers are aneuploid, due to a chromosomal instability (CIN) phenotype. Despite being hallmarks of cancer, however, the roles of CIN and aneuploidy in tumor formation have not unequivocally emerged from animal studies and are thus still unclear. Using a conditional mouse model for diverse degrees of CIN, we find that a particular range is sufficient to drive very early onset spontaneous adenoma formation in the intestine. In mice predisposed to intestinal cancer (ApcMin/+), moderate CIN causes a remarkable increase in adenoma burden in the entire intestinal tract and especially in the distal colon, which resembles human disease. Strikingly, a higher level of CIN promotes adenoma formation in the distal colon even more than moderate CIN does, but has no effect in the small intestine. Our results thus show that CIN can be potently oncogenic, but that certain levels of CIN can have contrasting effects in distinct tissues.

Intravital FRET imaging of tumor cell viability and mitosis during chemotherapy.

Taxanes, such as docetaxel, are microtubule-targeting chemotherapeutics that have been successfully used in the treatment of cancer. Based on data obtained from cell cultures, it is believed that taxanes induce tumor cell death by specifically perturbing mitotic progression. Here, we report on data that suggest that this generally accepted view may be too simplified. We describe a high-resolution intravital imaging method to simultaneously visualize mitotic progression and the onset of apoptosis. To directly compare in vitro and in vivo data, we have visualized the effect of docetaxel on mitotic progression in mouse and human colorectal tumor cell lines both in vitro and in isogenic tumors in mice. We show that docetaxel-induced apoptosis in vitro occurs via mitotic cell death, whereas the vast majority of tumor cells in their natural environment die independent of mitotic defects. This demonstrates that docetaxel exerts its anti-tumor effects in vivo through means other than mitotic perturbation. The differences between in vitro and in vivo mechanisms of action of chemotherapeutics may explain the limited response to many of the anti-mitotic agents that are currently validated in clinical trials. Our data illustrate the requirement and power of our intravital imaging technique to study and validate the mode of action of chemotherapeutic agents in vivo, which will be essential to understand and improve their clinical efficacy.

Entosis: aneuploidy by invasion.

Aneuploidy is one of the most prevalent phenotypes of human tumours, but the underlying cause of this phenomenon remains highly debated. Entosis, the invasion of a living cell into another cell's cytoplasm, is now shown to perturb cytokinesis and induce the formation of aneuploid cells.

Targeting the mitotic checkpoint to kill tumor cells.

One of the most common hallmarks of cancer cells is aneuploidy or an abnormal number of chromosomes. This abnormal chromosome content is a consequence of chromosome missegregation during mitosis, a defect that is seen more frequently in tumor cell divisions as in normal cell divisions. In fact, a large fraction of human tumors display a chromosome instable phenotype, meaning that they very frequently missegregate chromosomes. This can cause variegated aneuploidy within the tumor tissue. It has been argued that this hallmark of cancer could be exploited in anti-cancer therapies. Here we test this hypothesis by inactivation of the mitotic checkpoint through RNAi-mediated depletion of an essential checkpoint component, Mps1. The mitotic checkpoint delays segregation of chromosomes during mitosis until all chromosomes are properly attached to the mitotic spindle. Its inactivation will therefore lead to increased segregation errors. Indeed, we show that this can lead to increased cell death in tumor cells. We demonstrate that increased cell death is associated with a dramatic increase in segregation errors. This suggests that inhibition of the mitotic checkpoint might represent a useful anti-cancer strategy.

Chromosome segregation errors as a cause of DNA damage and structural chromosome aberrations.

Various types of chromosomal aberrations, including numerical (aneuploidy) and structural (e.g., translocations, deletions), are commonly found in human tumors and are linked to tumorigenesis. Aneuploidy is a direct consequence of chromosome segregation errors in mitosis, whereas structural aberrations are caused by improperly repaired DNA breaks. Here, we demonstrate that chromosome segregation errors can also result in structural chromosome aberrations. Chromosomes that missegregate are frequently damaged during cytokinesis, triggering a DNA double-strand break response in the respective daughter cells involving ATM, Chk2, and p53. We show that these double-strand breaks can lead to unbalanced translocations in the daughter cells. Our data show that segregation errors can cause translocations and provide insights into the role of whole-chromosome instability in tumorigenesis.

Kif15 cooperates with eg5 to promote bipolar spindle assembly.

BACKGROUND: The formation of a bipolar spindle is an essential step during cell division. Bipolar spindle assembly is driven by the highly conserved microtubule motor Eg5 (kinesin-5), which can slide antiparallel microtubules apart to drive centrosome separation. However, it is currently unclear whether and how additional motors can contribute to centrosome separation and bipolar spindle formation. RESULTS: We have developed a novel assay to identify motors involved in spindle bipolarity; via this assay, we identify Kif15/Hklp2 (kinesin-12, hereafter referred to as Kif15). Kif15 is not required for spindle bipolarity in cells with full Eg5 activity but becomes essential when Eg5 is partially inhibited. We show that the primary function of Kif15 is to promote spindle elongation and to ensure maintenance of spindle bipolarity. Nonetheless, ectopic expression of Kif15 can fully reconstitute bipolar spindle assembly in the absence of Eg5 activity, demonstrating that Kif15 can replace all essential functions of Eg5 in bipolar spindle assembly. Importantly, this activity of Kif15 depends on its interaction with the microtubule-associated protein TPX2, indicating that a Kif15-TPX2 complex promotes centrosome separation. CONCLUSIONS: These findings show that, similar to Eg5, Kif15 can drive centrosome separation during bipolar spindle assembly. For this activity, Kif15 requires both its motor domain and its interaction with TPX2. Based on these data, we propose that a complex of Kif15 and TPX2 can crosslink and slide two antiparallel microtubules apart, thereby driving centrosome separation.