Enrichment of Zα domains at cytoplasmic stress granules is due to their innate ability to bind to nucleic acids.

Zα domains recognize the left-handed helical Z conformation of double-stranded nucleic acids. They are found in proteins involved in the nucleic acid sensory pathway of the vertebrate innate immune system and host evasion by viral pathogens. Previously, it has been demonstrated that ADAR1 (encoded by ADAR in humans) and DAI (also known as ZBP1) localize to cytoplasmic stress granules (SGs), and this localization is mediated by their Zα domains. To investigate the mechanism, we determined the interactions and localization pattern for the N-terminal region of human DAI (ZαßDAI), which harbours two Zα domains, and for a ZαßDAI mutant deficient in nucleic acid binding. Electrophoretic mobility shift assays demonstrated the ability of ZαßDAI to bind to hyperedited nucleic acids, which are enriched in SGs. Furthermore, using immunofluorescence and immunoprecipitation coupled with mass spectrometry, we identified several interacting partners of the ZαßDAI-RNA complex in vivo under conditions of arsenite-induced stress. These interactions are lost upon loss of nucleic acid-binding ability or upon RNase treatment. Thus, we posit that the mechanism for the translocation of Zα domain-containing proteins to SGs is mainly mediated by the nucleic acid-binding ability of their Zα domains. This article has an associated First Person interview with Bharath Srinivasan, joint first author of the paper.

Genetic screening identifies a SUMO protease dynamically maintaining centromeric chromatin.

Centromeres are defined by a self-propagating chromatin structure based on stable inheritance of CENP-A containing nucleosomes. Here, we present a genetic screen coupled to pulse-chase labeling that allow us to identify proteins selectively involved in deposition of nascent CENP-A or in long-term transmission of chromatin-bound CENP-A. These include factors with known roles in DNA replication, repair, chromatin modification, and transcription, revealing a broad set of chromatin regulators that impact on CENP-A dynamics. We further identify the SUMO-protease SENP6 as a key factor, not only controlling CENP-A stability but virtually the entire centromere and kinetochore. Loss of SENP6 results in hyper-SUMOylation of CENP-C and CENP-I but not CENP-A itself. SENP6 activity is required throughout the cell cycle, suggesting that a dynamic SUMO cycle underlies a continuous surveillance of the centromere complex that in turn ensures stable transmission of CENP-A chromatin.

A Dual Inhibitory Mechanism Sufficient to Maintain Cell-Cycle-Restricted CENP-A Assembly.

Chromatin featuring the H3 variant CENP-A at the centromere is critical for its mitotic function and epigenetic maintenance. Assembly of centromeric chromatin is restricted to G1 phase through inhibitory action of Cdk1/2 kinases in other phases of the cell cycle. Here, we identify the two key targets sufficient to maintain cell-cycle control of CENP-A assembly. We uncovered a single phosphorylation site in the licensing factor M18BP1 and a cyclin A binding site in the CENP-A chaperone, HJURP, that mediated specific inhibitory phosphorylation. Simultaneous expression of mutant proteins lacking these residues results in complete uncoupling from the cell cycle. Consequently, CENP-A assembly is fully recapitulated under high Cdk activities, indistinguishable from G1 assembly. We find that Cdk-mediated inhibition is exerted by sequestering active factors away from the centromere. Finally, we show that displacement of M18BP1 from the centromere is critical for the assembly mechanism of CENP-A.

A two-step mechanism for epigenetic specification of centromere identity and function.

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either the amino- or carboxy-terminal tail of CENP-A for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C, respectively.

Assembly in G1 phase and long-term stability are unique intrinsic features of CENP-A nucleosomes.

Centromeres are the site of kinetochore formation during mitosis. Centromere protein A (CENP-A), the centromere-specific histone H3 variant, is essential for the epigenetic maintenance of centromere position. Previously we showed that newly synthesized CENP-A is targeted to centromeres exclusively during early G1 phase and is subsequently maintained across mitotic divisions. Using SNAP-based fluorescent pulse labeling, we now demonstrate that cell cycle-restricted chromatin assembly at centromeres is unique to CENP-A nucleosomes and does not involve assembly of other H3 variants. Strikingly, stable retention is restricted to the CENP-A/H4 core of the nucleosome, which we find to outlast general chromatin across several cell divisions. We further show that cell cycle timing of CENP-A assembly is independent of centromeric DNA sequences and instead is mediated by the CENP-A targeting domain. Unexpectedly, this domain also induces stable transmission of centromeric nucleosomes, independent of the CENP-A deposition factor HJURP. This demonstrates that intrinsic properties of the CENP-A protein direct its cell cycle-restricted assembly and induces quantitative mitotic transmission of the CENP-A/H4 nucleosome core, ensuring long-term stability and epigenetic maintenance of centromere position.

Human condensin function is essential for centromeric chromatin assembly and proper sister kinetochore orientation.

Condensins I and II in vertebrates are essential ATP-dependent complexes necessary for chromosome condensation in mitosis. Condensins depletion is known to perturb structure and function of centromeres, however the mechanism of this functional link remains elusive. Depletion of condensin activity is now shown to result in a significant loss of loading of CENP-A, the histone H3 variant found at active centromeres and the proposed epigenetic mark of centromere identity. Absence of condensins and/or CENP-A insufficiency produced a specific kinetochore defect, such that a functional mitotic checkpoint cannot prevent chromosome missegregation resulting from improper attachment of sister kinetochores to spindle microtubules. Spindle microtubule-dependent deformation of both inner kinetochores and the HEC1/Ndc80 microtubule-capturing module, then results in kinetochore separation from the Aurora B pool and ensuing reduced kinase activity at centromeres. Moreover, recovery from mitosis-inhibition by monastrol revealed a high incidence of merotelic attachment that was nearly identical with condensin depletion, Aurora B inactivation, or both, indicating that the Aurora B dysfunction is the key defect leading to chromosome missegregation in condensin-depleted cells. Thus, beyond a requirement for global chromosome condensation, condensins play a pivotal role in centromere assembly, proper spatial positioning of microtubule-capturing modules and positioning complexes of the inner centromere versus kinetochore plates.

Platinated DNA adducts enhance poisoning of DNA topoisomerase I by camptothecin.

Camptothecins constitute a novel class of chemotherapeutics that selectively target DNA topoisomerase I (Top1) by reversibly stabilizing a covalent enzyme-DNA intermediate. This cytotoxic mechanism contrasts with that of platinum drugs, such as cisplatin, which induce inter- and intrastrand DNA adducts. In vitro combination studies using platinum drugs combined with Top1 poisons, such as topotecan, showed a schedule-dependent synergistic activity, with promising results in the clinic. However, whereas the molecular mechanism of these single agents may be relatively well understood, the mode of action of these chemotherapeutic agents in combination necessitates a more complete understanding. Indeed, we recently reported that a functional homologous recombination pathway is required for cisplatin and topotecan synergy yet represses the synergistic toxicity of 1-beta-D-arabinofuranosyl cytidine in combination with topotecan (van Waardenburg, R. C., de Jong, L. A., van Delft, F., van Eijndhoven, M. A., Bohlander, M., Bjornsti, M. A., Brouwer, J., and Schellens, J. H. (2004) Mol. Cancer Ther. 3, 393-402). Here we provide direct evidence for Pt-1,3-d(GTG) poisoning of Top1 in vitro and demonstrate that persistent Pt-DNA adducts correlate with increased covalent Top1-DNA complexes in vivo. This contrasts with a lack of persistent lesions induced by the alkylating agent bis[chloroethyl]nitrosourea, which exhibits only additive activity with topotecan in a range of cell lines. In human IGROV-1 ovarian cancer cells, the synergistic activity of cisplatin with topotecan requires processive DNA polymerization, whereas overexpression of Top1 enhances yeast cell sensitivity to cisplatin. These results indicate that the cytotoxic activity of cisplatin is due, in part, to poisoning of Top1, which is exacerbated in the presence of topotecan.

Transcription elongation factor Spt4 mediates loss of phosphorylated RNA polymerase II transcription in response to DNA damage.

Previously, we found that Rad26, the yeast Cockayne syndrome B homolog and the transcription elongation factor Spt4 mediate transcription-coupled repair of UV-induced DNA damage. Here we studied the effect of DNA damage on transcription by directly analyzing the RNA polymerase II localization at active genes in vivo. A rad26 defect leads to loss of Ser5 phosphorylated RNA polymerase II localization to active genes, while localization is only transiently diminished in wild type cells. In contrast, loss of Ser5-P RNAP II localization is suppressed in spt4 cells. Interestingly, even when DNA damage is persistent the absence of Spt4 leads to a delayed loss of transcription suggesting that Spt4 is directly involved in mediating transcription shutdown. Comparative analysis of phosphorylated and non-phosphorylated RNA polymerase II localization revealed that Ser5-P RNAP II is preferentially lost in the presence of DNA damage. In addition, we found evidence for a transient Rad26 localization to active genes in response to DNA damage. These findings provide insight into the transcriptional response to DNA damage and the factors involved in communicating this response, which has direct implications for our understanding of transcription-repair coupling.