Cdc14 and Chromosome Condensation: Evaluation of the Recruitment of Condensin to Genomic Regions.

Chromosome condensation is an essential morphological event required for successful DNA segregation during mitosis. The high level of genome compaction achieved during this process is attained by the evolutionary conserved condensin complex. Recently, several lines of evidences have demonstrated that the mitotic phosphatase Cdc14 is required to ensure condensin loading onto chromosomes. To date several approaches have been used in order to characterize condensin activity and regulation, however these techniques are time-consuming and require complex equipment. In this chapter we described an easy and reliable protocol to analyze Cdc14-dependent condensin loading onto specific genomic DNA regions by using a chromatin immunoprecipitation (ChIP) technique.

Physical Proximity of Sister Chromatids Promotes Top2-Dependent Intertwining.

Sister chromatid intertwines (SCIs), or catenanes, are topological links between replicated chromatids that interfere with chromosome segregation. The formation of SCIs is thought to be a consequence of fork swiveling during DNA replication, and their removal is thought to occur because of the intrinsic feature of type II topoisomerases (Top2) to simplify DNA topology. Here, we report that SCIs are also formed independently of DNA replication during G2/M by Top2-dependent concatenation of cohesed chromatids due to their physical proximity. We demonstrate that, in contrast to G2/M, Top2 removes SCIs from cohesed chromatids at the anaphase onset. Importantly, SCI removal in anaphase requires condensin and coincides with the hyperactivation of condensin DNA supercoiling activity. This is consistent with the longstanding proposal that condensin provides a bias in Top2 function toward decatenation. A comprehensive model for the formation and resolution of toxic SCI entanglements on eukaryotic genomes is proposed.

Condensin Relocalization from Centromeres to Chromosome Arms Promotes Top2 Recruitment during Anaphase.

Condensin is a conserved chromosomal complex necessary to promote mitotic chromosome condensation and sister chromatid resolution during anaphase. Here, we report that yeast condensin binds to replicated centromere regions. We show that centromeric condensin relocalizes to chromosome arms as cells undergo anaphase segregation. We find that condensin relocalization is initiated immediately after the bipolar attachment of sister kinetochores to spindles and requires Polo kinase activity. Moreover, condensin localization during anaphase involves a higher binding rate on DNA and temporally overlaps with condensin's DNA overwinding activity. Finally, we demonstrate that topoisomerase 2 (Top2) is also recruited to chromosome arms during anaphase in a condensin-dependent manner. Our results uncover a functional relation between condensin and Top2 during anaphase to mediate chromosome segregation.

Divergence of Erv1-associated mitochondrial import and export pathways in trypanosomes and anaerobic protists.

In yeast (Saccharomyces cerevisiae) and animals, the sulfhydryl oxidase Erv1 functions with Mia40 in the import and oxidative folding of numerous cysteine-rich proteins in the mitochondrial intermembrane space (IMS). Erv1 is also required for Fe-S cluster assembly in the cytosol, which uses at least one mitochondrially derived precursor. Here, we characterize an essential Erv1 orthologue from the protist Trypanosoma brucei (TbERV1), which naturally lacks a Mia40 homolog. We report kinetic parameters for physiologically relevant oxidants cytochrome c and O(2), unexpectedly find O(2) and cytochrome c are reduced simultaneously, and demonstrate that efficient reduction of O(2) by TbERV1 is not dependent upon a simple O(2) channel defined by conserved histidine and tyrosine residues. Massive mitochondrial swelling following TbERV1 RNA interference (RNAi) provides evidence that trypanosome Erv1 functions in IMS protein import despite the natural absence of the key player in the yeast and animal import pathways, Mia40. This suggests significant evolutionary divergence from a recently established paradigm in mitochondrial cell biology. Phylogenomic profiling of genes also points to a conserved role for TbERV1 in cytosolic Fe-S cluster assembly. Conversely, loss of genes implicated in precursor delivery for cytosolic Fe-S assembly in Entamoeba, Trichomonas, and Giardia suggests fundamental differences in intracellular trafficking pathways for activated iron or sulfur species in anaerobic versus aerobic eukaryotes.

SUMOylation of the α-kleisin subunit of cohesin is required for DNA damage-induced cohesion.

BACKGROUND: Cohesion between sister chromatids is fundamental to ensure faithful chromosome segregation during mitosis and accurate repair of DNA damage postreplication. At the molecular level, cohesion establishment involves two defined events, a chromatin binding step and a chromatid entrapment event driven by posttranslational modifications on cohesin subunits. RESULTS: Here, we show that modification by the small ubiquitin-like protein (SUMO) is required for sister chromatid tethering after DNA damage. We find that all subunits of cohesin become SUMOylated upon exposure to DNA damaging agents or presence of a DNA double-strand break. We have mapped all lysine residues on cohesin's α-kleisin subunit Mcd1 (Scc1) where SUMO can conjugate. We demonstrate that Mcd1 SUMOylation-deficient alleles are still recruited to DSB-proximal regions but are defective in tethering sister chromatids and consequently fail to establish damage-induced cohesion both at DSBs and undamaged chromosomes. Moreover, we demonstrate that the bulk of Mcd1 SUMOylation in response to damage is carried out by the SUMO E3 ligase Nse2, a subunit of the related Smc5-Smc6 complex. SUMOylation occurs in cells with compromised Chk1 kinase activity, necessary for known posttranslational modifications on Mcd1, required for damage-induced cohesion. CONCLUSIONS: These findings demonstrate that SUMOylation of Mcd1 is a novel prerequisite for the establishment of DNA damage-induced cohesion at DSB-proximal regions and cohesion-associating regions (CARs) genome-wide.

Drug development for severe asthma: what are the metrics?

Although reversible airway obstruction in part defines asthma, lung function as measured by spirometry alone inadequately predicts the value of new therapeutic agents in the treatment of severe asthma. Our objectives are 1) to review whether pulmonary function and bronchodilator reversibility are endpoints for drug discovery and 2) to identify parameters that predict efficacy in drug development in severe asthma. An English language literature search using MedLine and PubMed was conducted from 1997 to present concerning pathophysiology, diagnosis and therapy of severe asthma using the terms "severe asthma," "irreversible asthma," "difficult asthma," "airway remodeling," "fixed airway obstruction," "reversibility" and "bronchodilator reversibility" as index terms. Eight studies were characterized that encompass 1424 subjects with asthma. Our review identified the limitations of using bronchodilator reversibility as a predictor in drug development for severe asthma. Neither improvement in lung function nor bronchodilator reversibility characterized the benefit of new drugs in the treatment of severe asthma. Newly approved drugs in the treatment of severe asthma show decreased asthma exacerbations and improved quality of life associated with steroid-sparing benefits without altering bronchodilator responsiveness or improving lung function. Although changes in lung function predict asthma control in mild/moderate asthma, lung function alone is inadequate to assess improvement in asthma control in severe asthma manifested by fixed airway obstruction. Endpoints that focus on asthma control, as defined by the Expert Panel Report 3 and GINA guidelines, may predict the value of new therapeutics in the management of severe asthma.

Nebulized dehydroepiandrosterone-3-sulfate improves asthma control in the moderate-to-severe asthma results of a 6-week, randomized, double-blind, placebo-controlled study.

Inhaled dehydroepiandrosterone-3-sulfate (DHEAS), but not dehydroepiandrosterone (DHEA), possesses anti-inflammatory activity in in vitro assays and in models of allergen and lipopolysaccharide challenges. We postulated whether an inhaled suspension of DHEAS delivered via nebulizer would improve asthma control in moderate-to-severe asthma patients. We also characterized the safety profile of an inhaled suspension of DHEAS. Patients receiving at least 500 µg of fluticasone equivalent plus long-acting beta-agonists (LABA) entered a 5-week run-in where the dose of inhaled corticosteroids was reduced to 200 µg of fluticasone plus LABA per day. Patients were randomized to 70 mg of DHEAS or placebo if their Asthma Control Questionnaire (ACQ) score was ≥2.0 and their FEV(1) ≥ 50%. When compared with control, a statistically significant improvement in ACQ in 6 weeks of treatment with 70 mg of DHEAS was observed. The median improvement in ACQ was -0.72 and -0.43 for the active and placebo groups, respectively (p = 0.0389); the percentage of patients with at least minimally clinically important difference of -0.50 from baseline was significantly greater in the DHEAS group versus the placebo, (59.4% versus 45.7%; p = 0.0236). Asthma symptom scores, the proportion of symptom-free days and symptom nights, although not statistically significant, had positive trends supporting the improvement in ACQ. Fewer patients were withdrawn from the study for respiratory events on DHEAS compared with placebo. There were few adverse events and no changes in sex hormones despite increases in circulating levels of DHEAS. An inhaled suspension of DHEAS delivered via nebulizer improved asthma control scores in subjects with poorly controlled moderate-to-severe asthma. AUSTRALIAN NEW ZEALAND CLINICAL TRIALS REGISTRY ANZCTR.ORG.AU IDENTIFIER: 012607000192482.