Stable kinetochore-microtubule attachment constrains centromere positioning in metaphase

With a single microtubule attachment, budding-yeast kinetochores provide an excellent system for understanding the coordinated linkage to dynamic microtubule plus ends for chromosome oscillation and positioning. Fluorescent tagging of kinetochore proteins indicates that, on average, all centromeres are clustered, distinctly separated from their sisters, and positioned equidistant from their respective spindle poles during metaphase. However, individual fluorescent chromosome markers near the centromere transiently reassociate with their sisters and oscillate from one spindle half to the other. To reconcile the apparent disparity between the average centromere position and individual centromere proximal markers, we utilized fluorescence recovery after photobleaching to measure stability of the histone-H3 variant Cse4p/CENP-A. Newly synthesized Cse4p replaces old protein during DNA replication. Once assembled, Cse4-GFP is a physically stable component of centromeres during mitosis. This allowed us to follow centromere dynamics within each spindle half. Kinetochores remain stably attached to dynamic microtubules and exhibit a low incidence of switching orientation or position between the spindle halves. Switching of sister chromatid attachment may be contemporaneous with Cse4p exchange and early kinetochore assembly during S phase; this would promote mixing of chromosome attachment to each spindle pole. Once biorientation is attained, centromeres rarely make excursions beyond their proximal half spindle.     

Accurate detection of aneuploidies in array CGH and gene expression microarray data

MOTIVATION: Chromosomal copy number changes (aneuploidies) are common in cell populations that undergo multiple cell divisions including yeast strains, cell lines and tumor cells. Identification of aneuploidies is critical in evolutionary studies, where changes in copy number serve an adaptive purpose, as well as in cancer studies, where amplifications and deletions of chromosomal regions have been identified as a major pathogenetic mechanism. Aneuploidies can be studied on whole-genome level using array CGH (a microarray-based method that measures the DNA content), but their presence also affects gene expression. In gene expression microarray analysis, identification of copy number changes is especially important in preventing aberrant biological conclusions based on spurious gene expression correlation or masked phenotypes that arise due to aneuploidies. Previously suggested approaches for aneuploidy detection from microarray data mostly focus on array CGH, address only whole-chromosome or whole-arm copy number changes, and rely on thresholds or other heuristics, making them unsuitable for fully automated general application to gene expression datasets. There is a need for a general and robust method for identification of aneuploidies of any size from both array CGH and gene expression microarray data. RESULTS: We present ChARM (Chromosomal Aberration Region Miner), a robust and accurate expectation-maximization based method for identification of segmental aneuploidies (partial chromosome changes) from gene expression and array CGH microarray data. Systematic evaluation of the algorithm on synthetic and biological data shows that the method is robust to noise, aneuploidal segment size and P-value cutoff. Using our approach, we identify known chromosomal changes and predict novel potential segmental aneuploidies in commonly used yeast deletion strains and in breast cancer. ChARM can be routinely used to identify aneuploidies in array CGH datasets and to screen gene expression data for aneuploidies or array biases. Our methodology is sensitive enough to detect statistically significant and biologically relevant aneuploidies even when expression or DNA content changes are subtle as in mixed populations of cells. AVAILABILITY: Code available by request from the authors and on Web supplement at http://function.cs.princeton.edu/ChARM/     

Molecular signatures of proliferation and quiescence in hematopoietic stem cells

Stem cells resident in adult tissues are principally quiescent, yet harbor enormous capacity for proliferation to achieve self renewal and to replenish their tissue constituents. Although a single hematopoietic stem cell (HSC) can generate sufficient primitive progeny to repopulate many recipients, little is known about the molecular mechanisms that maintain their potency or regulate their self renewal. Here we have examined the gene expression changes that occur over a time course when HSCs are induced to proliferate and return to quiescence in vivo. These data were compared to data representing differences between naturally proliferating fetal HSCs and their quiescent adult counterparts. Bioinformatic strategies were used to group time-ordered gene expression profiles generated from microarrays into signatures of quiescent and dividing stem cells. A novel method for calculating statistically significant enrichments in Gene Ontology groupings for our gene lists revealed elemental subgroups within the signatures that underlie HSC behavior, and allowed us to build a molecular model of the HSC activation cycle. Initially, quiescent HSCs evince a state of readiness. The proliferative signal induces a preparative state, which is followed by active proliferation divisible into early and late phases. Re-induction of quiescence involves changes in migratory molecule expression, prior to reestablishment of homeostasis. We also identified two genes that increase in both gene and protein expression during activation, and potentially represent new markers for proliferating stem cells. These data will be of use in attempts to recapitulate the HSC self renewal process for therapeutic expansion of stem cells, and our model may correlate with acquisition of self renewal characteristics by cancer stem cells.     

Targeted ubiquitination of CDT1 by the DDB1-CUL4A-ROC1 ligase in response to DNA damage

Cullins assemble a potentially large number of ubiquitin ligases by binding to the RING protein ROC1 to catalyse polyubiquitination, as well as binding to various specificity factors to recruit substrates. The Cul4A gene is amplified in human breast and liver cancers, and loss-of-function of Cul4 results in the accumulation of the replication licensing factor CDT1 in Caenorhabditis elegans embryos and ultraviolet (UV)-irradiated human cells. Here, we report that human UV-damaged DNA-binding protein DDB1 associates stoichiometrically with CUL4A in vivo, and binds to an amino-terminal region in CUL4A in a manner analogous to SKP1, SOCS and BTB binding to CUL1, CUL2 and CUL3, respectively. As with SKP1-CUL1, the DDB1-CUL4A association is negatively regulated by the cullin-associated and neddylation-dissociated protein, CAND1. Recombinant DDB1 and CDT1 bind directly to each other in vitro, and ectopically expressed DDB1 bridges CDT1 to CUL4A in vivo. Silencing DDB1 prevented UV-induced rapid CDT1 degradation in vivo and CUL4A-mediated CDT1 ubiquitination in vitro. We suggest that DDB1 targets CDT1 for ubiquitination by a CUL4A-dependent ubiquitin ligase, CDL4A(DDB1), in response to UV irradiation.     

Investigation of the nature of the methionine-pi interaction in beta-hairpin peptide model systems

There are frequent contacts between aromatic rings and sulfur atoms in proteins. However, it is unclear to what degree this putative interaction is stabilizing and what the nature of the interaction is. We have investigated the aryl-sulfur interaction by placing a methionine residue diagonal to an aromatic ring on the same face of a beta-hairpin, which places the methionine side chain in close proximity to the aryl side chain. The methionine (Met)-aryl interaction was compared with an equivalent hydrophobic and cation-pi interaction in the context of the beta-hairpin. The interaction between phenylalanine (Phe), tryptophan (Trp), or cyclohexylalanine (Cha) and Met stabilized the beta-hairpin by -0.3 to -0.5 kcal mole(-1), as determined by double-mutant cycles. The peptides were subjected to thermal denaturations that suggest a hydrophobic driving force for the interactions between Met and Trp or Cha. The observed interaction of Met or norleucine (Nle) with Trp or Cha are quite similar, implying a hydrophobic driving force for the Met-pi interaction. However, the thermodynamic data suggest that there may be some differences between the interaction of Met with Trp and Phe and that there may be a small thermodynamic component to the Met...Phe interaction.     

Host-symbiont stability and fast evolutionary rates in an ant-bacterium association: cospeciation of camponotus species and their endosymbionts, candidatus blochmannia

Bacterial endosymbionts are widespread across several insect orders and are involved in interactions ranging from obligate mutualism to reproductive parasitism. Candidatus Blochmannia gen. nov. (Blochmannia) is an obligate bacterial associate of Camponotus and related ant genera (Hymenoptera: Formicidae). The occurrence of Blochmannia in all Camponotus species sampled from field populations and its maternal transmission to host offspring suggest that this bacterium is engaged in a long-term, stable association with its ant hosts. However, evidence for cospeciation in this system is equivocal because previous phylogenetic studies were based on limited gene sampling, lacked statistical analysis of congruence, and have even suggested host switching. We compared phylogenies of host genes (the nuclear EF-1alphaF2 and mitochondrial COI/II) and Blochmannia genes (16S ribosomal DNA [rDNA], groEL, gidA, and rpsB), totaling more than 7 kilobases for each of 16 Camponotus species. Each data set was analyzed using maximum likelihood and Bayesian phylogenetic reconstruction methods. We found minimal conflict among host and symbiont phylogenies, and the few areas of discordance occurred at deep nodes that were poorly supported by individual data sets. Concatenated protein-coding genes produced a very well-resolved tree that, based on the Shimodaira-Hasegawa test, did not conflict with any host or symbiont data set. Correlated rates of synonymous substitution (d(S)) along corresponding branches of host and symbiont phylogenies further supported the hypothesis of cospeciation. These findings indicate that Blochmannia-Camponotus symbiosis has been evolutionarily stable throughout tens of millions of years. Based on inferred divergence times among the ant hosts, we estimated rates of sequence evolution of Blochmannia to be approximately 0.0024 substitutions per site per million years (s/s/MY) for the 16S rDNA gene and approximately 0.1094 s/s/MY at synonymous positions of the genes sampled. These rates are several-fold higher than those for related bacteria Buchnera aphidicola and Escherichia coli. Phylogenetic congruence among Blochmannia genes indicates genome stability that typifies primary endosymbionts of insects.     

Uniaxial strain upregulates matrix-degrading enzymes produced by human vascular smooth muscle cells

Arteries remodel in response to environmental changes. We investigated whether mechanical strain modulates production of matrix metalloproteinase (MMP)-2 and -9 by cultured vascular smooth muscle cells (SMC). MMP-2 and MMP-9 expression were tested using human saphenous vein SMC cultured on silicone membranes at rest or subjected to physiological levels (5%) of stationary or cyclical (1 Hz) uniaxial strain. Compared with control, stationary strain significantly increased MMP-2 mRNA levels at all time points, whereas cyclic strain decreased it after 48 h. Both secreted and cell-associated pro-MMP-2 levels were increased by stationary strain at all times (P < 0.01), whereas cyclic strain decreased secreted levels after 48 h (P < 0.02). MMP-9 mRNA levels and pro-MMP-9 protein were increased after 48 h of stationary stretch (P < 0.01) compared with both no strain and cyclic strain. Our study indicates that vascular SMC show a selective response to different types of strain. We suggest that local increases in stationary mechanical strain resulting from stenting, hypertension, or atherosclerosis may lead to enhanced matrix degradation by SMC.     

The pro-apoptotic Ras effector Nore1 may serve as a Ras-regulated tumor suppressor in the lung

Ras oncoproteins mediate multiple biological effects by activating multiple effectors. Classically, Ras activation has been associated with enhanced cellular growth and transformation. However, activated forms of Ras may also inhibit growth by inducing senescence, apoptosis, and differentiation. Induction of apoptosis by Ras may be mediated by its effector RASSF1, which appears to function as a tumor suppressor. We now show that the Ras effector Nore1, which is structurally related to RASSF1, can also mediate a Ras-dependent apoptosis. Moreover, an analysis of Nore1 protein expression showed that it is frequently down-regulated in lung tumor cell lines and primary lung tumors. Like RASSF1, this correlates with methylation of the Nore1 promoter rather than gene deletion. Finally, re-introduction of Nore1, driven by its own promoter, impairs the growth in soft agar of a human lung tumor cell line. Consequently, we propose that the Ras effector Nore1 is a member of a family of Ras effector/tumor suppressors that includes RASSF1.