Easy detection of chromatin binding proteins by the histone association assay

The Histone Association Assay provides an easy approach for detecting proteins that bind chromatin in vivo. This technique is based on a chromatin immunoprecipitation protocol using histone H3-specific antibodies to precipitate bulk chromatin from crosslinked whole cell extracts. Proteins that co-precipitate with chromatin are subsequently detected by conventional SDS-PAGE and Western blot analysis. Unlike techniques that separate chromatin and non-chromatin interacting proteins by centrifugation, this method can be used to delineate whether a protein is chromatin associated regardless of its innate solubility. Moreover, the relative amount of protein bound to DNA can be ascertained under quantitative conditions. Therefore, this technique may be utilized for analyzing the chromatin association of proteins involved in diverse cellular processes

Bub1 overexpression induces aneuploidy and tumor formation through Aurora B kinase hyperactivation

Hyperactivated Aurora B kinase is a primary mediator of Bub1 overexpression-induced aneuploidy and tumorigenesis in mice

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Interaction between PCNA and Diubiquitinated Mcm10 Is Essential for Cell Growth in Budding Yeast

The minichromosome maintenance protein 10 (Mcm10) is an evolutionarily conserved factor that is essential for replication initiation and elongation. Mcm10 is part of the eukaryotic replication fork and interacts with a variety of proteins, including the Mcm2-7 helicase and DNA polymerase alpha/primase complexes. A motif search revealed a match to the proliferating cell nuclear antigen (PCNA)-interacting protein (PIP) box in Mcm10. Here, we demonstrate a direct interaction between Mcm10 and PCNA that is alleviated by mutations in conserved residues of the PIP box. Interestingly, only the diubiquitinated form of Mcm10 binds to PCNA. Diubiquitination of Mcm10 is cell cycle regulated; it first appears in late G(1) and persists throughout S phase. During this time, diubiquitinated Mcm10 is associated with chromatin, suggesting a direct role in DNA replication. Surprisingly, a Y245A substitution in the PIP box of Mcm10 that inhibits the interaction with PCNA abolishes cell proliferation. This severe-growth phenotype, which has not been observed for analogous mutations in other PCNA-interacting proteins, is rescued by a compensatory mutation in PCNA that restores interaction with Mcm10-Y245A. Taken together, our results suggest that diubiquitinated Mcm10 interacts with PCNA to facilitate an essential step in DNA elongation

Aurora B hyperactivation by Bub1 overexpression promotes chromosome missegregation.

Item does not contain fulltextHigh expression of the mitotic kinase Bub1 is associated with a variety of human cancers and correlates with poor clinical prognosis, but whether Bub1 alone can drive tumorigenesis was unknown. We provided conclusive evidence that Bub1 has oncogenic properties by generating transgenic mice that overexpress Bub1 in a wide variety of tissues, resulting in aneuploidization. Consistently, Bub1 transgenic mice developed various kinds of spontaneous tumors as well as accelerated Myc-induced lymphomagenesis. While the mitotic checkpoint was robust in Bub1 overexpressing cells, misaligned and lagging chromosomes were observed. These defects originated from increased Aurora B activity and could be suppressed by inhibition of Aurora B. Taken together, this indicates that Bub1 has oncogenic properties and imply that aneuploidization and tumorigenesis result from Aurora B-dependent missegregation. Here, we focus on the complex relationship between Bub1 and Aurora B and discuss the broader implications of Bub1-dependent Aurora B activation in mediating error correction

Bub1 kinase activity drives error correction and mitotic checkpoint control but not tumor suppression

Item does not contain fulltextThe mitotic checkpoint protein Bub1 is essential for embryogenesis and survival of proliferating cells, and bidirectional deviations from its normal level of expression cause chromosome missegregation, aneuploidy, and cancer predisposition in mice. To provide insight into the physiological significance of this critical mitotic regulator at a modular level, we generated Bub1 mutant mice that lack kinase activity using a knockin gene-targeting approach that preserves normal protein abundance. In this paper, we uncover that Bub1 kinase activity integrates attachment error correction and mitotic checkpoint signaling by controlling the localization and activity of Aurora B kinase through phosphorylation of histone H2A at threonine 121. Strikingly, despite substantial chromosome segregation errors and aneuploidization, mice deficient for Bub1 kinase activity do not exhibit increased susceptibility to spontaneous or carcinogen-induced tumorigenesis. These findings provide a unique example of a modular mitotic activity orchestrating two distinct networks that safeguard against whole chromosome instability and reveal the differential importance of distinct aneuploidy-causing Bub1 defects in tumor suppression

Aneuploidy in health, disease, and aging

Item does not contain fulltextAneuploidy, an aberrant number of chromosomes, has been recognized as a feature of human malignancies for over a century, but compelling evidence for causality was largely lacking until mouse models for chromosome number instability were used. These in vivo studies have not only uncovered important new insights into the extremely complex aneuploidy-cancer relationship but also into the molecular mechanisms underlying proper and aberrant chromosome segregation. A series of diverse mouse models for the mitotic checkpoint protein BubR1 has provided evidence for a provocative novel link between aneuploidization and the development of age-related pathologies