Project description:Using genetic mutations to study protein functions in vivo is a central paradigm of modern biology. Single-domain camelid antibodies generated against GFP have been engineered as nanobodies or GFP-binding proteins (GBPs) that can bind GFP as well as some GFP variants with high affinity and selectivity. In this study, we have used GBP-mCherry fusion protein as a tool to perturb the natural functions of a few kinetochore proteins in the fission yeast Schizosaccharomyces pombe. We found that cells simultaneously expressing GBP-mCherry and the GFP-tagged inner kinetochore protein Cnp1 are sensitive to high temperature and microtubule drug thiabendazole (TBZ). In addition, kinetochore-targeted GBP-mCherry by a few major kinetochore proteins with GFP tags causes defects in faithful chromosome segregation. Thus, this setting compromises the functions of kinetochores and renders cells to behave like conditional mutants. Our study highlights the potential of using GBP as a general tool to perturb the function of some GFP-tagged proteins in vivo with the objective of understanding their functional relevance to certain physiological processes, not only in yeasts, but also potentially in other model systems.
Project description:TOP2B is involved in transcriptional initiation in response to nuclear hormone ligands and plays a role in transcriptional elongation. Whole genome TOP2B ChIP-seq was carried out on human MCF7 cells in the presence and absence of the nuclear hormone estradiol. Three peak calling methods were used and the peaks identified by at least two methods were analyzed further. Approximately half of the peaks fell either within a gene or within 5Kb of a transcription start site. The coincidence of TOP2B peaks and gene promoters was analyzed; TOP2B peaks were less frequently associated with promoters in estradiol treated than in control cells, suggesting a role of TOP2B in repression of transcription or a transient role in estradiol induced transcriptional changes. Whole genome TOP2B ChIP-seq was carried out on human MCF7 cells in the presence (30 mins exposure) and absence of the nuclear hormone estradiol. "Input" control samples were also sequenced for background detection and comparison.
Project description:TOP2B is involved in transcriptional initiation in response to nuclear hormone ligands and plays a role in transcriptional elongation. Whole genome TOP2B ChIP-seq was carried out on human MCF7 cells in the presence and absence of the nuclear hormone estradiol. Three peak calling methods were used and the peaks identified by at least two methods were analyzed further. Approximately half of the peaks fell either within a gene or within 5Kb of a transcription start site. The coincidence of TOP2B peaks and gene promoters was analyzed; TOP2B peaks were less frequently associated with promoters in estradiol treated than in control cells, suggesting a role of TOP2B in repression of transcription or a transient role in estradiol induced transcriptional changes.
Project description:ChIP-chip analyses of H3K9me2 (in WT, erh1â??, mmi1â?? and ccr4â??), Erh1-GFP (in WT and mmi1â??) and CFP-Mmi1 (in WT) H3K9me2: WT vs erh1â??, WT vs mmi1â?? and WT vs ccr4â??; Erh1-GFP: WT vs mmi1â??; CFP-Mmi1 in WT, Biological replicates: 2
Project description:Chromosomes undergo extensive conformational rearrangements in preparation for their segregation during cell divisions. Insights into the molecular mechanisms behind this still poorly understood condensation process require the development of new approaches to quantitatively assess chromosome formation in vivo. In this study, we present a live-cell microscopy-based chromosome condensation assay in the fission yeast Schizosaccharomyces pombe. By automatically tracking the three-dimensional distance changes between fluorescently marked chromosome loci at high temporal and spatial resolution, we analyze chromosome condensation during mitosis and meiosis and deduct defined parameters to describe condensation dynamics. We demonstrate that this method can determine the contributions of condensin, topoisomerase II, and Aurora kinase to mitotic chromosome condensation. We furthermore show that the assay can identify proteins required for mitotic chromosome formation de novo by isolating mutants in condensin, DNA polymerase ?, and F-box DNA helicase I that are specifically defective in pro-/metaphase condensation. Thus, the chromosome condensation assay provides a direct and sensitive system for the discovery and characterization of components of the chromosome condensation machinery in a genetically tractable eukaryote.
