The present invention relates to a method of identifying a region (one or more) of a genome of a cell to which a protein of interest binds. In the methods described herein, DNA binding protein of a cell is linked (e.g., covalently crosslinked) to genomic DNA of a cell. The genomic DNA to which the DNA binding protein is linked is removed and combined or contacted with DNA comprising a sequence complementary to genomic DNA of the cell under conditions in which hybridization between the identified genomic DNA and the sequence complementary to genomic DNA occurs. Region(s) of hybridization are region(s) of the genome of the cell to which the protein of binds. A method of identifying a set of genes where cell cycle regulator binding correlates with gene expression and of identifying genomic targets of cell cycle transcription activators in living cells is also encompassed.

https://science.sciencemag.org/content/sci/290/5500/2306.full.pdf

Genome-wide location and function of dna binding proteins

Cohesins: chromosomal proteins that prevent premature separation of sister chromatids

Reactive oxygen species are required for cell proliferation but can also induce apoptosis1. In proliferating cells this paradox is solved by the activation of protein kinase B (PKB; also known as c-Akt), which protects cells from apoptosis2. By contrast, it is unknown how quiescent cells that lack PKB activity are protected against cell death induced by reactive oxygen species. Here we show that the PKB-regulated Forkhead transcription factor FOXO3a (also known as FKHR-L1) protects quiescent cells from oxidative stress by directly increasing their quantities of manganese superoxide dismutase (MnSOD) messenger RNA and protein. This increase in protection from reactive oxygen species antagonizes apoptosis caused by glucose deprivation. In quiescent cells that lack the protective mechanism of PKB-mediated signalling, an alternative mechanism is induced as a consequence of PKB inactivity. This mechanism entails the activation of Forkhead transcription factors, the transcriptional activation of MnSOD and the subsequent reduction of reactive oxygen species. Increased resistance to oxidative stress is associated with longevity. The model of Forkhead involvement in regulating longevity stems from genetic analysis in Caenorhabditis elegans3,4,5,6, and we conclude that this model also extends to mammalian systems.

/pdf/forkhead-transcription-factor-foxo3a-protects-quiescent-12yj48oz0s.pdf

Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress

A Direct Link between Sister Chromatid Cohesion and Chromosome Condensation Revealed through the Analysis of MCD1 in S. cerevisiae

https://www.cell.com/cell/pdf/S0092-8674(05)00442-3.pdf

RNA degradation by the exosome is promoted by a nuclear polyadenylation complex

Fission yeast temperature-sensitive mutants cut3-477 and cut14-208 fail to condense chromosomes but small portions of the chromosomes can separate along the spindle during mitosis, producing phi-shaped chromosomes. Septation and cell division occur in the absence of normal nuclear division, causing the cut phenotype. Fluorescence in situ hybridization demonstrated that the contraction of the chromosome arm during mitosis was defective. Mutant chromosomes are apparently not rigid enough to be transported poleward by the spindle. Loss of the cut3 protein by gene disruption fails to maintain the nuclear chromatin architecture even in interphase. Both cut3 and cut14 proteins contain a putative nucleoside triphosphate (NTP)-binding domain and belong to the same ubiquitous protein family which includes the budding yeast Smc1 protein. The cut3 mutant was suppressed by an increase in the cut14+ gene dosage. The cut3 protein, having the highest similarity to the mouse protein, is localized in the nucleus throughout the cell cycle. Plasmids carrying the DNA topoisomerase I gene partly suppressed the temperature sensitive phenotype of cut3-477, suggesting that the cut3 protein might be involved in chromosome DNA topology.

Fission yeast cut3 and cut14, members of a ubiquitous protein family, are required for chromosome condensation and segregation in mitosis.

Mis6, a Fission Yeast Inner Centromere Protein, Acts during G1/S and Forms Specialized Chromatin Required for Equal Segregation

Cid13 Is a Cytoplasmic Poly(A) Polymerase that Regulates Ribonucleotide Reductase mRNA

Two fission yeast temperature-sensitive mutants, cut6 and lsd1, show a defect in nuclear division. The daughter nuclei differ dramatically in size (the phenotype designated lsd, large and small daughter). Fluorescence in situ hybridization (FISH) revealed that sister chromatids were separated in the lsd cells, but appeared highly compact in one of the two daughter nuclei. EM showed asymmetric nuclear elongation followed by unequal separation of nonchromosomal nuclear structures in these mutant nuclei. The small nuclei lacked electron-dense nuclear materials and contained highly compacted chromatin. The cut6+ and lsd1+ genes are essential for viability and encode, respectively, acetyl CoA carboxylase and fatty acid synthetase, the key enzymes for fatty acid synthesis. Gene disruption of lsd1+ led to the lsd phenotype. Palmitate in medium fully suppressed the phenotypes of lsd1. Cerulenin, an inhibitor for fatty acid synthesis, produced the lsd phenotype in wild type. The drug caused cell inviability during mitosis but not during the G2-arrest induced by the cdc25 mutation. A reduced level of fatty acid thus led to impaired separation of non-chromosomal nuclear components. We propose that fatty acid is directly or indirectly required for separating the mother nucleus into two equal daughters.

/pdf/aberrant-mitosis-in-fission-yeast-mutants-defective-in-fatty-2k157tudgx.pdf

Aberrant mitosis in fission yeast mutants defective in fatty acid synthetase and acetyl CoA carboxylase.

The chromatin immunoprecipitation (ChIP) method provides an ideal tool for detecting direct or indirect interactions between proteins of interest and DNAs with known sequences. Here, we introduce the ChIP protocol used in our laboratory to identify in vivo protein-DNA association in the fission yeast Schizosaccharomyces pombe. The cytological and genetic merits of the fission yeast for studying control of the eukaryotic cell cycle and chromosome dynamics are reinforced by application of this ChIP method.

Shigeaki Saitoh

Papers

Fission yeast cut3 and cut14, members of a ubiquitous protein family, are required for chromosome condensation and segregation in mitosis.

Mis6, a Fission Yeast Inner Centromere Protein, Acts during G1/S and Forms Specialized Chromatin Required for Equal Segregation

Cid13 Is a Cytoplasmic Poly(A) Polymerase that Regulates Ribonucleotide Reductase mRNA

Aberrant mitosis in fission yeast mutants defective in fatty acid synthetase and acetyl CoA carboxylase.

Application of the chromatin immunoprecipitation method to identify in vivo protein-DNA associations in fission yeast.