Search

The cell cycle is strictly ordered to ensure faithful genome duplication and chromosome segregation. Control mechanisms establish this order by dictating when a cell transitions from one phase to the next. Much is known about the control of the G1/S, G2/M, and metaphase/anaphase transitions, but thus far, no control mechanism has been identified for the S/G2 transition. Here we show that cells transactivate the mitotic gene network as they exit the S phase through a CDK1 (cyclin-dependent kinase 1)-directed FOXM1 phosphorylation switch. During normal DNA replication, the checkpoint kinase ATR (ataxia-telangiectasia and Rad3-related) is activated by ETAA1 to block this switch until the S phase ends. ATR inhibition prematurely activates FOXM1, deregulating the S/G2 transition and leading to early mitosis, underreplicated DNA, and DNA damage. Thus, ATR couples DNA replication with mitosis and preserves genome integrity by enforcing an S/G2 checkpoint.

Chromatin is the template for replication and tran- scription in the eukaryotic nucleus, which needs to be defined in composition and structure before these processes can be fully understood. We report an isolation protocol for the targeted purifi- cation of specific genomic regions in their native chromatin context from Saccharomyces cerevisiae . Subdomains of the multicopy ribosomal DNA locus containing transcription units of RNA polymerases I, II or III or an autonomous replication sequence were independently purified in sufficient amounts and purity to analyze protein composition and histone modifications by mass spectrometry. We present and discuss the proteomic data sets obtained for chromatin in different functional states. The native chromatin was further amenable to electron mi- croscopy analysis yielding information about nucleosome occupancy and positioning at the single-molecule level. We also provide evidence that chromatin from virtually every single copy genomic locus of interest can be purified and analyzed by this technique.

International audience; Eukaryotic ribosome biogenesis requires more than 150 auxiliary proteins, which transiently interact with pre-ribosomal particles. Previous studies suggest that several of these biogenesis factors function together as modules. Using a heterologous expression system, we show that the large ribosomal subunit (LSU) biogenesis factor Noc1p of Saccharomyces cerevisiae can simultaneously interact with the LSU biogenesis factor Noc2p and Rrp5p, a factor required for biogenesis of the large and the small ribosomal subunit. Proteome analysis of RNA polymerase-I-associated chromatin and chromatin immunopurification experiments indicated that all members of this protein module and a specific set of LSU biogenesis factors are co-transcriptionally recruited to nascent ribosomal RNA (rRNA) precursors in yeast cells. Further ex vivo analyses showed that all module members predominantly interact with early pre-LSU particles after the initial pre-rRNA processing events have occurred. In yeast strains depleted of Noc1p, Noc2p or Rrp5p, levels of the major LSU pre-rRNAs decreased and the respective other module members were associated with accumulating aberrant rRNA fragments. Therefore, we conclude that the module exhibits several binding interfaces with pre-ribosomes. Taken together, our results suggest a co-and post-transcriptional role of the yeast Rrp5p-Noc1p-Noc2p module in the structural organization of early LSU precursors protecting them from non-productive RNase activity.

In eukaryotic genomes, chromatin is the template of all nuclear processes including transcription, recombination and replication. Besides the wrapping of DNA in nucleosome core particles, eukaryotic chromatin is associated, interpreted and modified by numerous protein complexes including transcription factors, DNA and RNA metabolizing machineries, architectural proteins and chromatin remodeling and modifying enzymes. To understand how specific genomic loci adopt different functional states, it is critical to characterize the corresponding compositional changes in the local chromatin structure. In this work, a previously established technique based on site specific recombination at defined genomic locations was used to purify selected chromosomal domains from Saccharomyces cerevisiae under native conditions. After improvement of yield and purity of the chromatin preparation, the proteomes co-purifying with domains derived from the multi-copy ribosomal DNA locus transcribed by RNA polymerases I, II and III and at an autonomous replication sequence could be defined by comparative mass spectrometry (MS). Many protein components known to interact with the respective chromatin domains were identified as well as several new factors, for which association with rDNA chromatin could be confirmed in vivo. Mass spectrometric analysis allowed further to assess the posttranslational modifications of histones associated with the individual domains. In addition, electron microscopic analysis provided single molecule information about nucleosome configurations at 5S ribosomal RNA genes. First statistical analyses indicate a heterogeneous population of chromatin states likely correlating with different stages of transcriptional activity. Finally, the improved protocol was applied to the PHO5 gene in order to explore the potential of purifying genes that are only present in one copy per cell. The results indicate that PHO5 associated histone molecules can be enriched in sufficient amounts for MS analysis, opening the door to fully define the specific posttranslational histone modification state at virtually every gene in yeast., In eukaryotischen Genomen stellt Chromatin die Matrize aller nukleären Prozesse wie Transkription, Rekombination und Replikation dar. Neben dem Aufwickeln der DNA in nukleosomale Kernpartikel, ist eukaryotisches Chromatin assoziiert und wird interpretiert und modifiziert durch zahlreiche Proteinkomplexe, darunter Transkriptionsfaktoren, DNA- und RNA-metabolisierende Maschinerien, strukturelle Proteine und Chromatin-remodulierende und modifizierende Enzyme. Um zu verstehen wie spezifische genomische Loci verschiedene funktionelle Zustände einnehmen, ist es entscheidend, die entsprechenden kompositionellen Änderungen in der lokalen Chromatinstruktur zu charakterisieren. In dieser Arbeit wurde eine bereits etablierte Technik, basierend auf ortspezifischer Rekombination an spezifischen genomischen Loci, verwendet um bestimmte chromosomale Domänen aus der Bäckerhefe Saccharomyces cerevisiae unter nativen Bedingungen zu reinigen. Nach Verbesserung von Ausbeute und Reinheit der Chromatin-Präparationen konnten die assoziierten Proteome mit spezifischen Teilbereichen des Multikopien-ribosomalen DNA Lokus, welche von RNA Polymerase I, II und III transkribiert werden, sowie mit einer autonomen Replikationssequenz durch vergleichende Massenspektrometrie (MS) bestimmt werden. Neben vielen bekannten Proteinkomponenten der entsprechenden Chromatindomänen konnten auch mehrere neue Faktoren identifiziert werden, deren Assoziation mit rDNA Chromatin in vivo bestätigt werden konnte. Massenspektrometrische Analysen erlaubten weiter die Bestimmung der posttranslationalen Modifikationen von Histonen, die mit den verschiedenen Domänen assoziiert waren. Zusätzlich erlaubten elektronenmikroskopische Analysen Einzelmolekül-Informationen über die Nukleosomenkonfigurationen an 5S ribosomalen RNA Genen zu gewinnen. Erste statistische Analysen deuten auf eine heterogene Verteilung hin, die wahrscheinlich mit verschiedenen transkriptionellen Zuständen korrelieren. Schließlich wurde das verbesserte Protokoll auf das PHO5-Gen angewendet, um potentiell Chromatindomänen zu reinigen, die nur einmal pro Zelle existieren. Die Ergebnisse deuten an, dass PHO5-assoziierte Histonmoleküle in ausreichenden Mengen für MS-Analysen angereichert werden können. Dies eröffnet die Möglichkeit, den vollständigen posttranslationalen Histonmodifikationszustand jedes Gens in der Hefe zu definieren.