Project description:To define the molecular regulators required for differential pattern of H3K79 methylation by Dot1, we performed a GPS screen and discovered that the components of the cell cycle-regulated SBF complex were required for normal levels of H3K79 di- but not trimethylation. Genome-wide mapping revealed that H3K79 di- and trimethylation to present a mutually exclusive pattern on chromatin with M/G1 cell-cycle-regulated genes significantly enriched for H3K79 dimethylation. Since H3K79 trimethylation requires prior monoubiquitination of H2B, we performed genome-wide profiling of H2BK123 monoubiquitination and showed that H2BK123 monoubiquitination is excluded from cell cycle regulated genes and sites containing H3K79me2 but not from H3K79me3 containing regions. A genome-wide screen for factors responsible for the establishment/removal of H3K79 dimethylation resulted in the identification of several genes including NRM1 and WHI3, which both impact the transcription by the SBF, and MBF complexes, further linking the regulation of H3K79's methylation status to the cell cycle.
Project description:Experiment to obtain the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation and tiling microarray (DRIP-chip). Samples: wild type, Rnase H deletion mutant, hpr1 deletion mutant, sen1-1 temperature sensitive mutant.
Project description:Sgs1 is a DNA helicase with roles in DNA replication and repair. DNA repair proteins have been linked to genome instability in part by altering the landscape of DNA:RNA hybrids. Here, we mapped the Sgs1 binding profile along with the profile of DNA:RNA hybrids and gammaH2A genome-wide in an effort to identify direct vs indirect effects of Sgs1 in DNA:RNA hybrid and gammaH2A distribution. The wild type DNA:RNA hybrid sample was reported previously in E-MTAB-2388.
Project description:ChIP-chip by array of Rpb3 localization in yeast with different CTD lenghts and with or without CDK8 deletion to determine the role of CTD length and Cdk8 on RNAPII localization
Project description:Rad50 is a component of the conserved MRE11-RAD50-NBS1 (MRN) complex, which functions in genome stability and the cell’s ability to deal with stalled DNA replication forks. We identified Rad50 as a factor important for R-loop tolerance and thus mapped DNA:RNA hybrids in Rad50KO cells and compare them to previously reported wild-type and Sgs1KO profiles.
Project description:ChIP-chip by array of S. cerevisiae cells to investigate the genome wide occupancy of phospho-S2 and phospho-S5 forms of the RNAPII-CTD. ChIP-chip by array of S. cerevisiae cells to investigate the genome wide occupancy of Ste12 and Tec1.
Project description:The NuA4 histone acetyltransferase (HAT) complex is required for gene specific regulation, cell cycle progression, and DNA repair. Dissection of the 13-subunit complex reveals that the Eaf7 subunit bridges Eaf5 with Eaf3, a H3K36me3-binding chromodomain protein, and this Eaf5/7/3 trimer is anchored to NuA4 through Eaf5. This subcomplex represents a functional module as deletions of these genes create similar phenotypes and a large portion of the trimer exists in a native form outside the NuA4 complex. Gene-specific and genome-wide location analyses indicate that the Eaf5/7/3 trimer correlates with transcription activity and is enriched over the coding region. In agreement with a role in transcription elongation, the Eaf5/7/3 trimer interacts with phosphorylated RNA polymerase II and helps its progression. In addition, loss of Eaf5/7/3 partially suppresses intragenic cryptic transcription arising in set2 mutant cells, suggesting a role in nucleosome destabilization. Such a function is supported by genetic interactions with the FACT histone chaperone. On the other hand, loss of the trimer leads to an increase of replication-independent histone exchange over the coding region of transcribed genes. Taken together, these results lead to a model where Eaf5/7/3 associates with elongating polymerase and is involved in the disassembly of nucleosomes in front of the polymerase, but also in their recycling in its wake.
Project description:The most widely used method for detecting genome-wide protein-DNA interactions is chromatin immunoprecipitation on tiling microarrays, commonly known as ChIP-chip. Here, we conducted the first objective analysis of tiling array platforms, amplification procedures, and signal detection algorithms in a simulated ChIP-chip experiment. Mixtures of human genomic DNA and "spike-ins" comprised of nearly 100 human sequences at various concentrations were hybridized to four tiling array platforms by eight independent groups. Blind to the number of spike-ins, their locations, and the range of concentrations, each group made predictions of the spike-in locations. We found that microarray platform choice is not the primary determinant of overall performance. In fact, variation in performance between labs, protocols and algorithms within the same array platform was greater than the variation in performance between array platforms. However, each array platform had unique performance characteristics that varied with tiling resolution and the number of replicates, which have implications for cost versus detection power. Long oligonucleotide arrays were slightly more sensitive at detecting very low enrichment. On all platforms, simple sequence repeats and genome redundancy tended to result in false positives. LM-PCR and WGA, the most popular sample amplification techniques, reproduced relative enrichment levels with high fidelity. Performance among signal detection algorithms was heavily dependent on array platform. The spike-in DNA samples and the data presented here provide a stable benchmark against which future ChIP platforms, protocol improvements, and analysis methods can be evaluated. Keywords: ChIP-chip For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf 9 spike-in replicates and 9 genomic input control replicates