The availability of complete genome sequences is ushering in a new era in the analysis of gene regulation. For example, the full genome sequence of the yeast Saccharomyces cerevisiae is ideal for such studies. Many yeast biochemical and genetic pathways are well understood, and its genome contains a manageable number (6000) of genes which are arranged in a compact fashion. Previously, we have studied the sequences of all potential yeast promoters in order to identify genes whose transcription is co-regulated during the yeast cell cycle. Such work may result in a better understanding of the complex regulatory networks that orchestrate correct quantitative and temporal patterns of gene expression. In a newer experiment with yeast, we have identified biological features associated with double stranded break frequencies during meiotic recombination. Using a feature selection approach, we identified five features that distinguish hot from cold recombination hotspots in Saccharomyces cerevisiae with high accuracy. These are the histone marks H3K4me3, H3K14ac, H3K36me3, and H3K79me3; and GC content. We have also addressed the role that histone modifications play in chromatin organization in yeast. We have shown that there are strong positional preferences for sequence-specific chromatin modifying protein-binding motifs in potential regulatory regions. We have used DNA-binding motifs recognition algorithms and gene ontology enrichment tools to make these discoveries. We continue to interrogate many datasets to establish common properties of chromatin during a variety of active states. In a new collaboration, we are analyzing the mouse genome using new analytical tools and statistics to compare the results of several next generation sequencing (NGS) experiments. Data from ChIPseq, microarray and RNAseq experiments were included for analysis in order to further assess the role of HMGN1 and HMGN2 proteins in chromatin organization and gene expression. We developed analysis pipelines for ChIP-seq experiments of DNA sequences bound to HMGN1 and HMGN2 in wildtype and double knockout mice. The outcome of these collaborations is that we have developed an efficient and adjustable pipeline for the analysis of many NGS datasets in a reasonable time and can easily interrogate the data to further develop biological interpretations and devise new questions. In a separate study, we have performed ChIP followed by massively parallel sequencing (ChIPseq) against Mediator subunits from head (Med17), middle/tail (Med14), tail (Med15 and Med2), and CDK (Cdk8) modules in budding yeast. To allow better distinction of low levels of association from experimental noise or artifacts accompanying ChIP or library amplification prior to sequencing, we compared ChIP-seq profiles from wild type yeast to med17 ts yeast after 45 min at 37C. In yeast harboring this mutation, the head module is disrupted at 37C and mRNA transcription is greatly reduced genome-wide within 30 minutes. Furthermore, comparison of ChIP against Mediator subunits and Pol II in wild type and med17 ts yeast allowed detection of decreased association of Mediator and Pol II even at constitutively active promoters having relatively low amounts of Mediator association, while the relatively short temperature shift mitigates against the likelihood of indirect effects. We also compare association of Mediator subunits and Pol II in wild type and med3 med15 yeast, which lack two of the three subunits from the tail module triad of Med2/Med3/Med15, thus providing insight into the genome-wide function of the tail module in Mediator recruitment. These experiments are currently being extended with a new set of mutants to further understand the activities of the Mediator complex in gene regulation.
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