Regulation of chromatin function underlies many epigenetic processes and is a crucial determinant of gene regulation in all eukaryotic organisms. However, crosstalk between chromatin modification pathways in metazoans is not well understood. To address this, we have developed a high-throughput robotic platform with a automated microscopy readout for siRNA library screening of epigenetic marks. We termed this CROSS for Chromatin Regulation Ontology SiRNA Screening. We will apply CROSS to delineate key pathways of chromatin methylation in human cells. Genes involved in crosstalk with methylation of histone H3 lysine-4 or lysine-79 and (hydroxy)methylation of cytosines in DNA will be identified. Their involvement in gene expression will be investigated by mRNA profiling and by in vitro histone modification assays. Taken together, these approaches will yield unprecedented insight into crosstalk at the level of chromatin modification and function in relation to gene expression. The results will pave the way for effective intervention therapies in epigenetic pathways involved in human development and disease.

Regulation of gene expression in eukaryotes is a highly dynamic process. We have developed an experimental system to measure turnover of the transcription machinery on a genome-wide scale in budding yeast as model system. Our studies revealed a cyclic behavior of active and inactive transcription complexes on gene promoters. In the current project we will investigate the parameters regulating the dynamic behavior of transcription complexes and relate this to the mRNA output of an eukaryotic genome. We will make use of well-characterized mutant versions of the TATA-binding protein and exploit our technological advantages in genome-wide mapping. In addition, a novel approach to inactivate essential proteins will be applied to determine their contributions to the activation/deactivation cycles of the transcription machinery. The combination of approaches will provide quantitative information on the contributions of DNA sequence and transcription factor action on the dynamic parameters of transcription complexes. This information will greatly contribute to a systems-biology based description of the eukaryotic transcription process.

Understanding how genes are regulated is pivotal for understanding many cellular processes. Regulation of gene expression through transcription is complicated, involving interplay between gene-specific transcription factors, DNA packaged into chromatin, chromatin remodelers/modifiers, elongation and RNA processing factors. Intricate signal transduction pathways control these components through modifications such as phosphorylation, ubiquitination and acetylation. Past studies have yielded many details on how a minority of genes are regulated. This knowledge is too fragmented to form complete models of the entire system. Comprehensive knowledge is lacking about which genes are controlled by which regulatory proteins, how these factors are themselves regulated, which cooperate, how and on which genes. Such information, systematically generated, would result in genome control maps: genomic wiring-diagrams that describe in molecular detail how each gene is controlled by each regulatory component. My long-term aim is to develop such genome control maps since this would have wide-ranging benefits for a variety of research areas. Using a robotic facility we will first finish generating DNA microarray expression-profiles of targeted mutations in virtually all known and putative components of the transcription and signaling machinery in Saccharomyces cerevisiae, an important model organism for eukaryotic transcription. The data will be analyzed in a variety of ways, including analysis as detailed molecular phenotypes to determine regulatory relationships. Analysis alongside publicly available genome-scale data will further help uncover systems-level characteristics and the nature of the regulatory interactions observed. This will lead to biochemical and genetic follow-up experiments targeting the most interesting new regulatory mechanisms. Besides objectively assessing the role of many evolutionarily conserved regulators in parallel for the first time, the project will address key questions regarding general mechanisms of transcription regulation, such as redundancy, cross-talk, recruitment and specificity. The data will be useful for a variety of other purposes and allow functional mapping of the signaling and transcription machinery onto the genome comprehensively for the first time.

Although much effort is invested to identify and characterize histone modifying enzymes and their genomic targets, little is known about their downstream effects. One particular interesting modification that plays a role in regulating many processes including activation of transcription as well as the establishment and maintenance of silent heterochromatin is lysine methylation of core histones. An important function of these lysine methylations is thought to be the recruitment or stabilization of protein complexes that subsequently can exert their function at the site of recruitment. Indeed, a number of such chromatin ?readers? have been identified in recent years. However, these most likely only represent the tip of the iceberg. I therefore propose to: 1. Decipher the histone methyl lysine interactome using high accuracy quantitative mass spectrometry 2. Establish Chromatin ImmunoPrecipitation combined with quantitative mass Spectrometry (ChIP-qSpec) to determine the proteomic environment of histone methyl lysine sites and their interactors in vivo. The approach involves a combination of SILAC based histone peptide pull-downs and GFP purifications, ChIP-sequencing experiments, immunofluorescence and novel methodology that involves quantitative mass spectrometric analysis of proteins that are brought down in chromatin immunoprecipitation experiments. These studies will uncover a large number of novel methyl lysine readers and therefore will provide novel insights into the biology of different histone lysine methylation sites and their role in regulating cell growth and differentiation.

A layer of epithelial cells protects your body and various organs from the outside environment. This layer is continuously being renewed due to cell loss and the gain of new cells. The researcher investigates how cells sense when they need to divide to ensure a proper balance with cell loss.