GENE REGULATION AND EUKARIOTIC CHEMOGENOMICA
Learning outcomes of the course unit
The aim is to provide an in-depth knowledge on the mechanisms controlling eukaryotic gene expression at both the transcriptional and post-transcriptional level, taking into account the information recently made available by genomics. This will be pursued through a unifying conceptual framework pointing to general regulatory strategies shared by prokaryotes and eukaryotes and by different phases of the genetic information transfer process (transcription, mRNA splicing and other post-transcriptional events, controlled modification/degradation and subcellular localization of proteins, signal transduction to the cell nucleus). Both theoretical and practical aspects of gene expression control will be considered, with special emphasis on its biomedical and molecular diagnostics applications/implications. A related aim, specifically regarding Chemogenomics, is to provide basic information and specific application examples on one of the most advanced fields of post-genomic research, with special reference to drug discovery and “systems biology”, and other applications of biotechnology in the (bio)pharmaceutical field.
ACQUIRING KNOWLEDGE AND UNDERSTANDING.
Students are expected to acquire a detailed knowledge of some of the most important cellular and molecular processes underlying the elaboration of eukaryotic genome information and related applications, especially with regard to bio/molecular medicine. Special emphasis will be placed on post-genomic experimental strategies that are being employed to discover and/or functionally characterize bio-active compounds (“Chemogenomics”).
APPLYING KNOWLEDGE AND UNDERSTANDING.
Through the guided analysis of key experiments that have allowed to understand some of the above described processes, students will acquire the basic knowledge and competence required for the study of genetic information transfer/elaboration processes at the molecular level and for the exploitation of genomics as a tool for the functional characterization of bio-active small-molecules and the identification of novel compounds.
Basic knowledge in chemistry, biochemistry, molecular biology and genetics.
Course contents summary
Structure of eukaryotic genomes.
Regulatory strategies relying upon “regulated recruitment”.
Eukaryotic RNA polymerases, transcriptional regulators and promoters.
Chromatin and nucleosomes.
Enhancers and different modes of control of eukaryotic transcriptional regulators.
Other cellular processes relying on “regulated recruitment”.
Goals and fields of application of chemogenomics.
Bioactive compounds of molecular-biological origin: recombinant proteins/peptides, nucleotide (RNA/DNA) and peptide aptamers.
Chemogenomic technologies in the yeast S. cerevisiae.
Structure of eukaryotic genomes: repeated sequences and gene duplication, (retro)transposable elements, simple and complex transcription units, multigene families, regulatory sequences and differential gene expression.
Regulatory strategies relying upon “regulated recruitment” (key examples from prokaryotic systems); activator bypass experiments and the “two-hybrid” technology; “squelching” and transcription factor decoys; regulated recruitment and cooperativity; other types of gene regulation processes (RNA polymerase modification; DNA modification).
Eukaryotic RNA polymerases, transcriptional regulators and promoters. General transcription factors (GTFs); pre-initiation complex formation; “mediator”; (co)activators and (co)repressors; the Saccharomyces cerevisiae activator Gal4; the negative regulators Gal80 and Mig1; the other eukaryotic transcription systems (RNA pol I and RNA pol III); post-initiation events (capping, splicing and polyadenylation); the “spliceosome, splicing regulator elements (ESE, ESS and ISS) and splicing regulation.
Chromatin and nucleosomes; chromatin remodeling and histone modification (HAT, HDAC, HMT, HDMT); regulatory roles of histone tail modification (acetylation/deacetylation; methylation/demethylation); chromatin immunoprecipitation (ChIP); combinatorial control: mating type regulation in Saccharomyces cerevisiae; telomeres and their associated regulatory effects; DNA methylation; insulator sequences and other higher-order control elements.
Enhancers; different modes of control of transcriptional regulators; nuclear receptors; SREBP; Tubby; Notch and APP; NF-kB, TAT/TAR, Rb and cyclins.
Other cellular processes relying on “regulated recruitment”: ubiquitination and controlled degradation of selected target proteins, hormone-receptor interaction and signal transduction pathways directed to the nucleus.
Goals and fields of application of chemogenomics, as applied to “drug discovery”, “target discovery/mode of action identification”, “target validation” and evaluation of off-target effects.
Chemogenomics based on transcriptomic, proteomic and “phenomic” analyses (e..g., Synthetic Genetic Arrays, SGA, and “genomic phenotyping”. Chimeric gene constructs and the “reporter gene” approach: homologous promoter/heterologous coding sequence; heterologous promoter/heterologous regulatory protein/heterologous coding sequence (“reporter gene”) applied to drug discovery and toxicity profiling; the yeast two-hybrid system and its variants; “growth interference” screenings.
Bioactive compounds of molecular-biological origin: e.g., recombinant proteins/peptides, nucleotide (RNA/DNA) and peptide aptamers to be used for target validation (and as prototype drugs). Presentation and analysis of specific (bio)technological, chemogenomic platforms (SPLINT, Discoverex, NASCA, Aptanomics and others).
Chemogenomic technologies in the yeast S. cerevisiae. Microarray/transcriptome analyses: comparison of drug-treated and mutant yeast strains and the “compendium” approach; target/mechanism of action and side-effects discovery.
Systematic gene disruption in yeast (and gene silencing by shRNAs in other organisms). The yeast gene deletion mutant collections (knockout mutant arrays) and “genomic phenotyping” (HOP, HOP/SGA, HIP, MSP) of small-molecule drugs (or toxicants) in yeast: mechanism of action, off-target effects and new applications for “old drugs” or “lead compounds” abandoned in late phases of (pre)clinical development, novel validation systems. Use of functional genomics/interactomics databases (e.g., SGD) for data analysis.
Lodish H., Kaiser C.A., Bretscher A., Amon A., Berk A., Krieger M., Ploegh H., Scott M.P.
MOLECULAR BIOLOGY OF THE CELL (Seventh Edition)
Macmillan Higher Education/Freeman and Company Publishers
Watson J.D., Backer T.A., Bell S. P., Gann A., Levine M., Losick R.
BIOLOGIA MOLECOLARE DEL GENE/MOLECULAR BIOLOGY OF THE GENE (Sixth Edition)
Zanichelli/CSHL Press; Pearson/Benjamin Cummings Publishers
Ptashne M., Gann A. "GENES AND SIGNALS", Ed. Zanichelli
Reviews, selected articles and powerpoint presentations from companies in the field of Chemogenomics, which will be made available to the students in an electronic format.
The course is mainly based on lectures, but also includes exercises, the examination/discussion of original scientific articles and examples of data analysis.
Assessment methods and criteria
Evaluation of the expected learning achievements will be based on an oral examination that will also include the discussion of specific regulatory schemes, application examples/case-studies and discovery strategies presented in the course. This will allow a detailed evaluation of the theoretical and practical knowledge on the various genetic information transfer/elaboration processes illustrated in the course as well as the ability to apply such knowledge to address and solve specific experimental problems.