EPIGENETICS AND GENE REGULATION
Final oral exam
The main objective of the course is to provide the student with knowledge in the field of basic and applied molecular biology, from the understanding of the mechanisms controlling gene expression in eukaryotic at the transcriptional and post-transcriptional level, to the design of new molecular therapies, such as epigenetic drugs or stem cells.
The student will also be competent on the main and most modern experimental technologies for the study of complex biological phenomena. In addition, the course aims to make the student independent in the analysis of experimental data, applying their knowledge, and refine their communication skills, through the analysis and presentation of scientific data in the literature. At the end of the course the student will have the necessary tools to acquire independent judgment. In particular, the student will be able to:
- analyze and discuss topics of regulation of gene expression with mastery;
- propose strategies for investigating gene expression;
- understand and critically analyze the biomolecular literature in all its aspects;
- design detailed experimental approaches on general biomolecular problems.
In particular, the student through lessons, Journal Clubs and practical work, will acquire advanced knowledge on the topics covered in the duration of the course.
A. TECHNIQUES OF ANALYSIS OF GENE EXPRESSION
Basic techniques in the study of gene function.
Gene targeting 1.0: Genetic manipulation in animals. Transgenic mice. Site-specific recombination and inducible knock-out mice.
Gene targeting 2.0: Genome-editing nuclease: ZFN, TALEN, CRISPR-Cas9.
B. REGULATION OF GENE EXPRESSION IN EUKARYOTES
GENE EXPRESSION REGULATION AT: CHROMATIN AND TRANSCRIPTIONAL LEVEL
Role of chromatin modifications in gene expression regulation
Chromatin structure and organization
ATP-dependent chromatin remodeling systems.
Post-translational modifications of histones: acetylation, phosphorylation and methylation. Histone-modifying enzymes and their structural domains.
Histone phosphorylation is implicated in multiple biological processes. The H2A and H3 histone variants.
Eterochromatin and epigenetic regulation of gene expression
Genomic Imprinting. Monoallelic gene expression. Epigenetic control of transposable elements
POST-TRANSCRIPTIONAL LEVEL
- Primary, secondary and tertiary RNA structure; proteins that bind to RNA
-RNA interference and genes encoding miRNAs
- RNA coding genes: gene organization and biological activity of snoRNA;
- lncRNA and circRNA
- Non-coding RNAs and epigenetic modifications. The example of centromeric DNA.
- Splicing: modality and regulation of alternative splicing. The alternative splicing in the determination of Drosophila sex
- Editing: Editing by base inversion / deletion. RNA role guide Editing by modification of bases. Cytidine deaminase and adenosine deaminase. RNA editing and human diseases
- Transport and degradation of mRNAs
TRANSLATIONAL AND POST- TRANSLATIONAL LEVEL
Translation: principles and regulations
Post-translational modifications of proteins
The ubiquitin-mediated degradation system.
C. GENE EXPRESSION IN PHYSIOLOGICAL AND PATHOLOGICAL CONTEXTS
EPIGENETICS, CELL CYCLE AND CANCER
Regulation of gene expression during the cell cycle. The cyclin-kinase complexes and the regulation of their activity. Cell cycle control mechanisms: the role of retinoblastoma and p53 proteins.
Alterations of the cell cycle and epigenetic modifications involved in tumor transformation
LONG TERM REGULATION OF GENE EXPRESSION: DEVELOPMENT AND DIFFERENTIATION
Drosophila to model gene expression regulation during develpoment.
Maternal effect genes that effect the anterior-posterior and the and the dorsal-ventral polarity.
The segmentation genes and the zygotic effect. hunchback a transcriptional repressor. Eve promoter structure.
The homeotic genes and the definition of homeotic mutation. The complex antennipedia and bithorax. The homeodomain. the polycomb group proteins and the "freezing of decisions taken".
STEM CELLS: PRINCIPLES AND APPLICATIONS
Biological characteristics of stem cells: definition and characteristics of embryonic, amniotic and adult stem cells. Transcriptional regulation circuits associated with pluripotency: the triad Oct4, Nanog and Sox2; the polycomb group proteins and the chromatinic structure. Epigentica and cell identity in stem cells. Nuclear reprogramming and pluripotency induction methods (iPS). Applications of iPS. Tumor stem cells: biomolecular characteristics. Therapeutic potential of stem cells.
THERAPEUTIC RNA
-Attamers: Pegaptanib (macugen) in the treatment of degenerative maculapathy antisense RNA: characteristics and role in the regulation of gene expression
-RNA splicing modifiers: RNA to stimulate exon exclusion in Duchenne muscular dystrophy; to stimulate the exon inclusion in muscular spinal atrophy. Blocking a cryptic splicing site and the beta-globin gene
-Technology antimiR: the example of the miraven; mir122 and HCV.
LABORATORY
ChIP Analysis of c-fos promoter in human cells upon stimulation with serum.
1) L. Armstrong "Epigenetics" Garland Science
2) Epigenetics © 2006 Cold Spring Harbor Laboratory Press
3) Molecular Biology of the Gene- Watson et al.,
4) Extra reading material - a list of review articles
The slides and other materials used for lectures is made available to the students on the online moodle platform.
Lectures, using slides and video. Theoretical and practical exercises. Journal Club
1) Readiness to offer individual assistance also in English language to incoming students
2) Availability of supporting material and bibliographic references also in english language.
List:.Epigenetics. Allis, Jenuwein, Reinberg and Caparros. Cold Spring Harbour Laboratory Press. ISBN-13:978-0879697242 |Edition:1
3) Readiness to accept examination of incoming students also in a foreign language (English)