Themes
The aim of this two-week course for M2 students is to present gene expression as a dynamic and flexible system, through the influence of not only (1) environmental factors, but also (2) the cellular neighborhood during development.
1) The course progresses from understanding the general mechanisms of gene expression within the nucleus to the integration of these phenomena in an eukaryotic cell. This study helps to clarify the nature of the coordinated mechanisms set up by the cell in order to respond rapidly to variations in the environment (variations in temperature or pH, deficiencies..).
2) Like isolated cells, which are capable of modifying the expression of their genes, the cells of a future pluricellular organism "respond" to external signals. These signals, however, do not come from the "external environment" but rather from their immediate cellular neighborhood, not merely during the early stages of development due to their maternal inheritance, but also during later stages when they implement structures that give rise to a given organization plan. The course therefore endeavours to show that pluricellular organisms (Metazoans) develop according to a precise programme that requires differential and selective expression of genes. Some genes are inhibited, others are activated, and this sequence of events is not (or only very slightly) modulated by the external environment.
The course is divided into 3 parts: "The Actors of Gene Expression ", " Influence of the External Environment " and " Influence of the Cellular Neighborhood ".
PART 1:
THE ACTORS OF GENE EXPRESSION
Study of gene expression in cell cultures
In a cellular context in vitro, the molecules and structures at the basis of gene expression can be observed indirectly by analyzing the reshaping of chromatin.
The objectives are therefore:
1) To show that chromatin constitutes a dynamic system and that histones, through their relationship with DNA, intervene in the regulation of gene transcription.
2) To analyze the consequences of the expression of genes according to the subcellular localization and quantity of their products.
Theoretical aspects (lectures)
Chromatin : organization, structure and dynamics of chromatin, mechanisms of action, transcriptional regulation, stochastic aspects of the molecular organization.
Proteomics: characterization of the variations in the production of vast groups of proteins (proteome), identification and analysis of proteins by mass spectrometry (MALDI-TOF and ESI), functional analysis (SELDI-TOF).
Imaging: modern techniques of cellular imaging, video microscopy, scanning confocal microscopy, spinning-disc confocal microscopy, multiphoton multifocal microscopy (TriMscope), fluorescence resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging (FLIM).
Practical workshop (TPs)
1. An H3-GFP fusion gene, under the control of an inducible promoter, is the tool used to analyse the organization and the dynamics of chromatin in the living cell. It is possible 1) to obtain a nuclear cartography of the transcriptionally active regions of chromatin (by using the histone variant H3.3), 2) to observe the modifications of this cartography during the cell cycle and during gene activation, 3) to measure the dynamics of nucleosomal proteins by combining FRAP and FRET measurements between the histone fused to the GFP and the DNA stained with ethidium bromide or having incorporated fluorescent nucleotides, 4) to measure by immunoprecipitation of chromatin the incorporation of histones by genes during the course of transcription.
2. A transcriptional analysis led in parallel consists of a qualitative and quantitative evaluation of proteins expressed after stimulation of cell lines. These variations are analyzed by 2D-electrophoresis and mass spectrometry.
PART 2 :
INFLUENCE OF THE EXTERNAL ENVIRONMENT
Modifications of gene expression in the yeast Saccharomyces cerevisiae
Unicellular organisms respond to changes in their environment by modifying the expression of their genes, which leads in certain cases to phenotypic alterations.
The objectives are therefore:
1) to highlight the various stages of perception of a change in environment leading to the modification of gene expression.
2) to understand how the regulation of gene expression constitutes a response to this change.
Theoretical aspects (Lectures)
Study of simultaneous adaptations by transcriptional regulation and modification of intracellular traffic in the yeast S. cerevisiae. S. cerevisiae contains a large number of genes coding for plasma membrane transporters and can respond very quickly to environmental changes by the appearance or disappearance of transporters (e.g. sugars or amino acids) at the cell surface. This modulation occurs according to a double regulation: a transcriptional regulation and a regulation of the intracellular traffic of these transporters, when their presence at the plasma membrane is no longer necessary.
Practical workshop (TPs)
A gene coding the uracil permease (Fur4p), fused with the GFP and under the control of a promoter that is inducible by galactose serves as a model to monitor in vivo the intracellular traffic of a plasma membrane transporter. Both the delivery to the plasma membrane of the uracil permease and its endocytosis are very strictly regulated. Examples of this regulation are illustrated throughout the workshop.
PART 3:
INFLUENCE OF THE CELLULAR NEIGHBORHOOD
Study of gene expression during the development of a pluricellular organism
The morphogenesis of organisms is a continuous event whose information is passed down through the generations from the moment oogenesis takes place. A wide range of informative elements are involved, because not only transcription factors but also signalling proteins are concerned.
The objectives are therefore:
1) To highlight, through examples of the formation of the dorso-ventral axis in amphibians, and of the antero-posterior axis and laterality in mouse, the various strategies that are used by vertebrates to establish the first heterogeneities in gene expression - heterogeneities that are at the origin of the tissular and morphological differentiation of the embryo.
2) To study the notion of the establishment of polarity in the embryo.
3) To apprehend the diversity of representation of a signalling pathway.
4) To comprehend the notion of a regulatory network that connects the signalling pathways to one another.
Example 1: Role of maternal information in the control of gene expression during the formation of the dorso-ventral axis in the amphibian
Theoretical aspects (Lectures)
Study of the role of the transcription factor of VegT (Vegetal cortex T-box protein) during the first zygotic transcriptions in the amphibian.
Analysis of Nodal targets and induction of the mesoderm.
Presentation of ß-catenin, a crucial element for polarity of the embryo.
Practical workshop (TPs)
Distinction between maternal and zygotic information by means of GFP expression in Xenopus laevis embryos. The gene coding GFP is microinjected in the form of either mRNA or cDNA under the control of the CMV promoter. The period of protein expression is monitored during various stages of embryonic development by GFP fluorescence.
Example 2: Regulation of gene expression during development of the mouse: example of the gene Nodal
Theoretical aspects (Lectures)
Historical overview of the study of Nodal in the mouse: its gene expression regulators as an example of fine regulation of a developmental gene.
Practical workshop (TPs)
Observation of embryos of transgenic mice, 6 to 8 days post-coïtum, expressing the LacZ gene under the control of various transcriptional regulators of Nodal. Determination of the inserted transgene according to the expression profile observed.
Last modified 03/14/2011