Interactions between chromatin and the transcriptional co-activators TFIID, ATAC and SAGA – ChromAct

We aim at describing the shape and the movements of molecular assemblies controlling gene expresssion. We use an electron microscope to visualize these assemblies and image analysis methods to determine their three-dimensional shape. To prevent the dehydration of the molecules, they are frozen before being introduced into the vacuum of the microscope.

To be described

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To be described

This research project aims at understanding the fundamental mechanisms of eukaryotic gene expression at the initiation step of gene transcription. The efforts of molecular biologists, structural biologists and biological chemists will be joined to explore this question in a multidisciplinary approach. Transcription of protein coding genes is regulated by coactivators; large macromolecular complexes whose action results in the assembly on the gene promoter of a preinitiation complex that contains the catalytic RNA polymerase II molecule and the general transcription factors. To activate gene expression, small activator proteins bind upstream of gene promoters to specific DNA sequences where they recruit the transcriptional coactivators. Interestingly activator binding sites may be located a few hundreds of base pairs upstream of the transcription start site or within enhancer sequences placed several thousands of base pairs away. How these long distance effects are mediated is presently poorly understood. Coactivators not only bridge activators with the preinitiation complex but also modify the chromatin organization of the gene promoters in order to give access to the transcriptional machinery and in addition read specific histone post translational modifications. Little is known on the structural organization of such large chromatin assemblies and on the complex network of interactions between activators, coactivators, chromatin and general transcription factors. Moreover evidence accumulates showing that coactivators collaborate to turn on a gene, suggesting that they play complementary roles. The exact cellular role of coactivators is poorly understood as they could participate in the long distance physical organization of the genome within the cell such as enhancer-promoter interactions or interactions between the 5’ and 3’ ends of a gene resulting in looping out of the gene body.
To answer these questions, the TFIID, SAGA and ATAC coactivators will be studied; each having specific activator, enhancer and promoter recognition characteristics. We plan to use a multi-resolution approach to analyze the molecular architecture of activated transcription initiation complexes that are formed in vitro or in vivo on nucleosome-containing DNA templates. We will combine state-of-the-art macromolecular purification methods, different high resolution electron microscopy and light microscopy imaging modalities, advanced image analysis protocols, high throughput Chromatin ImmunoPrecipitation methods as well as new antibody conjugation and delivery methods to address this biological question by an integrated structural biology approach. High resolution cryo-electron microscopy associated with single particle image analysis protocols will be used to determine structural models of the purified coactivator complexes. These models will be used as references to identify and position the coactivators in larger assemblies that will be either assembled in vitro from purified reagents or formed in vivo. The combination of Chromatin ImmunoPrecipitation and electron microscopy imaging will provide new insights in the role and promoter recognition specificity for each type of coactivator and shed new light on the local chromatin environment of coactivator-bound gene promoters as well as on long range interactions. Finally we aim at delivering antibodies coupled to electron dense particles into living cells in order to label and identify the co-activators in their cellular environment. The challenge of this part of the project is to detect specifically the coactivator complexes which are present in low copy numbers in each cell. The combined use of electron tomography and pre-embedding immunolabelling will constitute a breakthrough for many biological applications since it will permit the detection of nuclear proteins in their 3-D cellular environment at 3-4 nm resolution.