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BIRTOACTION Report Summary

Project ID: 340551
Funded under: FP7-IDEAS-ERC
Country: France

Mid-Term Report Summary - BIRTOACTION (From birth to action: regulation of gene expression through transcription complex biogenesis)

Transcriptional regulation of protein coding genes in eukaryotic cells requires a complex interplay of sequence-specific DNA-binding factors, co-activators, general transcription factors (GTFs), RNA polymerase II (Pol II) and a given epigenetic status of target sequences. The majority of these nuclear transcription complexes function as large multiprotein assemblies and are often composed of functional modules. Several experiments suggest that an elaborate and regulated decision-making exists in cells governing the assembly and the allocation of factors to different transcription complexes to regulate distinct gene expression pathways. However, despite intensive studies on the subunit composition, the structure and the regulation of the activities of transcription complexes, very little is known about their biogenesis. To tackle this fundamental yet understudied question, we started to systematically analyse the regulated biogenesis of transcription complexes from their sites of translation in the cytoplasm, through their assembly intermediates and nuclear import, to their site of action in the nucleus. At present we started to study two types of key nuclear multiprotein complexes: i) one GTF (TFIID) and ii) one chromatin remodelling coactivator complex (SAGA), because we already know a lot about their structural, modular and functional organization. We have already made considerable advance in aims I and II:
I) Investigate their co-translation-driven assembly
The experiments show that for the co-translational assembly of transcription complexes, protein A with its interaction domain has to be fully synthetized, and thus can bind to factor B during its protein synthesis in the cytoplasm. Thus, we have discovered a fundamentally novel mechanism in transcription factor biogenesis. A manuscript presenting these data should be submitted early 2017.
II) Determine their cytoplasmic intermediates and factors required for assembly pathways
Our mass spec experiments combined with cellular approaches show that transcription factor complexes assemble and can be captured in the cytoplasm as building blocks (or functional modules) (see our Trowitzsch et al. Nature Communications 2015 paper), or as entirely assembled complexes. In contrast a third category of complexes cannot be captured from the cytoplasm, because of their extremely efficient nuclear import. These results show that the dynamics, nuclear import and the assembly pathways of these distinct transcription complexes are differentially regulated. A manuscript presenting these data should be submitted early 2017.
We have begun to analyse how the nuclear import of these complexes is regulated (Aim III).
To understand at the single molecule level the nuclear assembly and dynamics of transcription complexes we have started to use 3D-structured illumination microscopy (3D-SIM) super resolution technique (Aim IV). This technique allows the multicolour detection of labelled transcription factors in the 3D volume of the nucleus at 100 nm resolution either in fixed or live cells. We are also developing a labelled antibody-based tools (in collaboration with E. Weiss, ESBS, Illkirch, France) that can be delivered with a nearly 100% efficiency in nuclei of cells to visualize and track transcription processes in vivo. The comparison of the data sets obtained under different cellular transcription conditions will demonstrate how the assembly and integrity of the studied complexes is regulated during Pol II transcription.
In conclusion, we are on the way of successfully completing the aims of our ERC project, called Birtoaction. We are now refining and putting these results together for several visible publications. Next, we will push forward our efforts to investigate further how the cytoplasmic transcription (sub)complexes enter the into the nucleus and how they assemble in nucleus. Most certainly, the combination of several very complementary and cutting edge approaches involving molecular biology, super resolution imaging, mass spectrometry analyses orchestrated by state of the art biophysics and bioinformatics will be the hallmark for the success of our project. Thus, we anticipate that the results of our research will have a major impact on the field and will lead to a new paradigm for contemporary metazoan transcription regulation.

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