Final 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 have systematically analysed 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. We studied 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. Our experiments were centred around the following aims:
I) Investigate the co-translation-driven assembly of transcription complexes;
II) Determine their cytoplasmic intermediates and factors required for their assembly pathways;
III) Uncover their nuclear import;
IV) Understand at the single molecule level their nuclear assembly, their dynamics and their action at target genes.
We demonstrated that mammalian nuclear transcription complexes (TFIID, SAGA and TREX-2) composed of a large number of subunits but lacking precise architectural details are built co-translationally. We demonstrated that the dimerization domains and their positions in the interacting subunits of these complexes determine the co-translational assembly pathway (simultaneous or sequential). Our results indicate that protein translation and transcription complex assembly are linked in building mammalian multisubunit complexes and suggest that co-translational assembly is a general principle in cells to avoid non-specific interactions and protein aggregation.
We employed large scale proteomics screenings with an Orbitrap type instrument using immunoprecipitations for detecting TFIID or SAGA subunit partners from nuclear and cytomplasmic human cell extracts, and detecting rearrangement of complexes (or their submodules) when one of the subunits of these complexes is knocked down.
In Aim III) we used baculo-virus overexpression and cell based assays for either overexpression or knockdown of subunits of TFIID and SAGA to detect assembly pathways of endogenous complexes,
We have developed a versatile antibody-based imaging approach (VANIMA) to be able to precisely locate and track endogenous proteins in living cells using conventional or super resolution microscopy. The labelling is achieved by the efficient and harmless delivery of fluorescent dye-conjugated antibodies, or antibody fragments (Fabs) into living cells by electroporation and the specific binding of these antibodies to the target protein inside of the cell. Then live-cell imaging permits following the labelled probes bound to their endogenous targets. By using conventional and super-resolution imaging we showed dynamic changes in the distribution of several nuclear transcription factors (i.e. RNA polymerase II or TAF10), and for the first-time specific phosphorylated histones (γH2AX), upon distinct biological stimuli at the nanometer scale. Hence, VANIMA can now be used to uncover novel biological information based on the dynamic behaviour of transcription factors or posttranslational modifications in the nuclei of single live cells.
I) Investigate the co-translation-driven assembly of transcription complexes;
II) Determine their cytoplasmic intermediates and factors required for their assembly pathways;
III) Uncover their nuclear import;
IV) Understand at the single molecule level their nuclear assembly, their dynamics and their action at target genes.
We demonstrated that mammalian nuclear transcription complexes (TFIID, SAGA and TREX-2) composed of a large number of subunits but lacking precise architectural details are built co-translationally. We demonstrated that the dimerization domains and their positions in the interacting subunits of these complexes determine the co-translational assembly pathway (simultaneous or sequential). Our results indicate that protein translation and transcription complex assembly are linked in building mammalian multisubunit complexes and suggest that co-translational assembly is a general principle in cells to avoid non-specific interactions and protein aggregation.
We employed large scale proteomics screenings with an Orbitrap type instrument using immunoprecipitations for detecting TFIID or SAGA subunit partners from nuclear and cytomplasmic human cell extracts, and detecting rearrangement of complexes (or their submodules) when one of the subunits of these complexes is knocked down.
In Aim III) we used baculo-virus overexpression and cell based assays for either overexpression or knockdown of subunits of TFIID and SAGA to detect assembly pathways of endogenous complexes,
We have developed a versatile antibody-based imaging approach (VANIMA) to be able to precisely locate and track endogenous proteins in living cells using conventional or super resolution microscopy. The labelling is achieved by the efficient and harmless delivery of fluorescent dye-conjugated antibodies, or antibody fragments (Fabs) into living cells by electroporation and the specific binding of these antibodies to the target protein inside of the cell. Then live-cell imaging permits following the labelled probes bound to their endogenous targets. By using conventional and super-resolution imaging we showed dynamic changes in the distribution of several nuclear transcription factors (i.e. RNA polymerase II or TAF10), and for the first-time specific phosphorylated histones (γH2AX), upon distinct biological stimuli at the nanometer scale. Hence, VANIMA can now be used to uncover novel biological information based on the dynamic behaviour of transcription factors or posttranslational modifications in the nuclei of single live cells.