Understanding The Dynamic Role Of Co-Activator Complexes In The Regulation Of Genome-Wide Transcription In Stem Cells And Differentiated Mammalian Cells
Reference : PhD Laszlo TORA
Publication de l'offre : 6 avril 2016
The genome of eukaryotes is organized into chromatin by being wrapped around histone octamers forming nucleosomes. Nucleosomes consist of a histone H3-H4 tetramer, two histone H2A-H2B dimers and 146 base pairs of DNA. Nucleosomes can form an obstacle for the transcription initiation machinery.
Transcription regulators in eukaryotes can be divided into three functional classes: gene-specific transcription regulators, transcriptional co-activator complexes (TCCs) and the general RNA polymerase II (Pol II) transcription machinery. Their collaborative action is necessary to access specific loci in
chromatin and allow precise Pol II transcription initiation. Regulated transcription requires a highly concerted, stepwise assembly of transcription factor complexes that form the preinitiation complex, which consists of the Pol II and the corresponding general transcription factors (GTFs). TCCs make use of different mechanisms to mediate the effects of activators to the general transcription machinery. TCCs can facilitate access to DNA by post-translational modifications of histone tails, by nucleosome mobilization or by the incorporation of histone variants. Importantly, transcription regulation requires a complex interplay of sequence-specific DNA-binding factors, co-activators, Pol II and the epigenetic status of target sequences.
Distinct transcription networks determine the identity and function of different cell types with nodes consisting of key regulatory protein complexes. Acetylation of histone proteins is the most commonly known histone modification related to active transcription4. Gcn5/KAT2A represents the first described histone acetyl transferase (HAT) capable to acetylate lysines on histone proteins, mainly in their tail regions. To accomplish its full
acetylation activity, Gcn5 has to be incorporated into TCCs. The Spt-Ada-Gcn5-Acetyltransferase (SAGA) complex represents the most studied GCN5-containing TCC. Importantly, the structure and function of the eukaryotic SAGA complexes are highly conserved from yeast to human, as this complex contains about 20 conserved subunits organized in different functional modules, such as the HAT, the deubiquitination and the structural
modules. Furthermore, multicellular eukaryotes possess a second closely related Gcn5-containing TCC, the Ada Two-A containing (ATAC) complex.
The protein composition of the vertebrate ATAC complex has been identified, but little is currently known about its influence on the transcription of the metazoan genomes. In mice, disruption of genes coding for individual SAGA and/or ATAC subunits results often in embryonic phenotypes, demonstrating their importance
during development and differentiation. Moreover, several studies using different cellular model systems have demonstrated the involvement of Gcn5, Ada3 and Sgf29, common subunits of both SAGA and ATAC, in processes that can be linked to cancer.
The influence and the contribution of these TCCs on Pol II transcription of vertebrate genomes is not well-understood. Previous studies suggested that ATAC and SAGA regulate only a distinct group of genes. Recent results from our lab have shed new light on the potential dynamic genome-wide function of SAGA complexes. Considering only newly transcribed RNAs, we demonstrated that S. cerevisiae SAGA could influence the transcription initiation of all active genes.
Based on the above-described findings this PhD project will address the following questions:
1) Do the vertebrate SAGA and ATAC complexes influence the whole Pol II transcriptome?
2) Do SAGA and ATAC display redundant or specific functions in the regulation of transcription under distinct differentiation conditions?
3) Do SAGA or ATAC complexes exhibit different dynamical behaviours at target genes?
- COMPETENCES SOUHAITEES : Experience in: cell culture, biochemistry, immunoprecipitations, RNAi, RNA isolation, RT-qPCR, bioinformatics,
- EXPERTISES QUI SERONT ACQUISES AU COURS DE LA FORMATION : The PhD candidate will use/learn a combination of several very complementary approaches involving molecular biology, cutting-edge super resolution imaging, orchestrated by state of the art biophysics and bioinformatics (i.e. ChIP-seq and related bioinformatics, RNA-seq and related bioinformatics analyses, mouse embryonic stem cell culture and differentiation protocols) will be a hallmark for the success of this PhD project. Thus, the results of the proposed PhD project will have a major impact on the field and will lead to a new paradigm for contemporary metazoan transcription regulation
Date limite de candidature : 31 décembre 2016