This research proposal aims at understanding the fundamental mechanisms of human gene regulation at the initiation step of transcription. Molecular, structural and cell biologists will combined their efforts in a multidiscplinary approach to explore this central process. The enzyme that transcribes human genes into messenger RNA is RNA polymerase II. Transcription by this polymerase is regulated by a plethora of protein factors, including large and complicated machines with many subunits. The first general transcription factor (GTF) to bind gene promoters is TFIID. TFIID is a megadalton-sized multiprotein complex composed of TATA-box binding protein (TBP) and 13 TBP associated factors (TAFs). Despite its crucial role, the detailed molecular architecture and assembly mechanism of TFIID remain elusive. This is partly due to the low abundance and heterogeneity of TFIID in cells, and the difficulties associated with extracting TFIID to study its function. As a consequence, structural analysis of human holo-TFIID has been stalled for many years at moderate resolution (~35 Å) despite major efforts by some of the best laboratories world-wide. At the transcription start site, TFIID interacts with specific nucleosomes carrying defined post-translational modifications (epigenetic marks) that form the local chromatin template. Epigenetic modifications are an intense research focus, owing to the fact that they profoundly influence chromatin function, with direct impact on a large number of human diseases including cancer. TFIID contains reader domains that recognize epigenetic marks. The exact mechanism how TFIID binds to its chromatin template, and what the consequences are for the conformations of TFIID and the nucleosomes, is unknown to date. To activate gene expression, small activator proteins bind upstream of promoters, and several of these communicate with TFIID. Only low resolution data exists on the modalities of TFIID binding to activators, which are essential for properly directing gene expression. Which TAFs exactly may be involved in these interactions is not known, due to the lack of precise structural information on TAF geometries within TFIID, and the paucity of material due to the difficulties to get hold of purified TFIID. How TFIID is assembled in the cell is an unsolved mystery. Recent studies suggest that TFIID is composed of stable modules that may represent assembly intermediates. Unique transcriptional roles have been associated with these assembly intermediates. The identification of such intermediates and of assembly factors that may assist in the formation of holo-TFIID, has remained an unmet challenge. We have recently succeeded in producing, for the first time, fully recombinant, functional human holo-TFIID and its subassemblies in the quality and quantity required for structural and functional analysis. We had developed new expression methods for this purpose. In this proposal, we will exploit this break-through to determine the architecture of this essential complex by hybrid methods combining recombinant production of TFIID and its subcomplexes, cryo-EM, X-ray crystallography, cross-linking mass-spectrometry and multi-constraint modeling. Further, we will dissect the structure of TFIID bound to its epigenetically modified chromatin template, and we will elucidate the molecular determinants of selected transcriptional activators and a repressor binding to recombinant holo-TFIID. We will isolate stable TFIID assembly intermediates and non-canonical TFIID complexes directly from cells, and analyze their composition by using powerful new proteomics approaches. We will produce these assembly intermediates recombinantly and analyze their possible roles in transcription by a variety of means including protein transduction. We will identify putative assembly factors and study their activities in assisting holo-TFIID.

The ancient phylum Apicomplexa includes many of the world’s pre-eminent protozoan pathogens. Most deadly to humans is Plasmodium, the agent of malaria, which kills around a million people annually. As obligate intracellular parasites, they establish intimate interactions with their hosts. Toxoplasma gondii is an extreme example of this adaptation, able to replicate within nearly every cell type in any warm-blooded host. Not only the developmental program of this parasite in wildlife and livestock animals can result in potentially negative socio-economic impact, but remarkably, in about a third of the human population Toxoplasma also experiences prolonged quasi-silent persistence in tissues such as brain and retina. While the asymptomatic parasitism that proceeds typically offers life-long equilibrium and protection in immune competent hosts, sustained immune dysfunction is known to break parasite dormancy, promoting bradyzoite to tachyzoite transition and further T. gondii tachyzoite population expansion, these combined processes eventually resulting in encephalitis and meningitis as major damages. The strategy of T. gondii as a parasite is based on a quest for avirulence, a capacity to attenuate but not to fully counteract the immune defense of the host, thus securing the permanent residence required to await transmission. HostQuest focuses on elucidating the molecular mechanisms by which T. gondii is orchestrating immune evasion and lifelong persistence in hosts. Once intracellular, parasites actively reprogram gene expression of the immune cells they infect by subverting host transcription factors activity or by modulating the epigenetic status of target genes. Secreted effectors are involved. Those are singularly exported beyond the vacuole-containing parasites and reach the host cell nucleus to reshape the host genetic program. The discovery of new exported Toxoplasma effectors and the characterization of their activities, or the mechanisms by which they are recognized by the host immune system, continues to gather pace. Much has been learnt in recent years but we have only been chipping at the tip of the iceberg. We aim to study the modus operandi of these effectors and particularly their possible implications in immune evasion and parasite persistence. These effectors may adopt at least three alternative, although not mutually exclusive, strategies to subvert host gene expression. They may (i) modulate upstream signaling pathways (ii) directly target host transcription factor protein levels/activity and/or (iii) affect histone packing and chromatin configuration. HostQuest is an interdisciplinary project that aims to: i) Determine the full repertoire of GRA effectors and the magnitude of the changes they are eliciting in the infected cell; ii) Explore their synergistic and/or antagonist effects on gene regulation; iii) Decipher the extent to which they contribute to immune evasion and/or sustained parasitism; iv) Gain knowledge of the three-dimensional structure of effectors in complex with host cell factors in order to understand the protein function or to guide further experiments to investigate function. Studies of effectors also continue to offer opportunities for the development of tools to probe host cell biology in the absence of disease. In this respect, HostQuest is also poised to exploit Toxoplasma molecular intelligence developed over million years of co-evolution with its hosts to learn new lessons on the mechanisms regulating cell homeostasis and their alterations in host cells, including cancer cells.

The proper functioning of any living cell requires that approximately half of all proteins synthesized in the cytosol must be translocated across (in the case of exported proteins) or inserted into (in the case of membrane proteins) a cell membrane. Protein translocation at the membrane occurs through a proteinaceous channel, termed the translocon. The core of the translocon is a heterotrimeric integral membrane protein complex (SecY, SecE and SecG in eubacteria) and is conserved in all kingdoms of life. Little is known about the structure and function of the additional components of the holo-translocation machinery, SecD, SecF and YidC, which are essential for eubacteria. In spite of their central role, the function of these subunits is entirely unresolved to date. This is to a large part due to the lack of a purified, reconstituted SecYEG-DF-YidC holo-translocon, and the absence of any structural data on this large transmembrane multiprotein complex. Using a new recombineering-based vector system for expression of multi-protein complexes in E. coli, we now for the first time successfully over-expressed and purified the SecYEG-DF-YidC holo-translocon. With this purified holo-translocon in hand, we are now in a unique position to carry out a thorough functional and structural characterization of this vitally important membrane protein complex. Our project will be divided in five complementary parts: (1) We will solve the structure of the holo-translocon by cryo-electron microscopy and single particle analysis. (2) We will study co-translational translocation by solving the structure of the holo-translocon complex with a translating ribosome by cryo-electron microscopy and single particle analysis. Using the existing structures of the ribosome, SecYEG and the predicted homology of SecDF to AcrB, we will build a quasi-atomic model of the holo-translocon. (3) We will thoroughly assay the molecular level function of the holo-translocon in protein translocation, in a collaborative setup with Prof. Ian Collinson, Bristol, UK. The functional modulation of SecA-driven post-translational translocation by SecDF and the effect of the proton motive force will be investigated. (4) The stoichiometry of the subunits in the holo-translocon complex is an entirely unresolved issue to date. We will analyze the stoichiometry and the architecture of the holo-translocon by mass spectrometry, in collaboration with Prof. Carol V. Robinson, Cambridge, UK. (5) We will perform crystallization experiments of the holo-translocon for analysis by 2D electron crystallography (with Prof. Ian Collinson, Bristol) and for 3D X-ray crystallography by using the high-throughput crystallization facilities that are part of the Partnership in Structural Biology (PSB) here in Grenoble. A molecular understanding of protein export and membrane protein integration and folding will not only provide fundamental insight into a paramountly important biological process, but may also contribute significantly to the future design of novel, and urgently needed antibiotics.

Viral metagenomics is a fast evolving, new discipline emerging mainly due to the advent of next-generation sequencing techniques. Novel environmental, epidemiological, pathological samples from viral metagenomics projects are literally flooding databases with a wealth of totally novel virus sequence data. Recent metagenomic studies aiming at estimating virus prevalence and diversity in natural niches (“viromes”) have begun to cast light on what is now called a viral ‘dark matter’: viruses of outstanding genome sizes, gene/enzyme equipment, and gene organization are found ubiquitously throughout the environment, with multiple hosts, transmission-routes, and outcomes of infection, rising the legitimate question of the likelihood of future outbreaks involving novel agents. A novel viral world is being deciphered; Viral metagenomics represents an important landmark towards the detailed understanding of the biology of existing and up-coming RNA viruses. There is a caveat, though: sequence data is accumulating at a much faster pace than structural and functional data. Many viral enzyme gene and signature sequences are identified for which actual substrate, mechanism, role, regulation, plasticity, not to say crystal or Cryo-EM structure, are unknown. Immense viral sequence repositories are assembled for which individual mutations of polymorphisms have no signification yet. Entire families of major protein folds accumulate in the Protein Data Bank, but stay unannotated in major functional databanks (PFAM, UniProt, SCOP, etc...), nor have any enzyme activity nor mechanism of action assigned. This gap of knowledge is cruelly and increasingly missing when it is becomes necessary to understand eg., pathogenicity, tissue specificity, virulence determinants, drug resistance, genome evolution under selective pressure, or complex relationships of gene transfer between phyla. Consequently, a new field of science must emerge which, in characterizing this enormous wealth of virus data, would take advantage of novel bio-resources brought by RNA viromes, better integrate virus-related information in biological research at large, and also achieve scientific preparedness towards the control of viral diseases threatening the world. One reason contributing to the deepening gap appearing between big metagenomic data and its translation into useful and accessible knowledge is the current shortage of qualified scientists able to master the whole knowledge string, from massive raw sequencing data to its documented, publicly available form. Therefore, a new highly qualified expertise is in demand, which includes well trained scientists able to i) master virus hunting and discovery through biological and metagenomic techniques, ii) analyze and annotate in depth viral sequences by means of rapidly evolving software and the most upstream state-of-the-art gene/function prediction using, eg., artificial intelligence, and iii) discover, validate and refine enzyme activities through a deep understanding of what sequence/structure/activity and function means. This in turn would considerably aid to inform major databases that increasingly permeate the decision-making process in, eg., environmental policies, vaccine-, drug-design, and patient care. The project aims at training PhD students in Integrated Viral Metagenomics, for clinically relevant viruses but also environmental and biodiverse viruses, from which endless discoveries and understanding will have a great impact on the economies and sustainabilities of our societies. Trainees will master virus discovery, metagenomics and database/biobank handling/mining, and structural/functional analysis of viral enzymes of evolutionary, clinical and biotechnological interest. A special training on the integration of these fast-evolving scientific disciplines will be made so trainees become highly aware of market, societal, environmental and ethical issues associated with their future career.