The genomic DNA of all organisms must be condensed to fit within the confines of cells that are typically several thousand-fold smaller than the DNA itself. Compaction of bacterial chromosomes results in the formation of a structure called the nucleoid. Recent studies in E. coli have revealed a multilayered organization that includes long-range chromosome condensation based on large distinct regions termed macrodomains. There are four MDs (Ori, Right, Left, and Ter), which can be classified as separately organized portions of the nucleoid enclosed within cages of about 0.2 µm in diameter. Each of these domains contains approximately 1 Mbp of DNA. The Ori domain contains oriC. The Ter domain contains the replication termination site. The Left and Right MDs are adjacent to Ter and are separated from the Ori MD by two so-called nonstructured regions. Ongoing studies performed in the host laboratory have revealed that different strategies have been selected for structuring MDs. While the structuring of the Ori MD relies on a unique organizing centre, the organization of Ter, Left and Right MDs involves multiple specific determinants dispersed within the domains. In recent years, we revealed the molecular bases for structuring the Ter MD: the binding of the MatP protein to a 13 bp motif called matS repeated 23 times in the 800 kb long region is required to condense DNA and control the segregation of Ter MD. By performing a structure-function analysis of MatP, we further identified the molecular bases for MatP-mediated Ter condensation and MD formation. MatP contains a newly described tripartite fold that includes a four-helix bundle, a ribbon-helix-helix (RHH) domain and a C-terminal coiled-coil. While the four-helix bundle contains the DNA binding domain and the RHH the determinants for the formation of MatP dimer, the coiled-coils of MatP dimers form a bridged tetramer that flexibly link distant matS sites. Mutating the residues involved in the tetramerization affected DNA condensation within the Ter MD. We thus revealed for the first time a protein-mediated DNA-looping mechanism leading to chromosome condensation in bacteria. Studies showed that chromosome choreography and segregation are tightly controlled during the cell cycle and the MDs have defined positions in the cell throughout the cell cycle. Our results showed that the structuring of the Ter MD is important to ensure faithful chromosome distribution to daughter cells as MatP inactivation leads to severe defects in chromosome segregation. The persistent localization of the Ter MD at mid-cell before cell division relies on the specific interaction of MatP with the protein ZapB associated to the division apparatus. Remarkably, the anchoring of the Ter region to the division apparatus induces a constraining process that can spread in cis to the rest of the chromosome. A site-specific insulation involving 2 insulators flanking the Ter MD has been selected to restrict to the Ter region the consequences of anchoring the Ter MD to the divisome. These results demonstrated a tight coupling between chromosome organization and cell division in E. coli. The present project has three specific aims. Firstly we wish to characterize the in vivo organization of the Ter MD by analyzing the distribution of MatP molecules in the cell, by unveiling the spatial arrangement of the Ter MD DNA, by studying the consequences of altering MatP or matS sites distribution and by identifying the parameters required for the formation of the Ter MD. Secondly, we want to characterize the molecular bases responsible for the structuring of the Ori, Left and Right MDs by identifying the factors and the associated molecular mechanisms. A number of candidates which inactivation affects MD properties have already been identified. Finally, we want to characterize the role of MDs in chromosome management and study how these processes are integrated in the control of the cell cycle.

Transcription termination is essential, not only to allow the correct ending of functional transcripts but also to prevent interference between adjacent regions of transcription and between transcription and other cellular events such as replication or telomere maintenance. This is even more critical in a compact genome as that of S. cerevisiae. Termination is also one of the main strategies that the cell employs to control and limit the extent of pervasive/hidden transcription. In yeast, the main share of hidden transcripts is constituted by CUTs (Cryptic Unstable Transcripts) that we have contributed to discover and the transcription of which is terminated by the Nrd1 complex. In this proposal we aim at studying two aspects of transcription termination: i) the study of the mechanism of Nrd1-dependent transcription termination and ii) the analysis of a novel pathway of transcription termination that we have recently discovered. First we will exploit the biochemical tools that we set up as part of a previous ANR project. We have succeeded in obtaining transcription termination in vitro with purified RNAPII and Sen1p, a key component of the Nrd1 complex. We will explore the requirements for termination using essentially biochemical approaches with purified wild type and mutant Nrd1 complex components. In a second task, also derived from a previous ANR project, we will study the mechanism and functional significance of transcription termination induced by DNA-binding proteins, road-block termination. We have already shown that the DNA binding protein Reb1p (and possibly other proteins containing a similar DNA binding domain) can induce transcription termination in vivo, and have described the features that distinguish this pathway from the more “canonical” termination pathways. We will further study the mechanism and explore the genomewide distribution of road-block termination with an approach designed to specifically identify RNAPII pausing sites that are resolved by ubiquitination of the large subunit and proteasome degradation. Finally, we will investigate if road-block termination can be observed with other DNA-binding factors of the same family. We believe that this is an ambitious but realistic project in the light of the many, exciting preliminary results already obtained. These studies are expected to further our understanding of the control of pervasive transcription in yeast and possibly other organisms.

EAT (Embryonic AuTophagy) is a fundamental research project aiming in understanding the mechanism and function of selective autophagy during embryonic development. Autophagy is a general term for the degradation of cytoplasmic components within lysosomes. It is the major ubiquitous catabolic process which allows the bulk degradation of cytoplasmic constituents, generally by non selective sequestration during the formation of double membrane vesicles, the autophagosomes. However, recent data have identified that autophagy could also be a selective degradative process. Numerous studies have highlighted the large variety of physiological and pathophysiological roles of autophagy such as cell death, anti-aging, antigen presentation, elimination of microorganisms and tumor suppression. In addition, autophagy appears also to play a critical role in the remodeling of tissues during development. Whereas many studies in yeast and vertebrate cells in culture contributed to define the main steps of the autophagy pathway, studies in metazoans are critical to decipher its functions in development, ageing and reproduction as well as how regulation and specificity is achieved. EAT will be focused on the mechanisms and functions of selective autophagy during embryonic development and take advantage of C. elegans as simple animal model system. One main challenge in the field is to understand the function of the duplication of factors involved in autophagy. Our project is centered on the analysis of LGG-1 and LGG-2 proteins, the two homologues of the single Atg8 protein found in S. cerevisiae, in the formation, the maturation and the degradative function of autophagosomes. Having only two Atg8 homologues in C. elegans (seven in human) represents a simple configuration that will allow us to reveal the functional relevance of this duplication. Understanding the respective functions of LGG-1 and LGG-2 in the specificity, formation and maturation of the autophagosmes is essential but difficult to uncouple from the function of the autophagosomes themselves. Therefore, the mechanistic and functional implications of the two proteins will be conducted in parallel and at two different embryonic developmental stages. The EAT project is divided in three tasks and involves two complementary teams. Both teams will join their effort in the task devoted to the mechanistic analysis of formation and dynamics of LGG-1 and LGG-2 positive autophagosomes using a combination of cell biology, genetics and biochemical approaches. This task will be complemented and extended with the functional analysis of their respective roles in two specific developmental stages, in which we know they are strongly expressed. Each partner is an expert in one of this developmental stage and will be responsible for the task centered on the functional analysis of autophagy at this stage. The first stage is just after fertilization, with the autophagy of sperm-inherited components (allophagy). The identification of the mechanism(s) insuring specific paternal mitochondria degradation should in turn allow us to measure the impact of their stabilization on the progeny. This will be the first experimental system for testing the consequences of heteroplasmy establishment arising from the paternal mtDNA contribution. We will also test whether allophagy is conserved in the mouse embryo. The second one is during organogenesis and cell differentiation and polarity establishment in epidermis and intestine. The functional relevance of the enrichment of autophagy markers in the germ-cells precursors will be also analyzed. In conclusion, with the synergistic and rational approach proposed in the EAT project we expect to be able to draw general conclusions that could improve our understanding of other physiological and pathophysiological autophagy processes involving Atg8 homologous proteins across species.

Small non-coding RNAs make up a large family of ubiquitous regulators active in all domains of life. In higher eukaryotes, microRNAs participate in processes related to development, aging and disease. In prokaryotes, small RNAs (sRNAs) regulate mechanisms that promote adaptation to environmental changes and stress conditions. The largest class of bacterial sRNA regulators acts post-transcriptionally to affect translation or stability of target mRNAs. Due to their ability to regulate multiple targets at once, some sRNAs occupy nodal positions in regulatory networks and play key roles in rewiring gene expression in response to environmental changes. The present proposal originates from our discovery of a novel mechanism of sRNA-mediated regulation. Investigating the ability of a particular sRNA to downregulate the distal portion of a bicistronic operon in Salmonella enterica, we found that the sRNA acts by inducing Rho-dependent transcription termination within the proximal portion of the operon. By outcompeting the ribosome for binding to the nascent mRNA, the sRNA uncouples transcription and translation making the elongation complex susceptible to Rho-dependent termination. These findings have interesting implications and raise a number of questions: how general is this mechanism? Does it contribute to the coordinate repression of genes in polycistronic mRNAs? Are there specific features or players required for Rho recruitment by sRNA? Can there be situations where the sRNA competes with Rho for RNA binding? Answering these questions is the objective of the present proposal. A major part of the project aims at identifying additional examples of sRNA-regulated mechanisms involving a Rho-dependent step. Preliminary tests on loci tentatively identified by informed predictions are giving very encouraging results. Rho involvement in the regulation of these genes will be characterized in detail, by mutational approaches, by in vivo functional analysis and in vitro transcription and biochemical assays. In parallel, we will undertake a systematic, genome-wide approach to obtain a global view of the impact of Rho-dependent termination in sRNA-mediated regulation in Salmonella. This analysis, by high throughput RNA sequencing (RNA-seq) and cross-linking/immunoprecipitation (CLIP-seq) methodologies, will be specially designed to assess both positive and negative responses to Rho inhibition, and the participation of relevant players, notably Hfq. The new loci identified by this approach will also be characterized in detail in vivo and in vitro. In parallel with the above experiments, we will further dissect the mechanism of sRNA-induced Rho-dependent termination at the locus where it was initially uncovered, the chiPQ operon. At this site, we have established the participation of elongation effector NusG in the termination mechanism and plan to characterize its role in detail, using genetic and biochemical approaches. The involvement of other effectors of transcription-translation coupling, namely NusA and ribosomal protein S10 (NusE), will be explored as well. Finally, using chromatin immunoprecipitation (ChIP) techniques, we will probe the composition of the elongation complex as it moves along the chiP gene under conditions where chiPQ mRNA translation is unaffected, repressed by an sRNA, or prematurely terminated at a stop codon. We expect to obtain some fresh insight on basic features of prokaryotic transcription as well as on transcription-translation coupling. The past two years have seen a renewed interest in Rho-dependent termination, as new roles for Rho terminator are being uncovered. Our work has contributed to these emerging novel functions and the proposed research is expected to further increase this contribution by providing an exhaustive picture of the interplay of sRNA- and Rho-mediated activities. As an added bonus, results will generate the first catalog of Rho-dependent termination sites in the model pathogen Salmonella.

Recent developments in epigenetics suggest the widespread use of small-RNA pathways as a genomic immune system allowing eukaryotes to control molecular parasites such as transposable elements (TEs). Small RNAs have also been shown to mediate non-Mendelian inheritance in divergent species, resurrecting the Lamarckian idea of inheritance of acquired traits. To understand the basic biological logic that may underlie the connection between these themes, and identify fundamental aspects despite the confounding diversity of mechanisms uncovered in different phyla, we propose to address this issue from a phylogenetically unique angle. The ciliate Paramecium tetraurelia – at an equal evolutionary distance from plants and animals – was one of the first organisms in which transgenerational epigenetic inheritance was clearly demonstrated, and recent molecular studies have shown that many cases can be explained by the role of a meiosis-specific class of small RNAs, the scnRNAs, in epigenetic regulation of the genome rearrangements that occur during the development of the somatic macronucleus (MAC) from the germline micronucleus (MIC), in each sexual generation. Rearrangements include the elimination of all TEs as well as the precise excision of ~45.000 single-copy Internal Eliminated Sequences (IESs), which are believed to be degenerate remnants of ancient TE insertions. MIC centromeres may also be eliminated since they are not active in the MAC, which divides by a non-mitotic mechanism. During meiosis, scnRNAs are produced from the entire MIC genome and mediate a genome-wide comparison of germline and somatic sequences, allowing the zygotic MAC to eliminate any germline sequence not present in the maternal MAC. The system has probably evolved as an efficient mechanism to detect any new TE insertion in the germline, and has further been co-opted to ensure the non-Mendelian inheritance of essential phenotypic polymorphisms, such as mating types. But the mechanism should also in principle make any new insertion potentially lethal if it is introduced by conjugation into a naive cell - an effect that is reminiscent of hybrid dysgenesis in Drosophila, the devastating effects of TEs introduced by the male gamete when the mother cannot produce homologous piRNAs to repress transposition. By comparing the IES content of paralogous gene pairs of different ages derived from 3 successive whole-genome duplications, we recently obtained evidence that a substantial fraction of IESs have been acquired since the last duplication. This is the time when the Paramecium ancestor underwent numerous speciation events, resulting in a group of 15 sibling species that are morphologically indistinguishable but sexually incompatible. This raises the possibility that the scnRNA-based mechanism for recognition and elimination of TEs, which decreases the burden on host fitness and allows them to persist in the genome and degenerate into single-copy IESs, has been a major force driving speciation. The main objectives of this project are to reconstruct the evolutionary history of IESs and other MIC specific sequences such as TEs and centromeres, and to experimentally test the hypothesis that polymorphisms in these elements result in sexual incompatibility. We propose to (i) sequence and assemble the entire MIC genome of P. tetraurelia, since very little is currently known about the diversity and copy number of TEs; (ii) explore further the scnRNA pathway mechanism, by testing the role of known protein factors in the recognition of the genome-wide set of ~45,000 IESs, by identifying new ones, and by deep-sequencing scnRNA populations during sexual events to describe their dynamics; (iii) identify IESs and centromeres in different strains and species to study their evolution; and (iv) experimentally determine the effects of IES polymorphisms (presence/absence, divergence of sequences) on genome rearrangements in sexual progeny of interstrain or interspecies crosses.