François Képès

Topological and causal structure of the yeast transcriptional regulatory network.

Interpretation of high-throughput biological data requires a knowledge of the design principles underlying the networks that sustain cellular functions. Of particular importance is the genetic network, a set of genes that interact through directed transcriptional regulation. Genes that exert a regulatory role encode dedicated transcription factors (hereafter referred to as regulating proteins) that can bind to specific DNA control regions of regulated genes to activate or inhibit their transcription. Regulated genes may themselves act in a regulatory manner, in which case they participate in a causal pathway. Looping pathways form feedback circuits. Because a gene can have several connections, circuits and pathways may crosslink and thus represent connected components. We have created a graph of 909 genetically or biochemically established interactions among 491 yeast genes. The number of regulating proteins per regulated gene has a narrow distribution with an exponential decay. The number of regulated genes per regulating protein has a broader distribution with a decay resembling a power law. Assuming in computer-generated graphs that gene connections fulfill these distributions but are otherwise random, the local clustering of connections and the number of short feedback circuits are largely underestimated. This deviation from randomness probably reflects functional constraints that include biosynthetic cost, response delay and differentiative and homeostatic regulation.

Periodic epi-organization of the yeast genome revealed by the distribution of promoter sites.

The organization of transcription within the eukaryotic nucleus may be expected to both depend on and determine the structure of the chromosomes. This study shows that, in yeast, genes that are controlled by the same sequence-specific transcription factor tend to be regularly spaced along the chromosome arms; a similar period characterizes the spacing of origins of replication, although periodicity is less pronounced. The same period is found for most transcription factors within a chromosome arm. However, different periods are observed for different chromosome arms, making it unlikely that periodicity is caused by dedicated scaffolding proteins. Such regularities are consistent with a genome-wide loop model of chromosomes, in which coregulated genes tend to dynamically colocalize in 3D. This colocalization may also involve co-regulated genes belonging to different chromosomes, as suggested by partial conservation of the respective positioning of different transcription factors around the loops. Thus, binding at genuine regulatory sites on DNA would be optimized by locally increasing the concentration of multimeric transcription factors. In this model, self-organization of transcriptional initiation plays a major role in the functional nuclear architecture.

Effects of brefeldin A and nordihydroguaiaretic acid on endomembrane dynamics and lipid synthesis in plant cells

Effects of brefeldin A (BFA) and nordihydroguaiaretic acid (NDGA) on endomembrane structures and lipid synthesis were compared in maize root cells and tobacco Bright Yellow‐2 cells. Immunofluorescence and electron microscopy studies showed that NDGA altered the structure and distribution of the endoplasmic reticulum (ER) within 1 h but not of the Golgi apparatus whereas, as shown previously, BFA altered that organization of the Golgi apparatus and, only subsequently, of the ER. Biochemical studies revealed that both drugs and especially BFA led to a strong inhibition of the phytosterol biosynthetic pathway: BFA led to accumulation of sterol precursors. The importance of phytosterols in membrane architecture and membrane trafficking is discussed.

Characterization of a putative type IV aminophospholipid transporter P-type ATPase

The P-type ATPases comprise a well-studied family of proteins involved in the active transport of charged substrates across biological membranes. Starting from a mouse bone marrow-derived macrophage cDNA library and using a signal peptide trapping strategy, we identified a new P-type ATPase family member. We characterized the genomic structure of this gene, named Atp10d, as well as its human counterpart. The presence of P-type ATPase consensus motifs and phylogenetic analysis showed that this gene is a member of the type IV, putative amphipath transporters subfamily. We showed that this gene is expressed in kidney and placenta. We also found that the C57BL/6 strain carries a constitutive stop codon in the sequence of Atp10d exon 12, whereas 14 other inbred mouse strains show an uninterrupted reading frame at this location. This mutation in C57BL/6 should lead to a non-functional protein, suggesting that this gene may not be essential. We discuss the involvement of the Atp10d gene in the fat-prone phenotype of the C57BL/6 strain and its physical mapping within a QTL associated with HDL-cholesterol levels.n

Transcription-Based Solenoidal Model of Chromosomes

The organization of transcription within the eukaryotic nucleus or the prokaryotic nucleoid may be expected to both depend on and determine the structure of the chromosomes. In yeast and bacteria, genes that are controlled by the same sequence-specific transcription factor tend to be either clustered or regularly spaced along the chromosomes. The same spacing is found for most transcription factors within a chromosome. Furthermore, in bacteria, the gene encoding the transcription factor tends to locate at identical regular intervals from its targets. This periodicity is consistent with a solenoidal epi-organization of the chromosome, which would dynamically gather the interacting partners into foci. Binding at genuine regulatory sites on DNA would thus be optimized by locally increasing the concentration of transcription factors and their binding sites. As many transcription factors are simultaneously active and some share targets, the resulting collection of foci provides a potent self-organizational principle for the chromosome, and consequently for the functional nuclear architecture.

Secretory compartments as instances of dynamic self-evolving structures.

Biological objects are often “constructive dynamic systems” whose structures evolve as a consequence of their internal dynamics, which in turn is affected by the overall structure. As very few tools are presently adapted to tackle constructive dynamic systems, they constitute fascinating challenges for modeling/simulation. In cell biology, the secretory process in eukaryotic cells corresponds to this type of system, as it appears to autonomously generate new structures as a result of its molecular dynamics. Here I briefly review the only documented case of a membrane-bounded intracellular compartment whose very existence strictly depends on its continued functioning. Indeed, the Golgi apparatus of the yeast Saccharomyces cerevisiae appears at steady-state as a continuously renewed set of transitory membrane-bounded structures that self-mature, rather than as a permanent entity. On the basis of this case and of recent advances in related molecular studies, a detailed model is proposed, that encompasses the birth of a yeast Golgi element and bridges its molecular and morphogenetic aspects. This model is extended to briefly outline three evolutionary “inventions”, from S. cerevisiae to another yeast, Pichia pastoris, on to plant, and on to animal cells: stacking, stabilizing and aggregating the primary Golgi elements.

The PAUSE software for analysis of translational control over protein targeting: application to E. nidulans membrane proteins.

The PAUSE software has been developed as a new tool to study translational control over protein targeting. This makes it possible to correlate the position of clusters of rare codons in a gene, predicted to cause a translational pause, with the position of hydrophobic stretches in the encoded protein, predicted to span a membrane or to act as a cleavable signal for targeting to the secretory pathway. Furthermore, this software gathers these correlations over whole sets of genes. The PAUSE software is described here, and its use is illustrated on a set of membrane proteins from the fungus Emericella nidulans. Preferential distances of about 45 codons and of about 70 codons between putative transmembrane domains and predicted translational pauses were observed. Given that approximately 30 residues are required to span the large ribosomal subunit, the predicted pauses would therefore occur when the hydrophobic domain starts protruding from the ribosome (+45 pause), or fully protrudes as a hairpin (+70 pause). Thus, these specific pauses might reflect a translational control over membrane protein targeting or early recognition (+45 pause), and over insertion or folding (+70 pause).

Boosting binding sites prediction using gene's positions

Understanding transcriptional regulation requires a reliable identification of the DNA binding sites that are recognized by each transcription factor (TF). Building an accurate bioinformatic model of TF-DNA binding is an essential step to differentiate true binding targets from spurious ones. Conventional approches of binding site prediction are based on the notion of consensus sequences. They are formalized by the so-called position-specific weight matrices (PWM) and rely on the statistical analysis of DNA sequence of known binding sites. To improve these techniques, we propose to use genome organization knowledge about the optimal positioning of co-regulated genes along the whole chromosome. For this purpose, we use learning machine approaches to optimally combine sequence information with positioning information. We present a new learning algorithm called PreCisIon, which relies on a TF binding classifier that optimally combines a set of PWMs and chrommosal position based classifiers. This non-linear binding decision rule drastically reduces the rate of false positives so that PRECISION consistently outperforms sequence-based methods. This is shown by implementing a cross-validation analysis in two model organisms: Escherichia coli and Bacillus Subtilis. The analysis is based on the identification of binding sites for 24 TFs; PRECISION achieved on average an AUC (aera under the curve) of 70% and 60%, a sensitivity of 80% and 70%, and a specificity of 60% and 56% for B. subtilis and E. coli, respectively.

Membrane traffic in virtual reality.

Many of the past and present open questions in cell biology are inspired by morphological observations (1). An inadequate observation may spark years of misguided or misinterpreted research at the molecular level. It is therefore of utmost importance to provide correct structural descriptions of the biological objects and of their interactions. While it has become easier to visualize the cell in three dimensions with the advent of confocal light microscopes, many cell biological controversies cannot be solved without the spatial resolution afforded by the electron microscope (EM).

Morphodynamics of the yeast Golgi apparatus

Using metallic impregnation, Camillo Golgi (1898) discovered in the cytoplasm of nerve ganglion cells a new cell organelle that formed an extensive perinuclear network : the “appareil reticulaire interne”. With the empirical and non-specific methods used for its demonstration, the “Golgi apparatus” was not easily detected in living cells (reviews in Beams and Kessell (1968), Whaleyand Dauwalder(1979), Farquharand Palade (1981) Mollenhauer and Morre (1991), Berger (1997)). It was the merit of Dalton and Felix (1954) to demonstrate with the electron microscope that the Golgi apparatus was not an artefact. It appeared instead as a system of stacks of closely apposed lamellae or saccules usually encountered in the juxtanuclear area of mammalian cells. With the improvement of the fixation and staining techniques, it became clear that an organelle made up of stacked flattened saccules (cisternae) and vesicles could be seen in most cells.