Structural Kinetic Modeling of Metabolic Networks

To develop and investigate detailed mathematical models of cellular metabolic processes is one of the primary challenges in systems biology. However, despite considerable advance in the topological analysis of metabolic networks, explicit kinetic modeling based on differential equations is still often severely hampered by inadequate knowledge of the enzyme-kinetic rate laws and their associated parameter values. Here we propose a method that aims to give a detailed and quantitative account of the dynamical capabilities of metabolic systems, without requiring any explicit information about the particular functional form of the rate equations. Our approach is based on constructing a local linear model at each point in parameter space, such that each element of the model is either directly experimentally accessible, or amenable to a straightforward biochemical interpretation. This ensemble of local linear models, encompassing all possible explicit kinetic models, then allows for a systematic statistical exploration of the comprehensive parameter space. The method is applied to two paradigmatic examples: The glycolytic pathway of yeast and a realistic-scale representation of the photosynthetic Calvin cycle.Comment: 14 pages, 8 figures (color

Atrazine-Induced Aromatase Expression Is SF-1 Dependent: Implications for Endocrine Disruption in Wildlife and Reproductive Cancers in Humans

BACKGROUND: Atrazine is a potent endocrine disruptor that increases aromatase expression in some human cancer cell lines. The mechanism involves the inhibition of phosphodiesterase and subsequent elevation of cAMP. METHODS: We compared steroidogenic factor 1 (SF-1) expression in atrazine responsive and non-responsive cell lines and transfected SF-1 into nonresponsive cell lines to assess SF-1’s role in atrazine-induced aromatase. We used a luciferase reporter driven by the SF-1–dependent aromatase promoter (ArPII) to examine activation of this promoter by atrazine and the related simazine. We mutated the SF-1 binding site to confirm the role of SF-1. We also examined effects of 55 other chemicals. Finally, we examined the ability of atrazine and simazine to bind to SF-1 and enhance SF-1 binding to ArPII. RESULTS: Atrazine-responsive adrenal carcinoma cells (H295R) expressed 54 times more SF-1 than nonresponsive ovarian granulosa KGN cells. Exogenous SF-1 conveyed atrazine-responsiveness to otherwise nonresponsive KGN and NIH/3T3 cells. Atrazine induced binding of SF-1 to chromatin and mutation of the SF-1 binding site in ArPII eliminated SF-1 binding and atrazine-responsiveness in H295R cells. Out of 55 chemicals examined, only atrazine, simazine, and benzopyrene induced luciferase via ArPII. Atrazine bound directly to SF-1, showing that atrazine is a ligand for this “orphan” receptor. CONCLUSION: The current findings are consistent with atrazine’s endocrine-disrupting effects in fish, amphibians, and reptiles; the induction of mammary and prostate cancer in laboratory rodents; and correlations between atrazine and similar reproductive cancers in humans. This study highlights the importance of atrazine as a risk factor in endocrine disruption in wildlife and reproductive cancers in laboratory rodents and humans

Identical NR5A1 Missense Mutations in Two Unrelated 46,XX Individuals with Testicular Tissues

The role of monogenic mutations in the development of 46,XX testicular/ovotesticular disorders of sex development (DSD) remains speculative. Although mutations in NR5A1 are known to cause 46,XY gonadal dysgenesis and 46,XX ovarian insufficiency, such mutations have not been implicated in testicular development of 46,XX gonads. Here, we identified identical NR5A1 mutations in two unrelated Japanese patients with 46,XX testicular/ovotesticular DSD. The p.Arg92Trp mutation was absent from the clinically normal mothers and from 200 unaffected Japanese individuals. In silico analyses scored p.Arg92Trp as probably pathogenic. In vitro assays demonstrated that compared with wild‐type NR5A1, the mutant protein was less sensitive to NR0B1‐induced suppression on the SOX9 enhancer element. Other sequence variants found in the patients were unlikely to be associated with the phenotype. The results raise the possibility that specific mutations in NR5A1 underlie testicular development in genetic females

Combining ChIP-chip and Expression Profiling to Model the MoCRZ1 Mediated Circuit for Ca2+/Calcineurin Signaling in the Rice Blast Fungus

Significant progress has been made in defining the central signaling networks in many organisms, but collectively we know little about the downstream targets of these networks and the genes they regulate. To reconstruct the regulatory circuit of calcineurin signal transduction via MoCRZ1, a Magnaporthe oryzae C2H2 transcription factor activated by calcineurin dephosphorylation, we used a combined approach of chromatin immunoprecipitation - chip (ChIP-chip), coupled with microarray expression studies. One hundred forty genes were identified as being both a direct target of MoCRZ1 and having expression concurrently differentially regulated in a calcium/calcineurin/MoCRZ1 dependent manner. Highly represented were genes involved in calcium signaling, small molecule transport, ion homeostasis, cell wall synthesis/maintenance, and fungal virulence. Of particular note, genes involved in vesicle mediated secretion necessary for establishing host associations, were also found. MoCRZ1 itself was a target, suggesting a previously unreported autoregulation control point. The data also implicated a previously unreported feedback regulation mechanism of calcineurin activity. We propose that calcium/calcineurin regulated signal transduction circuits controlling development and pathogenicity manifest through multiple layers of regulation. We present results from the ChIP-chip and expression analysis along with a refined model of calcium/calcineurin signaling in this important plant pathogen

Nucleosome-coupled expression differences in closely-related species

<p>Abstract</p> <p>Background</p> <p>Genome-wide nucleosome occupancy is negatively related to the average level of transcription factor motif binding based on studies in yeast and several other model organisms. The degree to which nucleosome-motif interactions relate to phenotypic changes across species is, however, unknown.</p> <p>Results</p> <p>We address this challenge by generating nucleosome positioning and cell cycle expression data for <it>Saccharomyces bayanus </it>and show that differences in nucleosome occupancy reflect cell cycle expression divergence between two yeast species, <it>S. bayanus </it>and <it>S. cerevisiae</it>. Specifically, genes with nucleosome-depleted MBP1 motifs upstream of their coding sequence show periodic expression during the cell cycle, whereas genes with nucleosome-shielded motifs do not. In addition, conserved cell cycle regulatory motifs across these two species are more nucleosome-depleted compared to those that are not conserved, suggesting that the degree of conservation of regulatory sites varies, and is reflected by nucleosome occupancy patterns. Finally, many changes in cell cycle gene expression patterns across species can be correlated to changes in nucleosome occupancy on motifs (rather than to the presence or absence of motifs).</p> <p>Conclusions</p> <p>Our observations suggest that alteration of nucleosome occupancy is a previously uncharacterized feature related to the divergence of cell cycle expression between species.</p

Global Analysis of Dynamical Decision-Making Models through Local Computation around the Hidden Saddle

Bistable dynamical switches are frequently encountered in mathematical modeling of biological systems because binary decisions are at the core of many cellular processes. Bistable switches present two stable steady-states, each of them corresponding to a distinct decision. In response to a transient signal, the system can flip back and forth between these two stable steady-states, switching between both decisions. Understanding which parameters and states affect this switch between stable states may shed light on the mechanisms underlying the decision-making process. Yet, answering such a question involves analyzing the global dynamical (i.e., transient) behavior of a nonlinear, possibly high dimensional model. In this paper, we show how a local analysis at a particular equilibrium point of bistable systems is highly relevant to understand the global properties of the switching system. The local analysis is performed at the saddle point, an often disregarded equilibrium point of bistable models but which is shown to be a key ruler of the decision-making process. Results are illustrated on three previously published models of biological switches: two models of apoptosis, the programmed cell death and one model of long-term potentiation, a phenomenon underlying synaptic plasticity

Primed histone demethylation regulates shoot regenerative competency

Acquisition of pluripotency by somatic cells is a striking process that enables multicellular organisms to regenerate organs. This process includes silencing of genes to erase original tissue memory and priming of additional cell type specification genes, which are then poised for activation by external signal inputs. Here, through analysis of genome-wide histone modifications and gene expression profiles, we show that a gene priming mechanism involving LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 3 (LDL3) specifically eliminates H3K4me2 during formation of the intermediate pluripotent cell mass known as callus derived from Arabidopsis root cells. While LDL3-mediated H3K4me2 removal does not immediately affect gene expression, it does facilitate the later activation of genes that act to form shoot progenitors when external cues lead to shoot induction. These results give insights into the role of H3K4 methylation in plants, and into the primed state that provides plant cells with high regenerative competency

A Co-Opted DEAD-Box RNA Helicase Enhances Tombusvirus Plus-Strand Synthesis

Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. In this paper, we show that an essential translation factor, Ded1p DEAD-box RNA helicase of yeast, directly affects replication of Tomato bushy stunt virus (TBSV). To separate the role of Ded1p in viral protein translation from its putative replication function, we utilized a cell-free TBSV replication assay and recombinant Ded1p. The in vitro data show that Ded1p plays a role in enhancing plus-strand synthesis by the viral replicase. We also find that Ded1p is a component of the tombusvirus replicase complex and Ded1p binds to the 3′-end of the viral minus-stranded RNA. The data obtained with wt and ATPase deficient Ded1p mutants support the model that Ded1p unwinds local structures at the 3′-end of the TBSV (−)RNA, rendering the RNA compatible for initiation of (+)-strand synthesis. Interestingly, we find that Ded1p and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is another host factor for TBSV, play non-overlapping functions to enhance (+)-strand synthesis. Altogether, the two host factors enhance TBSV replication synergistically by interacting with the viral (−)RNA and the replication proteins. In addition, we have developed an in vitro assay for Flock house virus (FHV), a small RNA virus of insects, that also demonstrated positive effect on FHV replicase activity by the added Ded1p helicase. Thus, two small RNA viruses, which do not code for their own helicases, seems to recruit a host RNA helicase to aid their replication in infected cells

Primed histone demethylation regulates shoot regenerative competency

Acquisition of pluripotency by somatic cells is a striking process that enables multicellular organisms to regenerate organs. This process includes silencing of genes to erase original tissue memory and priming of additional cell type specification genes, which are then poised for activation by external signal inputs. Here, through analysis of genome-wide histone modifications and gene expression profiles, we show that a gene priming mechanism involving LYSINE-SPECIFIC DEMETHYLASE 1-LIKE 3 (LDL3) specifically eliminates H3K4me2 during formation of the intermediate pluripotent cell mass known as callus derived from Arabidopsis root cells. While LDL3-mediated H3K4me2 removal does not immediately affect gene expression, it does facilitate the later activation of genes that act to form shoot progenitors when external cues lead to shoot induction. These results give insights into the role of H3K4 methylation in plants, and into the primed state that provides plant cells with high regenerative competency