PHO4, a transcription factor required for induction of the PHO5 gene in response to phosphate starvation, is phosphorylated by the PHO80-PHO85 cyclin-CDK (cyclin-dependent kinase) complex when yeast are grown in phosphate-rich medium. PHO4 was shown to be concentrated in the nucleus when yeast were starved for phosphate and was predominantly cytoplasmic when yeast were grown in phosphate-rich medium. The sites of phosphorylation on PHO4 were identified, and phosphorylation was shown to be required for full repression of PHO5 transcription when yeast were grown in high phosphate. Thus, phosphorylation of PHO4 by PHO80-PHO85 turns off PHO5 transcription by regulating the nuclear localization of PHO4.

Background: Schistosome bloodflukes are complex trematodes responsible for 200 million cases of schistosomiasis worldwide. Their life cycle is characterized by a series of remarkable morphological and biochemical transitions between an invertebrate host, an aquatic environment, and a mammalian host. We report a global transcriptional analysis of how this parasite alters gene regulation to adapt to three distinct environments. Results: Utilizing a genomic microarray made of 12,000 45-50-mer oligonucleotides based on expressed sequence tags, three different developmental stages of the schistosome parasite were analyzed by pair-wise comparisons of transcript hybridization signals. This analysis resulted in the identification of 1,154 developmentally enriched transcripts. Conclusion: This study expands the repertoire of schistosome genes analyzed for stage-specific expression to over 70% of the predicted genome. Among the new associations identified are the roles of robust protein synthesis and programmed cell death in development of cercariae in the sporocyst stages, the relative paucity of cercarial gene expression outside of energy production, and the remarkable diversity of adult gene expression programs that reflect adaptation to the host bloodstream and an average lifespan that may approach 10 years.

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening free-swimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community.

The dynamic features of a genetic network's response to environmental fluctuations represent essential functional specifications and thus may constrain the possible choices of network architecture and kinetic parameters. To explore the connection between dynamics and network design, we have analyzed a general regulatory architecture that is commonly found in many metabolic pathways. Such architecture is characterized by a dual control mechanism, with end product feedback inhibition and transcriptional regulation mediated by an intermediate metabolite. As a case study, we measured with high temporal resolution the induction profiles of the enzymes in the leucine biosynthetic pathway in response to leucine depletion, using an automated system for monitoring protein expression levels in single cells. All the genes in the pathway are known to be coregulated by the same transcription factors, but we observed drastically different dynamic responses for enzymes upstream and immediately downstream of the key control point—the intermediate metabolite α-isopropylmalate (αIPM), which couples metabolic activity to transcriptional regulation. Analysis based on genetic perturbations suggests that the observed dynamics are due to differential regulation by the leucine branch-specific transcription factor Leu3, and that the downstream enzymes are strictly controlled and highly expressed only when αIPM is available. These observations allow us to build a simplified mathematical model that accounts for the observed dynamics and can correctly predict the pathway's response to new perturbations. Our model also suggests that transient dynamics and steady state can be separately tuned and that the high induction levels of the downstream enzymes are necessary for fast leucine recovery. It is likely that principles emerging from this work can reveal how gene regulation has evolved to optimize performance in other metabolic pathways with similar architecture. Single-cell organisms must constantly adjust their gene expression programs to survive in a changing environment. Interactions between different molecules form a regulatory network to mediate these changes. While the network connections are often known, figuring out how the network responds dynamically by looking at a static picture of its structure presents a significant challenge. Measuring the response at a finer time scales could reveal the link between the network's function and its structure. The architecture of the system we studied in this work—the leucine biosynthesis pathway in yeast—is shared by other metabolic pathways: a metabolic intermediate binds to a transcription factor to activate the pathway genes, creating an intricate feedback structure that links metabolism with gene expression. We measured protein abundance at high temporal resolution for genes in this pathway in response to leucine depletion and studied the effects of various genetic perturbations on gene expression dynamics. Our measurements and theoretical modeling show that only the genes immediately downstream from the intermediate are highly regulated by the metabolite, a feature that is essential to fast recovery from leucine depletion. Since the architecture we studied is common, we believe that our work may lead to general principles governing the dynamics of gene expression in other metabolic pathways.

Background A major challenge in computational genomics is the development of methodologies that allow accurate genome-wide prediction of the regulatory targets of a transcription factor. We present a method for target identification that combines experimental characterization of binding requirements with computational genomic analysis. Results Our method identified potential target genes of the transcription factor Ndt80, a key transcriptional regulator involved in yeast sporulation, using the combined information of binding affinity, positional distribution, and conservation of the binding sites across multiple species. We have also developed a mathematical approach to compute the false positive rate and the total number of targets in the genome based on the multiple selection criteria. Conclusion We have shown that combining biochemical characterization and computational genomic analysis leads to accurate identification of the genome-wide targets of a transcription factor. The method can be extended to other transcription factors and can complement other genomic approaches to transcriptional regulation.

Schistosomiasis is a debilitating disease that affects over 240 million people worldwide and is considered the most important neglected tropical disease following malaria. Free-swimming freshwater cercariae, one of the six morphologically distinct schistosome life stages, infect humans by directly penetrating through the skin. Cercariae identify and seek the host by sensing chemicals released from human skin. When they reach the host, they burrow into the skin with the help of proteases and other contents released from their acetabular glands and transform into schistosomula, the subsequent larval worm stage upon skin infection. Relative to host invasion, studies have primarily focused on the nature of the acetabular gland secretions, immune response of the host upon exposure to cercariae, and cercaria-schistosomulum transformation methods. However, the molecular signaling pathways involved from host-seeking through the decision to penetrate skin are not well understood. We recently observed that heat shock factor 1 (Hsf1) is localized to the acetabular glands of infectious schistosome cercariae, prompting us to investigate a potential role for heat shock proteins (HSPs) in cercarial invasion. In this study, we report that cercarial invasion behavior, similar to the behavior of cercariae exposed to human skin lipid, is regulated through an Hsp70-dependent process, which we show by using chemical agents that target Hsp70. The observation that biologically active protein activity modulators can elicit a direct and clear behavioral change in parasitic schistosome larvae is itself interesting and has not been previously observed. This finding suggests a novel role for Hsp70 to act as a switch in the cercaria-schistosomulum transformation, and it allows us to begin elucidating the pathways associated with cercarial host invasion. In addition, because the Hsp70 protein and its structure/function is highly conserved, the model that Hsp70 acts as a behavior transitional switch could be relevant to other parasites that also undergo an invasion process and can apply more broadly to other organisms during morphological transitions. Finally, it points to a new function for HSPs in parasite/host interactions. Parasitic schistosome worms cause morbid disease in over 240 million individuals worldwide. Acute infections with these worms can lead to Katayama fever, while chronic infections can lead to portal hypertension, enlarged abdomen, and liver damage. The infective larval stage, called cercariae, are free-swimming and can detect, seek, and penetrate human skin to enter the human host circulatory system, eventually developing into egg-laying adult worms that cause schistosomiasis. Molecular pathways associated with the initial cercarial invasion of the host, however, are largely unknown, especially with respect to the parasite-specific signals involved in host detection and subsequent decision to invade. Here, we describe a role for Hsp70 in cercarial invasion behavior. To date, only generic stimulation with skin lipid, linoleic acid or L-arginine are known to induce cercarial invasion behavior; thus, we can begin an initial investigation of molecular requirements for host invasion and environment transition for schistosomes and possibly other parasitic organisms.