Hormone-controlled cooperative binding of transcription factors drives synergistic induction of fasting-regulated genes.

During fasting, hepatocytes produce glucose in response to hormonal signals. Glucagon and glucocorticoids are principal fasting hormones that cooperate in regulating glucose production via gluconeogenesis. However, how these hormone signals are integrated and interpreted to a biological output is unknown. Here, we use genome-wide profiling of gene expression, enhancer dynamics and transcription factor (TF) binding in primary mouse hepatocytes to uncover the mode of cooperation between glucagon and glucocorticoids. We found that compared to a single treatment with each hormone, a dual treatment directs hepatocytes to a pro-gluconeogenic gene program by synergistically inducing gluconeogenic genes. The cooperative mechanism driving synergistic gene expression is based on 'assisted loading' whereby a glucagon-activated TF (cAMP responsive element binding protein; CREB) leads to enhancer activation which facilitates binding of the glucocorticoid receptor (GR) upon glucocorticoid stimulation. Glucagon does not only activate single enhancers but also activates enhancer clusters, thereby assisting the loading of GR also across enhancer units within the cluster. In summary, we show that cells integrate extracellular signals by an enhancer-specific mechanism: one hormone-activated TF activates enhancers, thereby assisting the loading of a TF stimulated by a second hormone, leading to synergistic gene induction and a tailored transcriptional response to fasting.

Fasting Hormones Synergistically Induce Amino Acid Catabolism Genes to Promote Gluconeogenesis.

BACKGROUND & AIMS: Gluconeogenesis from amino acids (AAs) maintains glucose homeostasis during fasting. Although glucagon is known to regulate AA catabolism, the contribution of other hormones to it and the scope of transcriptional regulation dictating AA catabolism are unknown. We explored the role of the fasting hormones glucagon and glucocorticoids in transcriptional regulation of AA catabolism genes and AA-dependent gluconeogenesis. METHODS: We tested the RNA expression of AA catabolism genes and glucose production in primary mouse hepatocytes treated with fasting hormones (glucagon, corticosterone) and feeding hormones (insulin, fibroblast growth factor 19). We analyzed genomic data of chromatin accessibility and chromatin immunoprecipitation in mice and primary mouse hepatocytes. We performed chromatin immunoprecipitation in livers of fasted mice to show binding of cAMP responsive element binding protein (CREB) and the glucocorticoid receptor (GR). RESULTS: Fasting induced the expression of 31 genes with various roles in AA catabolism. Of them, 15 were synergistically induced by co-treatment of glucagon and corticosterone. Synergistic gene expression relied on the activity of both CREB and GR and was abolished by treatment with either insulin or fibroblast growth factor 19. Enhancers adjacent to synergistically induced genes became more accessible and were bound by CREB and GR on fasting. Akin to the gene expression pattern, gluconeogenesis from AAs was synergistically induced by glucagon and corticosterone in a CREB- and GR-dependent manner. CONCLUSIONS: Transcriptional regulation of AA catabolism genes during fasting is widespread and is driven by glucagon (via CREB) and corticosterone (via GR). Glucose production in hepatocytes is also synergistically augmented, showing that glucagon alone is insufficient in fully activating gluconeogenesis.

Novel membrane-bound reporter molecule for sorting high producer cells by flow cytometry.

We developed a membrane bound reporter and selection molecule for sorting by fluorescence activated cell sorting (FACS) of cells producing a protein of interest. This molecule is composed of a transmembrane (TM) domain, fused on its extracellular end to a biotin mimetic peptide (BMP) and on its intracellular side to puromycin N-acetyl transferase (PAC). In this format BMP is displayed on the cell membrane surface and PAC faces the cell cytoplasm. BMP was detected and quantified on the cell surface by fluorescently labelled streptavidin, allowing cell sorting by FACS, according to the reporter expression level. The reporter and a gene of interest (GOI) were connected on the same transcript via an internal ribosomal entry site (IRES). The reporter expression level was found to correlate with that of the GOI, enabling sorting of high producer cells by FACS. Thus, the highest fluorescent cells sorted had also the highest protein of interest (POI) productivity level.

Characterization of extracellular lytic enzymes produced by the yeast biocontrol agent Candida oleophila.

The yeast Candida oleophila, the base of the commercial product Aspire, is recommended for the control of postharvest decay of citrus and pome fruit. Competition for nutrients and space is believed to be the major mode of action. Involvement of fungal cell wall-degrading enzymes is also suggested to play a role in the mechanism of action of yeast antagonists. The present study showed that the yeast C. oleophila is capable of producing and secreting various cell wall-degrading enzymes, including exo-beta-1,3-glucanase, chitinase and protease. Exo-beta-1,3-glucanase and chitinase were produced and maximized in the early stages of growth, whereas protease reached a maximum level only after 6-8 days. Production of exo-beta-1,3-glucanase, chitinase and protease was stimulated by the presence of cell wall fragments of Penicillium digitatum in the growth medium, in addition to glucose. This study also provided evidence that C. oleophila is capable of secreting exo-beta-1,3-glucanase into the wounded surface of grapefruit. The role of exo-beta-1,3-glucanase ( CoEXG1) in the biocontrol activity of C. oleophila was tested using CoEXG1-knockouts and double- CoEXG1 over-producing transformants. In vitro bioassays showed that wild-type C. oleophila and exo-beta-1,3-glucanase over-expressing transformants had similar inhibitory effects on spore germination and germ-tube elongation; and both were more inhibitory to the fungus than the knockout transformant. In experiments conducted on fruit to test the biocontrol activity against infection by P. digitatum, no significant difference in inhibition was observed between transformants and untransformed C. oleophila cells at the high concentrations of cells used, whereas at a lower concentration of yeast cells the knockout transformants appeared to be less effective.

Evidence for tryptophan residues in the cation transport path of the Na(+),K(+)-ATPase.

A family of aryl isothiouronium derivatives was designed as probes for cation binding sites of Na(+),K(+)-ATPase. Previous work showed that 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) acts as a competitive blocker of Na(+) or K(+) occlusion. In addition to a high-affinity cytoplasmic site (K(D) < 1 microM), a low-affinity site (K(D) approximately 10 microM) was detected, presumably extracellular. Here we describe properties of Br-TITU as a blocker at the extracellular surface. In human red blood cells Br-TITU inhibits ouabain-sensitive Na(+) transport (K(D) approximately 30 microM) in a manner antagonistic with respect to extracellular Na(+). In addition, Br-TITU impairs K(+)-stimulated dephosphorylation and Rb(+) occlusion from phosphorylated enzyme of renal Na(+),K(+)-ATPase, consistent with binding to an extracellular site. Incubation of renal Na(+),K(+)-ATPase with Br-TITU at pH 9 irreversibly inactivates Na(+),K(+)-ATPase activity and Rb(+) occlusion. Rb(+) or Na(+) ions protect. Preincubation of Br-TITU with red cells in a K(+)-free medium at pH 9 irreversibly inactivates ouabain-sensitive (22)Na(+) efflux, showing that inactivation occurs at an extracellular site. K(+), Cs(+), and Li(+) ions protect against this effect, but the apparent affinity for K(+), Cs(+), or Li(+) is similar (K(D) approximately 5 mM) despite their different affinities for external activation of the Na(+) pump. Br-TITU quenches tryptophan fluorescence of renal Na(+),K(+)-ATPase or of digested "19 kDa membranes". After incubation at pH 9 irreversible loss of tryptophan fluorescence is observed and Rb(+) or Na(+) ions protect. The Br-TITU appears to interact strongly with tryptophan residue(s) within the lipid or at the extracellular membrane-water interface and interfere with cation occlusion and Na(+),K(+)-ATPase activity.

The effect of under- and overexpressed CoEXG1-encoded exoglucanase secreted by Candida oleophila on the biocontrol of Penicillium digitatum.

The yeast, Candida oleophila, is acknowledged for its biocontrol activity against postharvest moulds. However, the mechanism of this activity is not fully understood. One of the conjectured modes of action is associated with extracellular lytic enzymes, such as beta-exoglucanase. The relationship of beta-exoglucanase in the biocontrol activity of C. oleophila was investigated by generating C. oleophila CoEXG1-knockouts and double-CoEXG1 transformants. The knockout transformants secreted 0-13% of the exoglucanase activity detected in the medium of the untransformed yeast (depending on the medium), indicating that CoEXG1 is the main gene responsible for the production of the secreted exoglucanase. Correspondingly, the double-CoEXG1 transformants secreted approximately twice as much 1,3-beta-exoglucanase as the untransformed C. oleophila. The biocontrol activity of the CoEXG1-knockout and the double-CoEXG1 transformants against Penicillium digitatum did not differ from that of the untransformed C. oleophila on kumquats. These results imply that the 1,3-beta-exoglucanase encoded by the gene CoEXG1 is not involved in the biocontrol activity of C. oleophila against P. digitatum under these experimental terms. However, these findings do not rule out the possibilities, that the participation of CoEXG1 in biocontrol is dependent on the activity of other gene products, or that its effect may be manifested under altered environmental conditions.