Continuous in vivo Metabolism by NMR

Dense time-series metabolomics data are essential for unraveling the underlying dynamic properties of metabolism. Here we extend high-resolution-magic angle spinning (HR-MAS) to enable continuous in vivo monitoring of metabolism by NMR (CIVM-NMR) and provide analysis tools for these data. First, we reproduced a result in human chronic lymphoid leukemia cells by using isotope-edited CIVM-NMR to rapidly and unambiguously demonstrate unidirectional flux in branched-chain amino acid metabolism. We then collected untargeted CIVM-NMR datasets for Neurospora crassa, a classic multicellular model organism, and uncovered dynamics between central carbon metabolism, amino acid metabolism, energy storage molecules, and lipid and cell wall precursors. Virtually no sample preparation was required to yield a dynamic metabolic fingerprint over hours to days at ~4-min temporal resolution with little noise. CIVM-NMR is simple and readily adapted to different types of cells and microorganisms, offering an experimental complement to kinetic models of metabolism for diverse biological systems

The <em>Caenorhabditis elegans</em> JNK Signaling Pathway Activates Expression of Stress Response Genes by Derepressing the Fos/HDAC Repressor Complex

<div><p>MAP kinases are integral to the mechanisms by which cells respond to a wide variety of environmental stresses. In <i>Caenorhabditis elegans</i>, the KGB-1 JNK signaling pathway regulates the response to heavy metal stress. In this study, we identified FOS-1, a bZIP transcription factor, as a target of KGB-1-mediated phosphorylation. We further identified two transcriptional targets of the KGB-1 pathway, <i>kreg-1</i> and <i>kreg-2</i>/<i>lys-3</i>, which are required for the defense against heavy metal stress. FOS-1 plays a critical role in the transcriptional repression of the <i>kreg-1</i> gene by recruiting histone deacetylase (HDAC) to its promoter. KGB-1 phosphorylation prevents FOS-1 dimerization and promoter binding, resulting in promoter derepression. Thus, HDAC behaves as a co-repressor modulating FOS-1-mediated transcriptional regulation. This study describes the direct link from JNK signaling, Fos phosphorylation, and regulation of <i>kreg</i> gene transcription, which modulates the stress response in <i>C. elegans</i>.</p> </div

The DNA binding activity of FOS-1 is inhibited by KGB-1-mediated phosphorylation.

<p>(A) FOS-1 binds to the TRE2 sites. HEK293 cells were co-transfected with the <i>Pkreg-1::venus</i> construct together with expression vectors encoding T7-FOS-1 or T7-hGrhl2 as indicated. For chromatin immunoprecipitation assays, immunoprecipitated complexes obtained with anti-T7 antibodies were analyzed by quantitative PCR. Data are compared using a one-way ANOVA. **P<0.01. Immunoprecipitated T7-FOS-1 and T7-hGrhl2 were monitored by Western blot. (B, C) Effect of FOS-1 phosphorylation by KGB-1 on the TRE2 binding activity. COS-7 cells were co-transfected with expression vectors encoding T7-FOS-1, HA-KGB-1 WT, HA-KGB-1 KN, and FLAG-MEK-1 as indicated. For gel retardation assays, cell extracts were incubated with the TRE2 retardation probes. Anti-T7 antibodies or normal mouse IgG were added in the binding reactions (B). Expression of T7-FOS-1, HA-KGB-1, and FLAG-MEK-1 was monitored by Western blot. Experiments were performed three times with similar results.</p

FOS-1 negatively regulates <i>kreg-1</i> expression via the TRE2 site.

<p>(A) Schematic representation of the structure of the <i>kreg-1</i> promoter. Two TRE sites are represented by dark boxes. (B, C) Effect of deletion of the TRE sites on expression of the <i>kreg-1</i> reporter. Wild-type and <i>kgb-1</i> mutant animals harboring the <i>Pkreg-1Δtre1::venus</i> or <i>Pkreg-1Δtre2::venus</i> transgene as an extrachromosomal array were cultured on plates seeded with a bacteria strain expressing the double-stranded RNA for <i>fos-1</i>. Fluorescent (Venus) views are shown in B. Scale bar: 100 µm. “Weak” refers to animals in which intestinal Venus was present at low levels. “Strong” indicates that Venus was present at high levels in most of the intestine. Percentages of animals in each expression category are listed in C. The numbers (n) of animals examined are shown.</p

HDA-1 functions cooperatively with FOS-1.

<p>(A, B) Effect of <i>hda-1</i> depletion on expression of the <i>kreg-1</i> reporter. Wild-type and <i>kgb-1</i> mutant animals harboring the <i>Pkreg-1::venus</i> transgenes as an extrachromosomal array were cultured on plates seeded with a bacteria strain expressing the double-stranded RNA for <i>hda-1</i>. Fluorescent (Venus) views are shown in A. Scale bar: 100 µm. “Weak” refers to animals in which intestinal Venus was present at low levels. “Strong” indicates that Venus was present at high levels in most of the intestine. Percentages of animals in each expression category are listed in B. The numbers (n) of animals examined are shown. (C) Interaction of HDA-1 with FOS-1. HEK293 cells were co-transfected with the <i>Pkreg-1::venus</i> construct and expression vectors encoding FLAG-HDA-1 and T7-FOS-1 as indicated. Whole cell extracts and immunoprecipitated complexes obtained with anti-FLAG antibodies were analyzed by Western blot. FOS-1 signal intensities in co-immunoprecipitates with HDA-1 were quantitated and normalized to those in whole cell extracts. Relative levels of immunoprecipitated FOS-1 are shown. Experiments were performed three times with similar results. (D) Suppression of the <i>kgb-1</i> heavy metal-sensitive phenotype by <i>hda-1</i> depletion. Each animal was cultured from embryogenesis on normal plates containing copper sulfate (40 µM) and seeded with a bacteria strain expressing the double-stranded RNA for <i>hda-1</i>. The relative viability is shown with standard errors. Error bars indicate 95% confidence interval. **P<0.01 as determined by Student's <i>t</i> test. NS, not significant.</p

Effect of <i>fos-1</i> inhibition on stress sensitivity.

<p>(A) Suppression of the <i>kgb-1</i> heavy metal-sensitive phenotype by <i>fos-1</i> depletion. Each animal was cultured from embryogenesis on normal plates containing copper sulfate (40 µM) and seeded with a bacteria strain expressing the double-stranded RNA for <i>fos-1</i>. The relative viability is shown with standard errors. Error bars indicate 95% confidence interval. **P<0.01 as determined by Student's <i>t</i> test. NS, not significant. (B) Heavy metal sensitivity caused by FOS-1(T304A) overexpression. Wild-type animals harboring the <i>Phsp-16::t7::fos-1</i> transgene as an extrachromosomal array were cultured from embryogenesis on normal plates containing copper sulfate (100 µM). The relative viability is shown with standard errors. **P<0.01 as determined by Student's <i>t</i> test. NS, not significant.</p

FOS-1 represses <i>kreg-1</i> expression.

<p>Wild-type and <i>kgb-1</i> mutant animals harboring the <i>Pkreg-1::venus</i> transgene as an extrachromosomal array were cultured on plates seeded with a bacteria strain expressing the double-stranded RNA for <i>fos-1</i>. Fluorescent (Venus) views are shown in A. Scale bar: 100 µm. “Weak” refers to animals in which intestinal Venus was present at low levels. “Strong” indicates that Venus was present at high levels in most of the intestine. Percentages of animals in each expression category are listed in B. The numbers (n) of animals examined are shown.</p

Proposed model for the KGB-1 pathway in stress response.

<p>In the absence of heavy metal stress, FOS-1 forms homodimers and binds to the TRE2 motif in the <i>kreg</i> target promoter. FOS-1 dimerization potentiates recruitment of HDA-1 to the promoter. The FOS-1/HDA-1 repressor complex represses transcription of <i>kreg</i> target genes (left panel). In the presence of heavy metal stress, the KGB-1 pathway is activated and FOS-1 is phosphorylated by KGB-1. FOS-1 phosphorylation leads to a switch from dimer to monomer, resulting in dissociation of the FOS-1/HDA-1 repressor complex from the target promoter that activates transcription of <i>kreg</i> genes (right panel).</p

The KGB-1 pathway regulates expression of <i>kreg</i> genes.

<p>(A, B) Effect of copper ion on expression of <i>kreg-1</i> (A) and <i>kreg-2</i> (B). Wild-type and <i>kgb-1</i> mutant animals were cultured on plates seeded with a bacteria strain. At 3 days after hatching, animals were treated with copper sulfate (1 mM) for 1 hour and total RNA was isolated. Expression of genes was analyzed by qRT-PCR. Data are compared using a one-way ANOVA. **P<0.01. (C) Heavy metal sensitivity caused by inhibition of <i>kreg</i> genes. The <i>eri-1</i> mutant animals were cultured from embryogenesis on normal plates containing copper sulfate (100 µM) and seeded with bacteria strains expressing the indicated double-stranded RNA. The relative viability is shown with standard errors. Error bars indicate 95% confidence interval. **P<0.01 as determined by Student's <i>t</i> test. (D, E) Effect of copper ion on expression of the <i>kreg-1</i> reporter. Wild-type and <i>kgb-1</i> mutant animals harboring the <i>Pkreg-1::venus</i> transgene as an extrachromosomal array were cultured on plates seeded with a bacteria strain expressing the double-stranded RNA for <i>vhp-1</i>. At 3 days after hatching, animals were treated with copper sulfate (1 mM) for 1 hour. These animals were then transferred to NGM plates and incubated for 3 hours. Fluorescent (Venus) views are shown in D. Scale bar: 100 µm. “Weak” refers to animals in which intestinal Venus was present at low levels. “Strong” indicates that Venus was present at high levels in most of the intestine. Percentages of animals in each expression category are listed in E. The numbers (n) of animals examined are shown.</p