Expression of a truncated ATHB17 protein in maize increases ear weight at silking.

ATHB17 (AT2G01430) is an Arabidopsis gene encoding a member of the α-subclass of the homeodomain leucine zipper class II (HD-Zip II) family of transcription factors. The ATHB17 monomer contains four domains common to all class II HD-Zip proteins: a putative repression domain adjacent to a homeodomain, leucine zipper, and carboxy terminal domain. However, it also possesses a unique N-terminus not present in other members of the family. In this study we demonstrate that the unique 73 amino acid N-terminus is involved in regulation of cellular localization of ATHB17. The ATHB17 protein is shown to function as a transcriptional repressor and an EAR-like motif is identified within the putative repression domain of ATHB17. Transformation of maize with an ATHB17 expression construct leads to the expression of ATHB17Δ113, a truncated protein lacking the first 113 amino acids which encodes a significant portion of the repression domain. Because ATHB17Δ113 lacks the repression domain, the protein cannot directly affect the transcription of its target genes. ATHB17Δ113 can homodimerize, form heterodimers with maize endogenous HD-Zip II proteins, and bind to target DNA sequences; thus, ATHB17Δ113 may interfere with HD-Zip II mediated transcriptional activity via a dominant negative mechanism. We provide evidence that maize HD-Zip II proteins function as transcriptional repressors and that ATHB17Δ113 relieves this HD-Zip II mediated transcriptional repression activity. Expression of ATHB17Δ113 in maize leads to increased ear size at silking and, therefore, may enhance sink potential. We hypothesize that this phenotype could be a result of modulation of endogenous HD-Zip II pathways in maize.

Accurate and sensitive diagnosis of geminiviruses through enrichment, high-throughput sequencing and automated sequence identification.

Existing diagnostic techniques used to identify plant-infecting DNA viruses and their associated molecules are often limited in their specificity and can be challenged by samples containing multiple viruses. We adapted a simple method of amplifying circular viral DNA and, in combination with high-throughput sequencing and bioinformatic analysis, used it as a virus diagnostic method. We validated this diagnostic method with a plant sample infected with a tomato yellow leaf curl geminivirus infectious clone and also compared PCR- and high-throughput-sequencing diagnostics on a geminivirus-infected field sample, showing that both methods gave similar results. Finally, we analyzed infected field samples of pepper from Mexico and tomato from India using this approach, demonstrating that it is both sensitive and capable of simultaneously identifying multiple discrete DNA viruses and subviral DNA elements in densely infected samples.

Using small RNA sequences to diagnose, sequence, and investigate the infectivity characteristics of vegetable-infecting viruses.

In a virus-infected plant, small interfering RNAs (siRNAs) corresponding to the viral genome form a large proportion of the small RNA population. It is possible to reassemble significant portions of the virus sequence from overlapping siRNA sequences and use these to identify the virus. We tested this technique with a resistance-breaking and a non-resistance-breaking strain of tomato spotted wilt virus (TSWV). We were able to assemble contigs covering 99% of the genomes of both viruses. The abundance of TSWV siRNAs allowed us to detect TSWV at early time points before the onset of symptoms, at levels too low for conventional detection. Combining traditional and bioinformatic detection methods, we also measured how replication of the resistance-breaking strain differed from the non-resistance-breaking strain in susceptible and resistant tomato varieties. We repeated this technique in identification of a squash-infecting geminivirus and also used it to identify an unspecified tospovirus.

Butylhydroquinone protects cells genetically deficient in glutathione biosynthesis from arsenite-induced apoptosis without significantly changing their prooxidant status.

Arsenic, first among the top environmentally hazardous substances, is associated with skin, lung, liver, kidney, prostate, and bladder cancer. Arsenic is also a cardiovascular and a central nervous system toxicant, and it has genotoxic and immunotoxic effects. Paradoxically, arsenic trioxide is used successfully in the treatment of acute promyelocytic leukemia and multiple myeloma. Arsenic induces oxidative stress, and its toxicity is decreased by free thiols and increased by glutathione depletion. To further characterize the role of glutathione and oxidative stress in the toxicity of arsenic, we have used fetal fibroblasts from Gclm(-/-) mice, which lack the modifier subunit of glutamate-cysteine ligase, the rate-limiting enzyme in glutathione biosynthesis. Gclm(-/-) mouse embryo fibroblasts (MEFs) are eight times more sensitive to arsenite-induced apoptotic death. Because of a dramatic decrease in glutathione levels, Gclm(-/-) MEFs have a high prooxidant status that is not significantly relieved by treatment with the phenolic antioxidant tBHQ; however, tBHQ blocks arsenite-induced apoptosis in both Gclm(+/+) and Gclm(-/-) cells, although it raises a significant antioxidant response only in Gclm(+/+) cells. Global gene expression profiles indicate that tBHQ is significantly effective in reversing arsenite-induced gene deregulation in Gclm(+/+) but not in Gclm(-/-) MEFs. This effect of tBHQ is evident in the expression of metalloproteases and chaperones, and in the expression of genes involved in DNA damage and repair, protein biosynthesis, cell growth and maintenance, apoptosis, and cell cycle regulation. These results suggest that regulation of glutathione levels by GCLM determines the sensitivity to arsenic-induced apoptosis by setting the overall ability of the cells to mount an effective antioxidant response.

Arsenite-induced aryl hydrocarbon receptor nuclear translocation results in additive induction of phase I genes and synergistic induction of phase II genes.

Complex mixtures of carcinogenic metalloids, such as arsenic, and polycyclic aromatic hydrocarbons or halogenated aromatic hydrocarbons are common environmental contaminants. The biological consequences of exposure to these mixtures are unpredictable and, although the health effects of individual chemicals may be known, the toxicity of environmental mixtures is largely unexplored. Arsenic, not a potent mutagen by itself, is co-mutagenic with many DNA-damaging agents. Mixtures of arsenite plus benzo[a]pyrene (B[a]P) augment B[a]P mutagenicity, suggesting that arsenite might uncouple expression of phase I and II genes responsible for detoxification. We have studied the effects of arsenite exposure on the activation of the aryl hydrocarbon receptor (AHR) and its subsequent role in gene transactivation. Treatment of mouse Hepa-1 cells with arsenite induces AHR nuclear translocation and binding to the Cyp1a1 gene promoter with the same efficiency as tetrachlorodibenzo-p-dioxin (TCDD), the most potent ligand of the AHR; however, TCDD and B[a]P are an order of magnitude more potent than arsenite in up-regulating Cyp1a1 transcription. Global profiling analyses of cells treated with arsenite plus B[a]P indicate that several phase I and II detoxification genes are in some cases additively and in others synergistically deregulated by the mixtures. Real-time reverse transcription-polymerase chain reaction analyses of mouse embryonic fibroblasts showed that the mixtures had an additive effect on the mRNA levels of Cyp1b1, a prototypical phase I detoxification gene, and an AHR-dependent synergistic effect on the corresponding levels of Nqo1, a prototypical phase II gene. We conclude that exposure to arsenite/B[a]P mixtures causes regulatory changes in the expression of detoxification genes that ultimately affect the metabolic activation and disposition of toxicants.

Gene expression profiles of mouse aorta and cultured vascular smooth muscle cells differ widely, yet show common responses to dioxin exposure.

Exposure to environmental toxicants may play a role in the onset and progression of cardiovascular disease. Many environmental agents, such as dioxin, are risk factors for atherosclerosis because they may exacerbate an underlying disease by altering gene expression patterns. Expression profiling of vascular tissues allows the simultaneous analysis of thousands of genes and may provide predictive information particularly useful in early disease stages. Often, however, in vivo experiments are unfeasible for material or ethical reasons, and data from cultured cells must be used instead, even though it may not be known whether cultured cells and live tissues share common global responses to the same toxicant. In a search for genes responsive to dioxin exposure, we used oligonucleotide microarrays with DNA sequences from 13,433 genes to compare global gene expression profiles of C57BL/6 mice aortas with cultured vascular smooth muscle cells (vSMCs) of the same mice. Aorta segments and vSMCs differed in the expression of more than 4500 genes, many showing expression differences greater than 1000-fold. Integration of microarray data into Gene Ontology Project annotations showed that many of the genes differentially expressed belonged to the same biological process or metabolic pathway. Notwithstanding these results, a subset of 35 genes responded in the same fashion to dioxin exposure in both systems. Genes in this subset encoded phase I and phase II detoxification enzymes, signal transduction kinases and phosphatases, and proteins involved in DNA repair and the cell cycle. We conclude that vSMCS may be useful aorta surrogates to study early gene expression responses to dioxin exposure, provided that analyses focus on this subset of genes.

A critical role for MAP kinases in the control of Ah receptor complex activity.

The heterodimeric complex of aromatic hydrocarbon receptor (AHR) and Ah receptor nuclear translocator (ARNT) plays a pivotal role in controlling the expression of drug metabolism genes, such as the cytochromes p450 (Cyp) 1a1 and 1b1, believed to be responsible for most toxic effects of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this study, we show that activation of Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) modulates ARNT transcription activity and potentiates the transcriptional activity of AHR/ARNT complexes. Inhibition of ERK by chemical compounds and ablation of JNK caused significant decreases in CYP1A1 induction by TCDD. Compared to wild type, JNK2 ablation significantly reduced TCDD-stimulated CYP1A1 expression in mouse thymus and testis, but not in liver. In contrast, CYP1B1 expression was unaffected in all three tissues of the knockout mice. These data suggest that JNK and ERK modulate ARNT activity and AHR/ARNT-dependent gene expression, contributing to the gene-specific and tissue-specific toxicity of environmental contaminants.

Chromium inhibits transcription from polycyclic aromatic hydrocarbon-inducible promoters by blocking the release of histone deacetylase and preventing the binding of p300 to chromatin.

Co-contamination with complex mixtures of carcinogenic metals, such as chromium, and polycyclic aromatic hydrocarbons is a common environmental problem with multiple biological consequences. Chromium exposure alters inducible gene expression, forms chromium-DNA adducts and chromium-DNA cross-links, and disrupts transcriptional activator-co-activator complexes. We have shown previously that exposure of mouse hepatoma Hepa-1 cells to chromate inhibits the induction of the Cyp1a1 and Nqo1 genes by dioxin. Here we have tested the hypothesis that chromium blocks gene expression by interfering with the assembly of productive transcriptional complexes at the promoter of inducible genes. To this end, we have studied the effects of chromium on the expression of genes induced by benzo[a]pyrene (B[a]P), another aryl hydrocarbon receptor agonist, and characterized the disruption of Cyp1a1 transcriptional induction by chromium. Gene expression profiling by using high density microarray analysis revealed that the inhibitory effect of chromium on B[a]P-dependent gene induction was generalized, affecting the induction of over 50 different genes involved in a variety of signaling transduction pathways. The inhibitory effect of chromium on Cyp1a1 transcription was found to depend on the presence of promoter-proximal sequences and not on the cis-acting enhancer sequences that bind the aryl hydrocarbon receptor-aryl hydrocarbon receptor nuclear translocator complex. By using transient reporter assays and chromatin immunoprecipitation analyses, we found that chromium prevented the B[a]P-dependent release of HDAC-1 from Cyp1a1 chromatin and blocked p300 recruitment. These results provide a mechanistic explanation for the observation that chromium inhibits inducible but not constitutive gene expression.