Growth-regulated co-occupancy of Mediator and Lsm3 at intronic ribosomal protein genes.

Mediator is a well-known transcriptional co-regulator and serves as an adaptor between gene-specific regulatory proteins and RNA polymerase II. Studies on the chromatin-bound form of Mediator revealed interactions with additional protein complexes involved in various transcription-related processes, such as the Lsm2-8 complex that is part of the spliceosomal U6 small nuclear ribonucleoprotein complex. Here, we employ Chromatin Immunoprecipitation sequencing (ChIP-seq) of chromatin associated with the Lsm3 protein and the Med1 or Med15 Mediator subunits. We identify 86 genes co-occupied by both Lsm3 and Mediator, of which 73 were intron-containing ribosomal protein genes. In logarithmically growing cells, Mediator primarily binds to their promoter regions but also shows a second, less pronounced occupancy at their 3'-exons. During the late exponential phase, we observe a near-complete transition of Mediator from these promoters to a position in their 3'-ends, overlapping the Lsm3 binding sites ∼250 bp downstream of their last intron-exon boundaries. Using an unbiased RNA sequencing approach, we show that transition of Mediator from promoters to the last exon of these genes correlates to reduction of both their messenger RNA levels and splicing ratios, indicating that the Mediator and Lsm complexes cooperate to control growth-regulated expression of intron-containing ribosomal protein genes at the levels of transcription and splicing.

The Effects of Endurance Exercise and Diet on Atherosclerosis in Young and Aged ApoE-/- and Wild-Type Mice.

BACKGROUND: Atherosclerosis is the leading cause of death worldwide. The disease development is by and large driven by old age and lifestyle factors, such as diet, physical activity, and smoking. In the present study, we have investigated the effect of exercise and diet on the development of atherosclerosis in young and aged mice. OBJECTIVE: This study aimed at comparing multiple age-dependent factors that may influence atherosclerosis in a transgenic mouse model. METHODS: Young (14 weeks) and aged (49-52 weeks) C57BL/6 wild-type (WT) and atherosclerosis-prone ApoE-/- mice were subjected to physical endurance exercise on a treadmill, with or without a high-fat diet. Five weeks later, the frequencies of regulatory T cells (TREGs) in lymph nodes were assessed by flow cytometry, plasmatic cytokines (interleukin [IL]-1ß, IL-6, IL-10, IL-17, interferon-γ, tumor necrosis factor-α, and transforming growth factor [TGF]-ß1) levels were determined by Luminex assay. Lipids (cholesterol and triglycerides) and anti-heat shock protein 60 (HSP60) autoantibodies were measured by ELISA. Aortic lesion sizes were assessed by en face imaging. Microarray analysis and qPCR of skeletal muscle gene expression were also performed. RESULTS: Exercise leads to a reduction of aortic lesions in young ApoE-/- and aged WT mice independent of diet. In most groups, this reduction was followed by an increased proportion of TREGs and TGF-ß1 levels. Moreover, gene expression analysis showed that exercise seems to affect the AMPK signaling pathway. In particular, PGC-1α1 mRNA was induced in aged WT mice, whereas it was reduced in young ApoE-/- mice. In addition, GSEA analysis showed a marked reduction in the insulin signaling pathway in aged ApoE-/- mice. CONCLUSION: Practicing endurance exercise seems to be enough for reducing early aortic lesion formation, independent of diet. However, this was only true in mice with smaller aortic lesions, since mice with large, advanced, complicated atherosclerotic plaques did not show any reduction in lesion size with exercise training.

Gene dosage effects in yeast support broader roles for the LOG1, HAM1 and DUT1 genes in detoxification of nucleotide analogues.

Purine and pyrimidine analogues have important uses in chemotherapies against cancer, and a better understanding of the mechanisms that cause resistance to these drugs is therefore of importance in cancer treatment. In the yeast Saccharomyces cerevisiae, overexpression of the HAM1 gene encoding inosine triphosphate pyrophosphatase confers resistance to both the purine analogue 6-N-hydroxylaminopurine (HAP) and the pyrimidine analogue 5-fluorouracil (5-FU) (Carlsson et al., 2013, PLoS One 8, e52094). To find out more about the mechanisms of resistance to nucleotide analogues, and possible interdependencies between purine and pyrimidine analogue resistance mechanisms, we screened a plasmid library in yeast for genes that confer HAP resistance when overexpressed. We cloned four such genes: ADE4, DUT1, APT2, and ATR1. We further looked for genetic interactions between these genes and genes previously found to confer resistance to 5-FU. We found that HMS1, LOG1 (YJL055W), HAM1, and ATR1 confer resistance to both 5-FU and HAP, whereas ADE4, DUT1 and APT2 are specific for HAP resistance, and CPA1 and CPA2 specific for 5-FU resistance. Possible mechanisms for 5-FU and HAP detoxification are discussed based on the observed genetic interactions. Based on the effect of LOG1 against both 5-FU and HAP toxicity, we propose that the original function of the LOG (LONELY GUY) family of proteins likely was to degrade non-canonical nucleotides, and that their role in cytokinin production is a later development in some organisms.

The histone demethylase activity of Rph1 is not essential for its role in the transcriptional response to nutrient signaling.

Rph1 and Gis1 are two related yeast zinc finger proteins that function as downstream effectors in the Ras/PKA, TOR and Sch9 nutrient signaling pathways. Both proteins also contain JmjC histone demethylase domains, but only Rph1 is known to be an active enzyme, demethylating lysine 36 of histone H3. We have studied to what extent the demethylase activity of Rph1 contributes to its role in nutrient signaling by performing gene expression microarray experiments on a yeast strain containing a catalytically inactive allele of RPH1. We find that the enzymatic activity of Rph1 is not essential for its role in growth phase dependent gene regulation. However, the ability of Rph1 to both activate and repress transcription is partially impaired in the active site mutant, indicating that the demethylase activity may enhance its function in vivo. Consistent with this, we find that the Rph1 mutation and a deletion of the histone H3 methylase Set2 affect the same target genes in opposite directions. Genes that are differentially expressed in the Rph1 mutant are also enriched for binding of Rpd3, a downstream effector in silencing, to their promoters. The expression of some subtelomeric genes and genes involved in sporulation and meiosis are also affected by the mutation, suggesting a role for Rph1-dependent demethylation in regulating these genes. A small set of genes are more strongly affected by the active site mutation, indicating a more pronounced role for the demethylase activity in their regulation by Rph1.

A Ham1p-dependent mechanism and modulation of the pyrimidine biosynthetic pathway can both confer resistance to 5-fluorouracil in yeast.

5-Fluorouracil (5-FU) is an anticancer drug and pyrimidine analogue. A problem in 5-FU therapy is acquired resistance to the drug. To find out more about the mechanisms of resistance, we screened a plasmid library in yeast for genes that confer 5-FU resistance when overexpressed. We cloned five genes: CPA1, CPA2, HMS1, HAM1 and YJL055W. CPA1 and CPA2 encode a carbamoyl phosphate synthase involved in arginine biosynthesis and HMS1 a helix-loop-helix transcription factor. Our results suggest that CPA1, CPA2, and HMS1 confer 5-FU resistance by stimulating pyrimidine biosynthesis. Thus, they are unable to confer 5-FU resistance in a ura2 mutant, and inhibit the uptake and incorporation into RNA of both uracil and 5-FU. In contrast, HAM1 and YJL055W confer 5-FU resistance in a ura2 mutant, and selectively inhibit incorporation into RNA of 5-FU but not uracil. HAM1 is the strongest resistance gene, but it partially depends on YJL055W for its function. This suggests that HAM1 and YJL055W function together in mediating resistance to 5-FU. Ham1p encodes an inosine triphosphate pyrophosphatase that has been implicated in resistance to purine analogues. Our results suggest that Ham1p could have a broader specificity that includes 5-FUTP and other pyrimidine analogoue triphosphates.

Internet administered guided self-help versus individualized e-mail therapy: A randomized trial of two versions of CBT for major depression.

Internet-delivered psychological treatment of major depression has been investigated in several trials, but the role of personalized treatment is less investigated. Studies suggest that guidance is important and that automated computerized programmes without therapist support are less effective. Individualized e-mail therapy for depression has not been studied in a controlled trial. Eighty-eight individuals with major depression were randomized to two different forms of Internet-delivered cognitive behaviour therapy (CBT), or to a waiting-list control group. One form of Internet treatment consisted of guided self-help, with weekly modules and homework assignments. Standard CBT components were presented and brief support was provided during the treatment. The other group received e-mail therapy, which was tailored and did not use the self-help texts i.e., all e-mails were written for the unique patient. Both treatments lasted for 8 weeks. In the guided self-help 93% completed (27/29) and in the e-mail therapy 96% (29/30) completed the posttreatment assessment. Results showed significant symptom reductions in both treatment groups with moderate to large effect sizes. At posttreatment 34.5% of the guided self-help group and 30% of the e-mail therapy group reached the criteria of high-end-state functioning (Beck Depression Inventory score below 9). At six-month follow-up the corresponding figures were 47.4% and 43.3%. Overall, the difference between guided self-help and e-mail therapy was small, but in favour of the latter. These findings indicate that both guided self-help and individualized e-mail therapy can be effective.

Costs of alien invasive species in Sweden.

The purpose of this paper is to present calculations of total costs of 13 alien invasive species (AIS) in Sweden. All species are subject to control by Swedish public authorities, and estimates for most AIS include either damage cost or actual control cost. The results indicate a total annual cost between approximately 1620 and 5080 million SEK, which correspond to SEK 175 and SEK 565 per capita in Sweden. The estimates are well within the range of similar calculations for other countries, but differ with respect to the composition of costs of different AIS. Whereas costs for the agricultural and forestry sectors dominate in most other studies, the costs of AIS in Sweden are more equally divided among different categories. The results also indicate that the highest costs are attributable to unintentionally introduced AIS and that the most reliable cost estimates are related to human and animal health.

Adenovirus type 5 fiber knob domain has a critical role in fiber protein synthesis and encapsidation.

Adenovirus serotype 5 (Ad5) vectors carrying knobless fibers designed to remove their natural tropism were found to have a lower fiber content than recombinant Ad5 with wild-type (WT) capsid, implying a role for the knob-coding sequence or/and the knob domain in fiber encapsidation. Experimental data using a variety of fiber gene constructs showed that the defect did not occur at the fiber mRNA level, but at the protein level. Knobless fiber proteins were found to be synthesized at a significant slower rate compared with knob-carrying fibers, and the trimerization process of knobless fibers paralleled their slow rate of synthesis. A recombinant Ad5 diploid for the fiber gene (referred to as Ad5/R7-ZZ(wt)/E1 : WT-fiber) was constructed to analyse the possible rescue of the knobless low-fiber-content phenotype by co-expression of WT fiber. Ad5/R7-ZZ(wt)/E1 : WT-fiber contained a knobless fiber gene in its natural location (L5) in the viral genome and an additional WT fiber gene in an ectopic position in E1. Knobless fiber was still synthesized at low levels compared with the co-expressed E1 : WT fiber and the recovery of the two fiber species in virus progeny reflected their respective amounts in the infected cells. Our results suggested that deletion of the fiber knob domain had a negative effect on the translation of the fiber mRNA and on the intracellular concentration of fiber protein. They also suggested that the knob control of fiber protein synthesis and encapsidation occurred as a cis effect, which was not modified by WT fiber protein provided in trans by the same Ad5 genome.