Termination of RNA Polymerase II (Pol II) activity serves a vital cellular role by separating ubiquitous transcription units and influencing RNA fate and function. In the yeast Saccharomyces cerevisiae, Pol II termination is carried out by cleavage and polyadenylation factor (CPF-CF) and Nrd1-Nab3-Sen1 (NNS) complexes, which operate primarily at mRNA and non-coding RNA genes, respectively. Premature Pol II termination (attenuation) contributes to gene regulation, but there is limited knowledge of its prevalence and biological significance. In particular, it is unclear how much crosstalk occurs between CPF-CF and NNS complexes and how Pol II attenuation is modulated during stress adaptation. In this study, we have identified an attenuator in the DEF1 DNA repair gene, which includes a portion of the 5′-untranslated region (UTR) and upstream open reading frame (ORF). Using a plasmid-based reporter gene system, we conducted a genetic screen of 14 termination mutants and their ability to confer Pol II read-through defects. The DEF1 attenuator behaved as a hybrid terminator, relying heavily on CPF-CF and Sen1 but without Nrd1 and Nab3 involvement. Our genetic selection identified 22 cis-acting point mutations that clustered into four regions, including a polyadenylation site efficiency element that genetically interacts with its cognate binding-protein Hrp1. Outside of the reporter gene context, a DEF1 attenuator mutant increased mRNA and protein expression, exacerbating the toxicity of a constitutively active Def1 protein. Overall, our data support a biologically significant role for transcription attenuation in regulating DEF1 expression, which can be modulated during the DNA damage response.
Hyperhomocysteinemia (HHcy) is a risk factor for atherosclerosis through mechanisms which are still incompletely defined. One possible mechanism involves the hypomethylation of the nuclear histone proteins to favor the progression of atherosclerosis. In previous cell studies, hypomethylating stress decreased a specific epigenetic tag (the trimethylation of lysine 27 on histone H3, H3K27me3) to promote endothelial dysfunction and activation, i.e., an atherogenic phenotype. Here, we conducted a pilot study to investigate the impact of mild HHcy on vascular methylating index, atherosclerosis progression and H3K27me3 aortic content in apolipoprotein E-deficient (ApoE −/−) mice. In two different sets of experiments, male mice were fed high-fat, low in methyl donors (HFLM), or control (HF) diets for 16 (Study A) or 12 (Study B) weeks. At multiple time points, plasma was collected for (1) quantification of total homocysteine (tHcy) by high-performance liquid chromatography; or (2) the methylation index of S-adenosylmethionine to S-adenosylhomocysteine (SAM:SAH ratio) by liquid chromatography tandem-mass spectrometry; or (3) a panel of inflammatory cytokines previously implicated in atherosclerosis by a multiplex assay. At the end point, aortas were collected and used to assess (1) the methylating index (SAM:SAH ratio); (2) the volume of aortic atherosclerotic plaque assessed by high field magnetic resonance imaging; and (3) the vascular content of H3K27me3 by immunohistochemistry. The results showed that, in both studies, HFLM-fed mice, but not those mice fed control diets, accumulated mildly elevated tHcy plasmatic concentrations. However, the pattern of changes in the inflammatory cytokines did not support a major difference in systemic inflammation between these groups. Accordingly, in both studies, no significant differences were detected for the aortic methylating index, plaque burden, and H3K27me3 vascular content between HF and HFLM-fed mice. Surprisingly however, a decreased plasma SAM: SAH was also observed, suggesting that the plasma compartment does not always reflect the vascular concentrations of these two metabolites, at least in this model. Mild HHcy in vivo was not be sufficient to induce vascular hypomethylating stress or the progression of atherosclerosis, suggesting that only higher accumulations of plasma tHcy will exhibit vascular toxicity and promote specific epigenetic dysregulation.
Hyperhomocysteneinemia (HHcy) is common in the general population and is a risk factor for atherosclerosis by mechanisms that are still elusive. A hypomethylated status of epigenetically relevant targets may contribute to the vascular toxicity associated with HHcy. Ketogenic diets (KD) are diets with a severely restricted amount of carbohydrates that are being widely used, mainly for weight-loss purposes. However, studies associating nutritional ketosis and HHcy are lacking. This pilot study investigates the effects of mild HHcy induced by nutritional manipulation of the methionine metabolism in the absence of dietary carbohydrates on disease progression and specific epigenetic changes in the apolipoprotein-E deficient (apoE–/–) mouse model. ApoE–/– mice were either fed a KD, a diet with the same macronutrient composition but low in methyl donors (low methyl KD, LMKD), or control diet. After 4, 8 or 12 weeks plasma was collected for the quantification of: (1) nutritional ketosis, (i.e., the ketone body beta-hydroxybutyrate using a colorimetric assay); (2) homocysteine by HPLC; (3) the methylating potential S-adenosylmethionine to S-adenosylhomocysteine ratio (AdoHcy/AdoMet) by LC-MS/MS; and (4) the inflammatory cytokine monocyte chemoattractant protein 1 (MCP1) by ELISA. After 12 weeks, aortas were collected to assess: (1) the vascular AdoHcy/AdoMet ratio; (2) the volume of atherosclerotic lesions by high-field magnetic resonance imaging (14T-MRI); and (3) the content of specific epigenetic tags (H3K27me3 and H3K27ac) by immunofluorescence. The results confirmed the presence of nutritional ketosis in KD and LMKD mice but not in the control mice. As expected, mild HHcy was only detected in the LMKD-fed mice. Significantly decreased MCP1 plasma levels and plaque burden were observed in control mice versus the other two groups, together with an increased content of one of the investigated epigenetic tags (H3K27me3) but not of the other (H3K27ac). Moreover, we are unable to detect any significant differences at the p < 0.05 level for MCP1 plasma levels, vascular AdoMet:AdoHcy ratio levels, plaque burden, and specific epigenetic content between the latter two groups. Nevertheless, the systemic methylating index was significantly decreased in LMKD mice versus the other two groups, reinforcing the possibility that the levels of accumulated homocysteine were insufficient to affect vascular transmethylation reactions. Further studies addressing nutritional ketosis in the presence of mild HHcy should use a higher number of animals and are warranted to confirm these preliminary observations.
The dysfunction of vascular endothelial cells is profoundly implicated in the pathogenesis of atherosclerosis and cardiovascular disease, the global leading cause of death. Aquaporins (AQPs) are membrane channels that facilitate water and glycerol transport across cellular membranes recently implicated in the homeostasis of the cardiovascular system. Apolipoprotein-E deficient (apoE−/−) mice are a common model to study the progression of atherosclerosis. Nevertheless, the pattern of expression of AQPs in this atheroprone model is poorly characterized. In this study, apoE−/− mice were fed an atherogenic high-fat (HF) or a control diet. Plasma was collected at multiple time points to assess metabolic disturbances. At the endpoint, the aortic atherosclerotic burden was quantified using high field magnetic resonance imaging. Moreover, the transcriptional levels of several AQP isoforms were evaluated in the liver, white adipocyte tissue (WAT), and brown adipocyte tissue (BAT). The results revealed that HF-fed mice, when compared to controls, presented an exacerbated systemic inflammation and atherosclerotic phenotype, with no major differences in systemic methylation status, circulating amino acids, or plasma total glutathione. Moreover, an overexpression of the isoform AQP5 was detected in all studied tissues from HF-fed mice when compared to controls. These results suggest a novel role for AQP5 on diet-induced atherosclerosis that warrants further investigation.
Objectives Hyperhomocysteinemia is an independent risk factor for atherosclerosis and cardiovascular disease through mechanisms still incompletely defined. We investigated the impact of mild diet-induced accumulation of homocysteine on atherosclerotic plaque formation in apoE knockout (KO) mice, a model for atherosclerosis in humans. We hypothesize that diet induced hyperhomocysteinemia will promote plaque development. Methods 7 wk-old male apoE-KO mice (n = 5) were fed a methyl-deficient diet for 16 wk. The diet consisted of a purified high (40%)-fat high-methionine diet with restricted levels of B vitamins and choline (HypoMet). A second group of animals (control, n = 5) was fed a normal-methyl matched diet. After 4 and 12 wk, plasma homocysteine was quantified by reverse phase HPLC with fluorometric detection, and a panel of inflammatory cytokines (MCP-1; TNF-; IL-17A/F; IL-2; IL-6, IL-10; KC/GRO; MIP-1; IFN-) were assayed (U-plex, Meso Scale Discovery). After 16 wk, mice were euthanized and perfusion-fixed aortas were stained for lipids (Oil Red-O) and subjected to 2-D-quantification of stained plaque (Image J software), and to 3-D analysis by magnetic resonance imaging (MRI, Agilent 14-tesla microimaging system). Standard 3-D gradient echo imaging yielded a resolution of 20 microns isotropic. Data were reconstructed using Matlab and segmented to obtain plaque volumes using Avizo 9.0. Results HypoMet-mice had significantly higher plasma homocysteine (µM), when compared to controls, at 4 wk (10.6 ± 4.5 vs 2.3 ± 0.8, P < 0.05) and 12 wk (7.6 ± 1.5 vs 2.5 ± 1.1, P < 0.05). No significant differences were observed in inflammatory cytokines. Surprisingly, Oil Red-O staining revealed that HypoMet-mice did not display more plaque coverage (37 ± 6.9%) than controls (54.4 ± 10.4%). 14-T MRI results (Fig.1) confirmed that HypoMet-mice did not present higher plaque volumes than controls (0.61 ± 0.18 mm3 vs 1.2 ± 0.35 mm3, for HypoMet mice vs controls). Conclusions A mild accumulation of homocysteine, induced by a methyl-deficient diet, did not favor atherosclerosis formation in the aortas of apoE-KO mice. Future analysis of intracellular hypomethylation may explain the observed effects of mild hyperhomocysteinemia on plaque formation. Funding Sources Graduate Program and University Funding. Supporting Tables, Images and/or Graphs
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