Human native lipoprotein-induced de novo DNA methylation is associated with repression of inflammatory genes in THP-1 macrophages

<p>Abstract</p> <p>Background</p> <p>We previously showed that a VLDL- and LDL-rich mix of human native lipoproteins induces a set of repressive epigenetic marks, <it>i.e. de novo </it>DNA methylation, histone 4 hypoacetylation and histone 4 lysine 20 (H4K20) hypermethylation in THP-1 macrophages. Here, we: 1) ask what gene expression changes accompany these epigenetic responses; 2) test the involvement of candidate factors mediating the latter. We exploited genome expression arrays to identify target genes for lipoprotein-induced silencing, in addition to RNAi and expression studies to test the involvement of candidate mediating factors. The study was conducted in human THP-1 macrophages.</p> <p>Results</p> <p>Native lipoprotein-induced <it>de novo </it>DNA methylation was associated with a general repression of various critical genes for macrophage function, including pro-inflammatory genes. Lipoproteins showed differential effects on epigenetic marks, as <it>de novo </it>DNA methylation was induced by VLDL and to a lesser extent by LDL, but not by HDL, and VLDL induced H4K20 hypermethylation, while HDL caused H4 deacetylation. The analysis of candidate factors mediating VLDL-induced DNA hypermethylation revealed that this response was: 1) surprisingly, mediated exclusively by the canonical maintenance DNA methyltransferase DNMT1, and 2) independent of the Dicer/micro-RNA pathway.</p> <p>Conclusions</p> <p>Our work provides novel insights into epigenetic gene regulation by native lipoproteins. Furthermore, we provide an example of DNMT1 acting as a <it>de novo </it>DNA methyltransferase independently of canonical <it>de novo </it>enzymes, and show proof of principle that <it>de novo </it>DNA methylation can occur independently of a functional Dicer/micro-RNA pathway in mammals.</p

Some practical considerations for linearity assessment of calibration curves as function of concentration levels according to the fitness-for-purpose approach

7 Figurass.-- 1 TablaSince linear calibration is mostly preferred for analytical determinations, linearity in the calibration range is an important performance characteristic of any instrumental analytical method. Linearity can be proved by applying several graphical and numerical approaches. The principal graphical criteria are visual inspection of the calibration plot, the residuals plot, and the response factors plot, also called sensitivity or linearity plot. All of them must include confidence limits in order to visualize linearity deviations. In this work, the graphical representation of percent relative errors of back-calculated concentrations against the concentration of the calibration standards is proposed as linearity criterion. This graph considers a confidence interval based on the expected recovery related to the concentration level according to AOAC approach. To illustrate it, four calibration examples covering different analytical techniques and calibration situations have been studied. The proposed %RE graph was useful in all examples, helping to highlight problems related to non-linear behavior such as points with high leverage and deviations from linearity at the extremes of the calibration range. By this way, a numerical decision limit which takes into account the concentration of calibration standards can be easily included as linearity criterion in the form of %RE=2·C. Accordingly, this %RE parameter is accurate for the decision-making related to linearity assessment according to the fitness-for-purpose approach

Organomegaly and tumors in transgenic mice with targeted expression of HpaII methyltransferase in smooth muscle cells

Current data suggest that angiogenesis, smooth muscle cell migration, differentiation and proliferation may be epigenetically regulated. Prokaryotic DNA methyltransferases have been proposed as tools to modify mammalian DNA methylation. In order to assess the impact of DNA hypermethylation on smooth muscle pathophysiology, we expressed an HpaII site-specific methyltransferase transgene in smooth muscle cells in mice. The enzyme is expected to target only a subset (CCGG) of unmethylated CpG dinucleotides, thus avoiding possible deleterious effects of widespread hypermethylation. Transgenics of two independent lines were born at expected frequencies, showed no obvious abnormalities and were fertile. Nevertheless, ∼30% of >1 year-old transgenics developed organomegaly and ∼20% showed a range of tumors. Global DNA methylation was unchanged in transgenic tissue whether hyperplastic or normal, but tumor DNA showed a pronounced global hypermethylation. DNA hypermethylation was not indiscriminate, as five tested tumor suppressor genes showed promoter CpG and non-CpG hypermethylation and transcriptional downregulation, whereas the methylation status of one intergenic CpG islands, repeated elements (n = 2) and non-tumor suppressor gene promoters (n = 3) was unchanged. Our work is the first report on the effects of HpaII methyltransferase on endogenous chromatin and in a whole animal. Furthermore, our data expand previous findings that imply that global DNA hypomethylation is not an obligate oncogenic pathway at least in the tumor types examined here