Dimer Formation Enhances Structural Differences between Amyloid β-Protein (1–40) and (1–42): An Explicit-Solvent Molecular Dynamics Study

Amyloid -protein (A) is central to the pathology of Alzheimer's disease. A 5% difference in the primary structure of the two predominant alloforms, A and A, results in distinct assembly pathways and toxicity properties. Discrete molecular dynamics (DMD) studies of A and A assembly resulted in alloform-specific oligomer size distributions consistent with experimental findings. Here, a large ensemble of DMD–derived A and A monomers and dimers was subjected to fully atomistic molecular dynamics (MD) simulations using the OPLS-AA force field combined with two water models, SPCE and TIP3P. The resulting all-atom conformations were slightly larger, less compact, had similar turn and lower -strand propensities than those predicted by DMD. Fully atomistic A and A monomers populated qualitatively similar free energy landscapes. In contrast, the free energy landscape of A dimers indicated a larger conformational variability in comparison to that of A dimers. A dimers were characterized by an increased flexibility in the N-terminal region D1-R5 and a larger solvent exposure of charged amino acids relative to A dimers. Of the three positively charged amino acids, R5 was the most and K16 the least involved in salt bridge formation. This result was independent of the water model, alloform, and assembly state. Overall, salt bridge propensities increased upon dimer formation. An exception was the salt bridge propensity of K28, which decreased upon formation of A dimers and was significantly lower than in A dimers. The potential relevance of the three positively charged amino acids in mediating the A oligomer toxicity is discussed in the light of available experimental data

The importance of vibronic perturbations in ferrocytochrome c spectra: a re-evaluation of spectral properties based on low-temperature optical absorption, resonance Raman, and molecular-dynamics simulations.

We have measured and analyzed the low-temperature (T=10 K) absorption spectrum of reduced horse heart and yeast cytochrome c. Both spectra show split and asymmetric Q(0) and Q(v) bands. The spectra were first decomposed into the individual split vibronic sidebands assignable to B-1g (nu(15)) and A(2g) (nu(19), nu(21), and nu(22)) Herzberg-Teller active modes due to their strong intensity in resonance Raman spectra acquired with Q(0) and Q(v) excitations. The measured band splittings and asymmetries cannot be rationalized solely in terms of electronic perturbations of the heme macrocycle. On the contrary, they clearly point to the importance of considering not only electronic perturbations but vibronic perturbations as well. The former are most likely due to the heterogeneity of the electric field produced by charged side chains in the protein environment, whereas the latter reflect a perturbation potential due to multiple heme-protein interactions, which deform the heme structure in the ground and excited states. Additional information about vibronic perturbations and the associated ground-state deformations are inferred from the depolarization ratios of resonance Raman bands. The results of our analysis indicate that the heme group in yeast cytochrome c is more nonplanar and more distorted along a B-2g coordinate than in horse heart cytochrome c. This conclusion is supported by normal structural decomposition calculations performed on the heme extracted from molecular-dynamic simulations of the two investigated proteins. Interestingly, the latter are somewhat different from the respective deformations obtained from the x-ray structures

Coordinate Regulation of DNA Methylation and H3K27me3 in Mouse Embryonic Stem Cells

<div><p>Chromatin is separated into functional domains distinguished by combinatorial patterns of post-translational histone modifications and DNA methylation. Recent studies examining multiple histone modifications have found numerous chromatin states with distinct profiles of chromatin marks and functional enrichments. There are data showing coordinate regulation between DNAme and H3K27me3, which are both involved in the establishment and maintenance of epigenetic gene silencing, but the data are conflicting. Multiple studies have presented evidence to support the theory that PRC2 and DNAme cooperate to achieve silencing, or alternatively that H3K27me3 and DNAme act antagonistically. Here we examine the effect loss of either PRC2 or DNA methyltransferase activity has on the placement of the reciprocal mark in mouse ES cells. We find that DNAme is acting globally to antagonize the placement of H3K27me3, in accordance with recently published results. At least 471,011 domains in the mouse genome acquire H3K27me3 when DNAme is diminished. Of these 466,563 have been shown to be fully methylated in wildtype ES cells, indicating the effects of DNAme on H3K27me3 are direct. In a reciprocal experiment, we examine the effect loss of PRC2 has on the placement of DNAme. In contrast to the global antagonism DNAme has on the placement of H3K27me3, loss of H3K27me3 has a modest effect on DNAme, with only 4% of genes undergoing changes in DNAme, including 861 showing increases and 552 showing losses of overall DNAme. We anticipate that integrating genomic datasets where the effect of loss of a particular epigenetic mark has on the placement of other marks will help elucidate the rules governing epigenetic regulation and what role coordinate regulation of epigenetic marks plays in development and disease.</p> </div

Successful Computational Prediction of Novel Imprinted Genes from Epigenomic Features▿ †

Approximately 100 mouse genes undergo genomic imprinting, whereby one of the two parental alleles is epigenetically silenced. Imprinted genes influence processes including development, X chromosome inactivation, obesity, schizophrenia, and diabetes, motivating the identification of all imprinted loci. Local sequence features have been used to predict candidate imprinted genes, but rigorous testing using reciprocal crosses validated only three, one of which resided in previously identified imprinting clusters. Here we show that specific epigenetic features in mouse cells correlate with imprinting status in mice, and we identify hundreds of additional genes predicted to be imprinted in the mouse. We used a multitiered approach to validate imprinted expression, including use of a custom single nucleotide polymorphism array and traditional molecular methods. Of 65 candidates subjected to molecular assays for allele-specific expression, we found 10 novel imprinted genes that were maternally expressed in the placenta

<i>Eed^−/−</i> and <i>Dnmt^TKO</i> cells have similar gene expression changes relative to wildtype cells by RNA-seq.

<p><b>a,</b> Number of genes in <i>Dnmt^TKO</i> and <i>Eed^−/−</i> cells with significant changes in expression relative to wildtype cells. <b>b,</b> Boxplot of mean fold change in expression level relative to wildtype. <b>c,</b> Venn diagram showing number of genes with significant expression level changes common to both <i>Eed^−/−</i> and <i>Dnmt^TKO</i> cells. Significance of common genes determined by chi-square test, df = 1 (p<.0001). <b>d,</b> Gene ontology analysis of genes commonly misregulated in both <i>Eed^−/−</i> and <i>Dnmt^TKO</i> cells. <b>e,</b> Classification of genes commonly misregulated in <i>Dnmt^TKO</i> and <i>Eed^−/−</i> cells based on promoter CpG content, or H3K4me3 and H3K27me3 marks. Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053880#pone.0053880-Mikkelsen1" target="_blank">[5]</a>.</p

Loss of PRC2 activity leads to changes in DNA methylation. a,

<p>Relative fluorescence ratios for each probe from three independent MeDIP-chip experiments across the Nkx2-1 promoter. The peak of increased DNA methylation is indicated under the probes (grey bar) and the first 1 kb of the gene is indicated on the bottom. <b>b,</b> Validation of the peak of increased DNA methylation by bisulfite PCR. Each line represents an individual clone. Methylated CpGs are indicated by filled-in circles. <b>c,</b> A Fisher’s exact test was conducted for each CpG in (b) (** p<.01, *** p<.001). <b>d,</b> Profile of average DNA methylation relative to TSS calculated in 100 bp bins. <b>e,</b> Hierarchical clustering was performed on MeDIP-chip enrichment profiles to identify genes with similar profiles. 1,282 genes that passed the filtering step of the clustering software are on the y-axis. The x-axis is based on average fluorescent ratios in 100 bp bins from −3 kb (left) to +1 kb (right). Red indicates increased DNA methylation and green indicates decreased DNA methylation while black indicates unchanged DNA methylation. <b>f,</b> Gene ontology classifications for genes with increased (red) or decreased (green) DNAme. <b>g,</b> Gene ontology classifications for genes with increased DNAme upstream of the TSS (cluster 1, white) or across the entire promoter (cluster 2, grey). <b>h,</b> Classification of promoters based on CpG content. HCP, ICP & LCP, High-, Intermediate- & low CpG content promoter. <b>i,</b> Classification of promoters based on presence of H3K27me3 & H3K4me3. CpG and bivalent data used in (h) and (i) from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053880#pone.0053880-Mikkelsen1" target="_blank">[5] </a><b>j,</b> Boxplot of expression level change for genes enriched or depleted for DNAme in <i>Eed^−/−</i> cells as well as for each of the two clusters described in (e).</p

Global antagonism to H3K27me3 in <i>Dnmt^TKO</i> cells. a,

<p>Classification of promoters identified in ChIP-seq experiment based on presence of H3K27me3 and H3K4me3 in wildtype cells. H3K4 and H3K27 methylation data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053880#pone.0053880-Mikkelsen1" target="_blank">[5]</a>. <b>b,</b> Profile of enrichment of ChIP-seq tags in 100 bp bins across the promoter for all genes with or without peaks of increased H3K27me3 in <i>Dnmt^TKO</i> cells. <b>c,</b> Distribution of ChIP-seq reads according to genomic features. <b>d,</b> Number of ChIP-seq peaks intersecting with either fully-, low- or unmethylated regions according to data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053880#pone.0053880-Stadler1" target="_blank">[26]</a>. <b>e,</b> Expression level of <i>Eed</i> in v6.5 and <i>Dnmt^TKO</i> cells by qRT-PCR. <b>f,</b> Western blot analysis of EZH2 in v6.5 and <i>Dnmt^TKO</i> cells. Relative intensity of EZH2 band from calculated using ImageJ is shown on the bottom. Intensity levels of EZH2 are normalized to Tubulin. <b>h,</b> Boxplot of expression level change for genes enriched in H3K27me3 in <i>Dnmt^TKO</i> cells.</p