Diverged CTCF binding between <i>Drosophila</i> species.

<p>(A) Evolutionary dynamics of CTCF binding profiles at the <i>Bithorax complex</i> region. The four colored wiggle file tracks show the ChIP CDP enrichment scores estimated from our quantitative analysis pipeline for the four species: <i>D. melanogaster</i> (blue), <i>D. simulans</i> (green), <i>D. yakuba</i> (orange), and <i>D. pseudoobscura</i> (purple). The four tracks are at the same scale, with the height of each curve at each coordinate denoting the enrichment score values. In the top panel, the blue arrows point to examples of conserved binding events across the four species, and the red arrows point to examples of diverged binding events between species. The fifth track shows the boundaries of previously identified insulator elements (in sky blue). The last track shows the genes in the genomic region. (B) Number of conserved and diverged binding events. From left to right, the three bar plots show the number of <i>D. melanogaster</i>–specific (pink), shared (blue), and non–<i>D. melanogaster</i> (D.xxx, yellow) specific binding events between each of the species pairs (<i>D. melanogaster/D. simulans</i>, <i>D. melanogaster/D. yakuba</i>, and <i>D. melanogaster/D. pseudoobscura</i>) for all binding events possibly identified (All, left), Two-Way Orthologous Binding events (TWOB, middle), and Four-Way Orthologous Binding events (FWOB, right). TWOB is defined as a binding event identified in regions where the sequence identity between the two compared species is >50%. FWOB is defined as a binding event identified in regions where the sequence identity across all four species is >50%. (C) Linear increase of pair-wise binding divergence with species divergent time. The binding divergence is calculated as the percent of <i>D. melanogaster</i> binding events not shared with the non–<i>D. melanogaster</i> species in each pair-wise comparison. Different shaped and colored points represent different groups of binding events as indicated by the legend. The red dashed line depicts the fitted linear regression line of TWOB binding divergence with divergent time. (D) Evolutionary groups of CTCF binding events. Top panel, representative dynamic binding profiles in the four <i>Drosophila</i> species (<i>D. melanogaster</i>, blue; <i>D. simulans</i>, green; <i>D. yakuba</i>, orange; <i>D. pseudoobscura</i>, purple) illustrating examples of 15 mutually exclusive evolutionary groups of binding status. The height at each binding curve denotes the ChIP CDP enrichment score estimated from our analyses pipeline. For each evolutionary group, the <i>y</i>-axes of the four binding curves are at the same scale. The first row of the lower table shows the Boolean conservation score corresponding to the binding profiles, where 0 indicates absence of binding event and 1 indicates the presence of binding events. The second and third rows of the lower table summarize the number of all binding events (second row) and FWOB events (third row) falling into each evolutionary group. The last row of the lower table shows the inferred evolutionary age for different groups of <i>D. melanogaster</i> binding events using Parsimony methods. * As for the evolutionary group with boolean conservation score 0,1,1,1, there is no instance identified in our analyses, so the representative binding profile in the figure is generated by artificially modifying another binding profile to represent the specific category.</p

Selection on CTCF motif sites.

<p>(A) Proportion of binding sites with conserved motifs. The bar plots show proportions of <i>D. melanogaster</i>–specific (pink) and shared (green) binding sites that have conserved motifs between each species pair. A binding site is defined as having conserved motifs if there is at least one species-specific motif identified in the corresponding orthologous sequences. The <i>p</i> value cutoff for FIMO motif searching here is 0.005. For any species pair, the proportion of conserved (here shared) binding sites having conserved motifs is significantly higher than the diverged (here <i>D. melanogaster</i>–specific) binding sites. Significance levels: * <i>p</i><0.05; ** <i>p</i><0.01, two-sided Fisher's exact test. (B) Mean Tajima's D values for CTCF-motif sites. Tajima's D values were calculated using 37 <i>D. melanogaster</i> North American strains' polymorphism data for various groups of CTCF-motif sites, the synonymous and nonsynonymous sites of nearest genes, and randomly sampled 3′UTR, 5′UTR, and intergenic 9 bp sites. The center of each filled circle depicts the mean Tajima's D value for each group, with the error bar indicating 2 standard deviations. (C and D) Estimated shared proportion of adaptation with neutral reference to nearest gene synonymous sites (C) and a set of small introns (D). <i>D. yakuba</i> sequences were used as an out-group for estimating alpha values for different groups of CTCF-motif sites using an extension of the MK test framework. The filled colored circles depict the shared alpha value estimated within each group, with the error bar indicating the 95% confidence interval. Label abbreviations: Syn, synonymous sites of nearest genes of CTCF binding sites; Nonsyn, non-synonymous sites of nearest genes of CTCF binding sites; TWOB, CTCF-motif sites associated with two-way orthologous binding events between <i>D. melanogaster</i> and the out-group; conserved TWOB, CTCF-motif sites associated with conserved two-way orthologous binding events; diverged TWOB, CTCF-motif sites associated with <i>D. melanogaster</i>–specific two-way othologous binding events; FWOB binding, sites associated with four-way orthologous binding events; Young FWOB, sites associated with FWOBs, for which the age is estimated to be <2.5 Myr; old FWOB, sites associated with FWOBs, for which the age is estimated to be >6 Myr.</p

Functional consequences of CTCF binding evolution.

<p>(A–B) CTCF binding evolution is associated with gene expression evolution. The bar plots show the proportion of genes with diverged expression between (A) <i>D. melanogaster/D. simulans</i> and (B) <i>D. melanogaster/D. yakuba</i> comparisons associated with different groups of CTCF binding sites: Genome-wide (black), Conserved TWOB (pink), Diverged TWOB (green), Old FWOB (orange), and Young FWOB (light purple). The table below each bar plot shows the number of genes with diverged and conserved gene expression in the corresponding comparisons and associated with the corresponding CTCF binding sites. For each groups of CTCF binding sites, the associated genes are the union of the nearest gene to each binding site. The evolutionary status of gene expression (conserved or diverged) is determined using triplicate WPP mRNA-seq data through a generalized linear regression framework. Label abbreviations are the same as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001420#pbio-1001420-g003" target="_blank">Figure 3</a>. Significance levels: * <i>p</i><0.05; **<i>p</i><0.01; one-sided Fisher's exact test. (C–E) CTCF binding evolution is correlated with new gene origination. The four colored wiggle tracks in each of the plots show the ChIP CDP enrichment scores of the four species (<i>D. melanogaster</i>, blue; <i>D. simulans</i>, green; <i>D. yakuba</i>, orange; <i>D. pseudoobscura</i>, purple) across different genomic regions. CTCF binding peaks are observed in <i>D. melanogaster</i>, <i>D. simulans</i>, and <i>D. yakuba</i> at flanking genomic regions of newly evolved genes <i>TFII-A-S2</i> (C) and <i>CheB93a</i> (D). The two genes both originated after the split of the <i>melanogaster</i> group with the <i>pseudoobscura</i> group. CTCF binding peak is only observed in the <i>D. melanogaster</i> genome in the flanking genomic regions of <i>D. melanogaster</i> lineage-specific gene <i>sphinx</i> (E).</p

The evolution of courtship behaviours through the origination of a new gene in Drosophila

New genes can originate by the combination of sequences from unrelated genes or their duplicates to form a chimeric structure. These chimeric genes often evolve rapidly, suggesting that they undergo adaptive evolution and may therefore be involved in novel phenotypes. Their functions, however, are rarely known. Here, we describe the phenotypic effects of a chimeric gene, sphinx, that has recently evolved in Drosophila melanogaster. We show that a knockout of this gene leads to increased male¿male courtship in D. melanogaster, although it leaves other aspects of mating behavior unchanged. Comparative studies of courtship behavior in other closely related Drosophila species suggest that this mutant phenotype of male¿male courtship is the ancestral condition because these related species show much higher levels of male¿male courtship than D. melanogaster. D. melanogaster therefore seems to have evolved in its courtship behaviors by the recruitment of a new chimeric gene

A Combination of Let-7d, Let-7g and Let-7i Serves as a Stable Reference for Normalization of Serum microRNAs

<div><p>Recent studies have indicated that circulating microRNAs (miRNAs) in serum and plasma are stable and can serve as biomarkers of many human diseases. Measurement of circulating miRNAs with sufficient sensitivity and precision, however, faces some special challenges, among which proper normalization is the most critical but often an underappreciated issue. The primary aim of this study was to identify endogenous reference genes that maintain consistent levels under various conditions to serve as an internal control for quantification of serum miRNAs. We developed a strategy combining Illumina’s sequencing by synthesis (SBS) technology, reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay, literature screening and statistical analysis to screen and validate the most suitable reference genes. A combination of let-7d, let-7g and let-7i is selected as a reference for the normalization of serum miRNAs and it is statistically superior to the commonly used reference genes U6, RNU44, RNU48 and miR-16. This has important implications for proper experimental design and accurate data interpretation.</p> </div

Selection of the most stable reference genes by SBS technology.

<p>(<b>A</b>) Sera from cancer patients and healthy participants were pooled separately as described above, and miRNA levels were determined using SBS technology. SBS reads were converted to the log₂ scale. The average log₂-transformed read of each miRNA was plotted against the standard deviation of the log₂-transformed read. MiRNAs highlighted in red are those with higher abundance (log₂-transformed reads > 10) and lower standard deviations (< 1) in the dataset. (<b>B</b>) The average expression values (SBS reads ± standard errors) of the selected miRNAs were plotted. (<b>C</b>) Selection of the most stable reference genes from a panel of 25 genes using geNorm. The geNorm program calculates the average expression stability value (M) for each gene. Genes with the lowest M values are considered the most stable. The least stable gene with the highest M value was automatically excluded for the next calculation round. The x-axis from left to right indicates the ranking of the reference genes according to their expression stability from the least to the most stable, and the y-axis represents the M values of the remaining reference genes. (<b>D</b>) Identification of the optimal number of reference genes for accurate normalization using geNorm. V is the pairwise variation (V_n/V_n+1) between two sequential normalization factors (NF_n and NF_n+1). The magnitude of the change in the normalization factor after the inclusion of an additional reference gene reflects the improvement that is obtained. The authors of geNorm suggest that V > 0.15 should be considered the threshold for including an extra reference gene in the assay, and the least number of genes for each V < 0.15 is selected as the optimal set of genes for normalization. (<b>E</b>) Selection of the most stable reference gene or gene combinations using geNorm. In this case, geNorm indicated that the combination of let-7d, let-7g and let-7i was statistically superior to other combinations or each individually. (<b>F</b>) Identification of the most stable reference genes using NormFinder. The NormFinder algorithm ranks the set of candidate normalization genes according to their expression stability in different groups (e.g., disease versus normal). According to this algorithm, lower stability values of the individual genes indicate greater gene stability. In this case, 23 samples were divided into two groups (12 normal controls and 11 cancer patients). Blue bars represent the stability values of the candidate genes. </p

Characterization of the absolute concentration and the stability of let-7d/g/i in serum.

<p>(<b>A</b>) Dynamic range and sensitivity of the RT-qPCR assay for measuring let-7d/g/i (n = 5). Synthetic single-stranded let-7d/g/i ranging from 0.01 attomole (0.0033 attomole each, equivalent to 6×10³ copies in total) to 10 pmol (3.3 pmol each) were serially diluted over ten orders of magnitude and were assessed by the RT-qPCR assay. The resulting C_q values were plotted versus the amount of input let-7d/g/i to generate a standard curve. An assay using water instead of RNA for reverse-transcription was included as a negative control. (<b>B</b>) Correlation of serum volume to the C_q values (n = 5). Total RNA was extracted from different volumes of serum ranging from 10 µL to 400 µL. The levels of serum let-7d/g/i were assessed by RT-qPCR. The resulting C_q values were plotted versus the serum volume used for RNA extraction. An assay using water instead of RNA for reverse-transcription was included as a negative control. (<b>C</b>) Stability of let-7d/g/i in serum after extended storage (n = 5). Serum samples were equally divided and stored at room temperature, 4°C, -20°C or -80°C for 1, 2, 3, 7, 14 or 30 days. For each time point, total RNA was isolated and let-7d/g/i was measured by RT-qPCR assay. Storage at room temperature for 30 days yielded no apparent increase in C_q values. (<b>D</b>) Instability of other RNAs in serum (n = 5). Serum samples were equally divided and stored at room temperature for 1 to 24 h. For each time point, total RNA was isolated, and the levels of some large molecular weight RNA (β-actin, GAPDH and 28S rRNA) and snRNA/snoRNA (U6, RNU44, RNU48, SNORD24, SNORD38B, SNORD43, SNORA66 and SNORA74A) were measured by RT-qPCR. Storage at room temperature for 24 h resulted in an apparent increase of C_q values for these RNAs. (<b>E</b>) Stability of let-7d/g/i in serum after RNase digestion (n = 5). Serum samples were treated with 10 U/ml RNase A and 400 U/ml RNase T1 for 4 h at 37°C. After the treatment, the RNA was extracted from the serum, and the levels of let-7d/g/i were assessed by RT-qPCR. (<b>F</b>) Instability of other RNAs in serum after RNase digestion (n = 5). Serum samples were treated with 10 U/ml RNase A and 400 U/ml RNase T1 for 1, 2 or 4 h at 37°C. After the treatment, the RNA was extracted and the levels of the indicated RNAs were assessed by RT-qPCR assay. (<b>G</b> and <b>H</b>) Stability of let-7d/g/i under acidic or alkaline conditions (n = 5). Serum samples were incubated for 1 h under acidic (pH 2) or alkaline (pH 12) conditions. The levels of let-7d/g/i were assessed by RT-qPCR. (<b>I</b>) Stability of let-7d/g/i in serum following re-freezing and re-thawing of the samples (n = 6). Serum samples were subjected to eight freeze-thaw cycles.</p

Effect of different normalization approaches on the levels of serum miRNAs.

<p>(<b>A</b>) Serum samples from healthy controls, ulcerative colitis patients and colon cancer patients were divided into three groups (n =5 in each group). After the initial denaturation steps, 1, 2 and 4 attomole of synthetic artificial miRNA (5’-GUGGAUUCCGUCUCGUUAG-3’) were spiked into 100 μL of serum of each group (1 attomole for control group, 2 attomole for ulcerative colitis group and 4 attomole for colon cancer group). After isolation of total RNA, the levels of artificial miRNA were assessed by RT-qPCR assay and were normalized to serum volume, let-7d/g/i, U6 or miR-191, respectively. Relative levels were calculated using the 2^-△△Cq method and were shown by dot plots. Significance was calculated by t-test. (<b>B</b>) Expression levels of miR-25, miR-214, miR-223 and miR-483-5p were measured in serum from cancer patients (n = 84) and healthy controls (n = 41) by RT-qPCR and were normalized to serum volume, let-7d/g/i, U6 or miR-191. Relative levels were calculated using the 2^-△△Cq method and are presented as mean fold changes ± standard errors. Significance was calculated by t-test.</p

Strategy for the identification of stable reference genes for normalizing serum miRNAs.

<p>Strategy for the identification of stable reference genes for normalizing serum miRNAs.</p

Le Courrier

18 septembre 18371837/09/18 (N261)