Distribution of uniquely mapped reads on chromosome X of <i>Drosophila melanogaster</i>.

<p>A snapshot of the University of California, Santa Cruz (UCSC) genome browser shows the distribution of the mapped reads after scaling in reads number per million mapped reads. A and B: The mapped reads in the Dam-DsxM/+ and Dam only (control) genotypes, respectively. C: Reads distribution of Dam-DsxM/+ after the control signal subtraction. D: Distribution of the restriction enzyme <i>Dpnl</i>. Below this the annotated genes are displayed.</p

A Non-Parametric Peak Calling Algorithm for DamID-Seq

<div><p>Protein—DNA interactions play a significant role in gene regulation and expression. In order to identify transcription factor binding sites (TFBS) of double sex (DSX)—an important transcription factor in sex determination, we applied the DNA adenine methylation identification (DamID) technology to the fat body tissue of <i>Drosophila</i>, followed by deep sequencing (DamID-Seq). One feature of DamID-Seq data is that induced adenine methylation signals are not assured to be symmetrically distributed at TFBS, which renders the existing peak calling algorithms for ChIP-Seq, including SPP and MACS, inappropriate for DamID-Seq data. This challenged us to develop a new algorithm for peak calling. A challenge in peaking calling based on sequence data is estimating the averaged behavior of background signals. We applied a bootstrap resampling method to short sequence reads in the control (Dam only). After data quality check and mapping reads to a reference genome, the peaking calling procedure compromises the following steps: 1) reads resampling; 2) reads scaling (normalization) and computing signal-to-noise fold changes; 3) filtering; 4) Calling peaks based on a statistically significant threshold. This is a non-parametric method for peak calling (NPPC). We also used irreproducible discovery rate (IDR) analysis, as well as ChIP-Seq data to compare the peaks called by the NPPC. We identified approximately 6,000 peaks for DSX, which point to 1,225 genes related to the fat body tissue difference between female and male <i>Drosophila</i>. Statistical evidence from IDR analysis indicated that these peaks are reproducible across biological replicates. In addition, these peaks are comparable to those identified by use of ChIP-Seq on S2 cells, in terms of peak number, location, and peaks width.</p></div

Number of peaks validated by the IDR analysis.

<p>A&B: Using the NPPC algorithm, we identified 6,157 and 5,836 between-replicate shared peaks for DsxF and DsxM, respectively. C&D: IDR analysis between biological replicates in DsxF and DsxM, respectively. According to Li et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117415#pone.0117415.ref008" target="_blank">8</a>], a correspondence curve describes the function between a parameter of top t% ranked peaks and corresponding rank intersection between the two replicates. The slope (y-axis) is the first derivative of the function against the parameter. The slope change from 0 to larger than 0 represents the decay point of inconsistency. E: The relationship between ranked peaks and the overall IDR. Corresponding to the top 6,000 peaks, the IDR is 5%.</p

Relationships between local irreproducible discovery rate (idr) and log2 fold changes.

<p>Local idr is the probability that two peaks are irreproducible. We use two sets of peaks to illustrate the intrinsic association of local idr and log2 fold changes in DsxF: the set of 8,500 overlapping peaks (≥ 50% bp) between the two biological replicates (A through C), and the set of 6,000 reproducible peaks by IDR analysis (D through F). A and D: Plots of the log2 fold changes between the two replicates. B and E: Plots of the log2 fold changes against the local idr (in -log2 scale) in replicate 1. C and F: Similar plots in replicate 2. Comparisons of the two sets of peaks indicate that IDR analysis is essentially to find the low end cutoff of the log2 fold changes.</p

Distribution of peaks in gene features.

<p>A: On the basis of the <i>Drosophila melanogaster</i> gene annotation (Flybase.r5.38), we aligned the approximately 6,000 peaks of DsxF against the gene features. The majority of the peaks fall into less than one Kb promoters and intron regions, accounting for 61% together; followed by distal intergenic regions (9.7%) and 5′ UTR regions (5.1%). B: In order to identify the sequence motif of DSX binding sites, we performed a <i>de novo</i> search using the MEME algorithm (<a href="http://meme.nbcr.net/meme/" target="_blank">http://meme.nbcr.net/meme/</a>), based on the middle 200-bp DNA sequences of the reproducible peaks.</p

The unweighted pair group method average (UPGMA) based dendrogram of 41 white birch genotypes from six geographical locations, based on their allelic constitution at 111 SSR loci. H1-H3, Huanren provenance, China; L1-L4, Liangshui provenance, China; X1-X7, Xiaobeihu provenance, China; Q1-Q7, Qingyuan provenance, China; M1-M15, Maoershan provenance, China; F1-F5, Finland provenance, Finland.

<p>The unweighted pair group method average (UPGMA) based dendrogram of 41 white birch genotypes from six geographical locations, based on their allelic constitution at 111 SSR loci. H1-H3, Huanren provenance, China; L1-L4, Liangshui provenance, China; X1-X7, Xiaobeihu provenance, China; Q1-Q7, Qingyuan provenance, China; M1-M15, Maoershan provenance, China; F1-F5, Finland provenance, Finland.</p

An example of SSR variation at the BP-293 locus across 41 white birch genotypes.

<p>M, DL2000 DNA ladder Marker; 1–41, each represents one of the 41 white birch plants.</p

Multiple sequence alignment of BP-293 showing the position of SSR motifs, and expansion and contraction of the motif.

<p>Multiple sequence alignment of BP-293 showing the position of SSR motifs, and expansion and contraction of the motif.</p

Image_2_Isoprenoid biosynthesis regulation in poplars by methylerythritol phosphate and mevalonic acid pathways.TIF

It is critical to develop plant isoprenoid production when dealing with human-demanded industries such as flavoring, aroma, pigment, pharmaceuticals, and biomass used for biofuels. The methylerythritol phosphate (MEP) and mevalonic acid (MVA) plant pathways contribute to the dynamic production of isoprenoid compounds. Still, the cross-talk between MVA and MEP in isoprenoid biosynthesis is not quite recognized. Regarding the rate-limiting steps in the MEP pathway through catalyzing 1-deoxy-D-xylulose5-phosphate synthase and 1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR) and also the rate-limiting step in the MVA pathway through catalyzing 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the characterization and function of HMGR from Populus trichocarpa (PtHMGR) were analyzed. The results indicated that PtHMGR overexpressors (OEs) displayed various MEP and MVA-related gene expressions compared to NT poplars. The overexpression of PtDXR upregulated MEP-related genes and downregulated MVA-related genes. The overexpression of PtDXR and PtHMGR affected the isoprenoid production involved in both MVA and MEP pathways. Here, results illustrated that the PtHMGR and PtDXR play significant roles in regulating MEP and MVA-related genes and derived isoprenoids. This study clarifies cross-talk between MVA and MEP pathways. It demonstrates the key functions of HMGR and DXR in this cross-talk, which significantly contribute to regulate isoprenoid biosynthesis in poplars.</p

Table_1_Isoprenoid biosynthesis regulation in poplars by methylerythritol phosphate and mevalonic acid pathways.XLS

It is critical to develop plant isoprenoid production when dealing with human-demanded industries such as flavoring, aroma, pigment, pharmaceuticals, and biomass used for biofuels. The methylerythritol phosphate (MEP) and mevalonic acid (MVA) plant pathways contribute to the dynamic production of isoprenoid compounds. Still, the cross-talk between MVA and MEP in isoprenoid biosynthesis is not quite recognized. Regarding the rate-limiting steps in the MEP pathway through catalyzing 1-deoxy-D-xylulose5-phosphate synthase and 1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR) and also the rate-limiting step in the MVA pathway through catalyzing 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the characterization and function of HMGR from Populus trichocarpa (PtHMGR) were analyzed. The results indicated that PtHMGR overexpressors (OEs) displayed various MEP and MVA-related gene expressions compared to NT poplars. The overexpression of PtDXR upregulated MEP-related genes and downregulated MVA-related genes. The overexpression of PtDXR and PtHMGR affected the isoprenoid production involved in both MVA and MEP pathways. Here, results illustrated that the PtHMGR and PtDXR play significant roles in regulating MEP and MVA-related genes and derived isoprenoids. This study clarifies cross-talk between MVA and MEP pathways. It demonstrates the key functions of HMGR and DXR in this cross-talk, which significantly contribute to regulate isoprenoid biosynthesis in poplars.</p