Additional file 1 of Mechanisms of blood homeostasis: lineage tracking and a neutral model of cell populations in rhesus macaques

Mathematical appendices and data fitting. (PDF 327 kb

Base composition surrounding XMRV integration sites.

<p>Base compositions of the 4-bp target site duplication (positions D1 to D4; demarcated by the thick vertical lines) and 10 bp upstream (positions −1 to −10) and downstream (positions +1 to +10) of the direct repeat were calculated. The datasets include the 13 integration sites with correct 4-bp direct repeat (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010255#pone-0010255-t001" target="_blank">Table 1</a>), 472 integration sites from acutely infected DU145 cells (GenBank accession numbers EU981292 to EU981799) and 14 integration sites from human prostate cancer tissues (GenBank accession numbers EU981800 to EU981813) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010255#pone.0010255-Kim1" target="_blank">[14]</a>. Integration occurs between positions −1 and D1 on the top strand, and between positions D4 and +1 on the bottom strand (blue arrows). Any base in a position that is significantly overrepresented than the random dataset (<i>P</i><0.0001) is highlighted in green, while any base in a position that is significantly underrepresented than the random dataset (<i>P</i><0.0001) is highlighted in red.</p

Positions of XMRV integration sites and lengths of the target site sequence duplication.

<p>*The nucleotide position corresponds to the position of viral DNA insertion at the top strand of the chromosome indicated. Symbols + and – within the parenthesis indicate the orientation of the viral transcription is the same and opposite, respectively, to the polarity of the top strand. GenBank accession numbers for the integration site sequences are GU816075 to GU816104.</p><p>†The left LTR of the provirus contains a 5-bp deletion that includes the conserved CA dinucleotide at the viral end.</p><p>ψThe target DNA contains a T to A transversion immediately adjacent to the left LTR (position 4).</p

Integration of retroviral DNA and generation of short direct repeats flanking the provirus.

<p>(A) DNA breaking and joining steps during integration. Viral and target DNA strands are represented by thick black and parallel lines, respectively, and the viral long terminal repeats (LTRs) are depicted as grey boxes. Nucleotides at the top and bottom strands are denoted by uppercase and lowercase letters, respectively. During 3′-end processing, IN removes two nucleotides from the 3′ end of each strand of linear viral DNA so that the viral 3′ ends terminate with a conserved CA dinucleotide. Closed arrowheads denote the positions of strand transfer, a concerted cleavage-ligation reaction during which IN makes a staggered break in the target DNA. Host DNA repair enzymes fill in the resulting single-stranded gaps, denoted by D1 to D4 in the upper strand and d1 to d4 in the lower strand of target DNA, and remove the two unpaired nucleotides at the 5′ ends of the viral DNA (open arrowheads), thereby generating the short direct repeats flanking the provirus. (B) A potential pathway for generating a base transversion in the short direct repeat during XMRV integration. A coordinated integration of the two viral ends occurred at the 4-bp staggered positions as depicted by the closed arrowheads. During repair of the single-stranded gap adjacent to the upstream LTR, an adenine nucleotide was introduced at the D4 position either by misincorporation or aberrant processing of the unpaired AA-dinucleotide at the viral 5′ end. Subsequent repair of the mismatch resulted in the observed transversion (denoted by bold types).</p

L1 up-regulation is observed during the reprogramming process and is independent of the transducing vector.

<p>HFF were transduced with either the FRh11 lentiviral vector or the pMX murine γ-retroviral vector encoding <i>OCT4, C-MYC, SOX2</i> and <i>KLF4</i>. Three days post-transduction, the cells were then seeded onto a feeder layer of iMEFs and cultured under hESC conditions. Total cells were collected at 8, 14, 21 and 28 days post-seeding and iMEFs were removed by positive selection. Total RNA extracts were obtained from the remaining human cells which were then subjected to quantitative real-time RT-PCR to assess L1 expression. Total RNA extracts obtained from H1-hESC and iMEFs were used as positive and negative controls, respectively. Quantitative real-time RT-PCR results were normalized with respect to GAPDH content. Fold increase of L1 expression was then calculated with respect to the results of HFF. Results are shown as average ± standard deviation. Asterisks denote statistical significant increase in L1 expression when compared to the reference parental cells as assessed by the Wilcoxon rank sum test (p<0.05).</p

Schematic of PCR strategy for template preparation for 454 sequencing of L1Hs family members (adapted from Ewing et al) [33].

<p>L1Hs libraries were prepared as previously described, except that the 454 primers A and B were used instead of Illumina adapters and that high throughput sequencing was performed by using the primer A instead of the primer B, thus allowing the detection of the polyA (pA) sequence followed by the sequence of the new locus of insertion. The sequences were then processed for mapping on the genome to detect reference as well as non-reference L1Hs insertions. L1Hs reference insertion sequences would match the reference genome from their 3′UTR sequence to the end of their flanking sequence in one location only while non-reference insertion sequences will have their 3′UTR sequence and flanking sequence match the genome on two distinct locations.</p

L1 transcriptional up-regulation in human iPSC clones is independent of donors.

<p>L1 expression was evaluated by quantitative real-time RT-PCR on total RNA extracted from iPSC clones derived from (A) NHDF1 (B) HFF (C) IMR90 cell line. To evaluate the respective basal level of L1 expression, total RNA extracts from the respective parental cells were subjected to real-time PCR. Real-time RT-PCR results were normalized with respect to GAPDH content. Fold increase of L1 expression was then calculated with respect to the result obtained from the parental cells. Results are shown as average ± standard deviation. RNA extracts from the H1 human embryonic stem cell line was used a positive control. Asterisks denote statistical significant increase in L1 expression when compared to the reference parental cells as assessed by the Wilcoxon rank sum test (p<0.05).</p

Summary of the germline insertion results.

<p>Summary of the germline insertion results.</p

Deep Sequencing Reveals Low Incidence of Endogenous LINE-1 Retrotransposition in Human Induced Pluripotent Stem Cells

<div><p>Long interspersed element-1 (LINE-1 or L1) retrotransposition induces insertional mutations that can result in diseases. It was recently shown that the copy number of L1 and other retroelements is stable in induced pluripotent stem cells (iPSCs). However, by using an engineered reporter construct over-expressing L1, another study suggests that reprogramming activates L1 mobility in iPSCs. Given the potential of human iPSCs in therapeutic applications, it is important to clarify whether these cells harbor somatic insertions resulting from endogenous L1 retrotransposition. Here, we verified L1 expression during and after reprogramming as well as potential somatic insertions driven by the most active human endogenous L1 subfamily (L1Hs). Our results indicate that L1 over-expression is initiated during the reprogramming process and is subsequently sustained in isolated clones. To detect potential somatic insertions in iPSCs caused by L1Hs retotransposition, we used a novel sequencing strategy. As opposed to conventional sequencing direction, we sequenced from the 3′ end of L1Hs to the genomic DNA, thus enabling the direct detection of the polyA tail signature of retrotransposition for verification of true insertions. Deep coverage sequencing thus allowed us to detect seven potential somatic insertions with low read counts from two iPSC clones. Negative PCR amplification in parental cells, presence of a polyA tail and absence from seven L1 germline insertion databases highly suggested true somatic insertions in iPSCs. Furthermore, these insertions could not be detected in iPSCs by PCR, likely due to low abundance. We conclude that L1Hs retrotransposes at low levels in iPSCs and therefore warrants careful analyses for genotoxic effects.</p></div

Data_Sheet_1_Expression of vitamin D receptor, CYP24A1, and CYP27B1 in normal and inflamed canine pancreases.DOCX

Vitamin D plays a role in anti-inflammatory processes, and the alteration of its metabolism is associated with the inflammatory processes of pancreatitis. This study was performed to evaluate the expression of the vitamin D receptor (VDR) and the two major enzymes that regulate vitamin D metabolism, 1α-hydroxylase (CYP27B1) and 24-hydroxylase (CYP24A1), in the canine pancreas and to compare their degrees of immunoreactivity between normal and inflamed pancreases. Five normal and inflamed pancreatic tissues each were obtained from six dogs. The expression of VDR, CYP24A1, and CYP27B1 were determined immunohistochemically, and the degree of immunostaining was assessed semiquantitatively. The VDR was expressed in the ducts, acini, and islets of Langerhans of normal pancreases and in the ducts and acini of inflamed ones. There was a significant difference in the immunoreactivity score for VDR in the islets of Langerhans between normal (median, 3 [interquartile range, 2–7.5] score) and inflamed pancreatic tissues (0 [0–0.5] score, p = 0.03). CYP24A1 was expressed in the ducts and islets of Langerhans in both normal and inflamed pancreases, whereas CYP27B1 was expressed in the ducts and acini in only some normal and inflamed pancreatic tissues. This study showed that VDR expression decreased in inflamed pancreases and demonstrated CYP24A1 and CYP27B1 expression in the canine pancreas for the first time. These findings indicate that the pancreas could regulate the metabolism and biological activity of vitamin D and suggest that a decrease in these might be related to the pathophysiology of pancreatitis.</p