<i>In vitro</i> binding of h PARP-1 to the h <i>c-myc</i> GQ structure and the effect of TMPyP4 on that binding.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042690#pone-0042690-g001" target="_blank">Figure 1A</a> Effect of TMPyP4 on the binding of PARP-1 to the <i>c-myc</i> GQ structure. 0.45 pmol aliquots of h PARP-1 were incubated with 5 pmols of 5′-biotine end-labeled wild type h <i>c-myc</i> GQ oligonucleotide (wild type GQ, 5′-biotin-TGG GGA GGG TGG GGA GGG TGG GGA AGG) in the absence and in the presence of different concentrations of the cationic porphyrin compound TMPyP4 as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042690#s4" target="_blank">Materials and Methods</a>. Binding is expressed as % of binding measured in the absence of competing TMPyP4 and is shown on the ordinate. TMPyP4 concentrations (0, 0.58, 2.9, 14.4, and 72 µM) are shown on the abscissa. Asterisks represent samples where the extent of binding are significantly different (p<0.05, Student t-test). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042690#pone-0042690-g001" target="_blank">Figure 1B </a><i>In vitro</i> binding of h PARP-1 to various GQ structures and the competing effect of TMPyP4 on that binding. 0.45 pmol aliquots of h PARP-1 were incubated either with 5 pmols h telomeric GQ structure (h-telomeric GQ, 5′-biotin-TTA GGG TTA GGG TTA GGG TTA GGG) or with wild type <i>c-myc</i> GQ structure (wild type GQ, 5′-biotin-TGG GGA GGG TGG GGA GGG TGG GGA AGG) or its mutants (mutant-1 GQ, 5′-biotin-TGG GGA GGG TG<b>A</b> GGA GGG TGG GGA AGG, mutant-2 GQ, 5′-biotin-TG<b>A</b> GGA GGG TGG GGA G<b>A</b>G TGG GGA AGG). The sites of mutations are shown in bold. Binding assay was carried out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042690#s4" target="_blank">Materials and Methods</a>. Ordinate shows the binding of PARP-1 to GQ structures (telomeric GQ, <i>c-myc</i> GQ, G-131A and G-140, -126A) expressed in percentage. The amount of PARP-1 bound to telomeric GQ is taken as 100%. Insert shows the applied concentrations of TMPyP4. Asterisks represent samples where the difference in binding is significantly different (p<0.05).</p

ChIP-qPCR analysis of <i>in vivo</i> binding of h PARP-1 to the NHE III₁ region present in the promoter of the h <i>c-myc</i> gene, and which is presumably either in the GQ form or in the double stranded B-DNA form.

<p>ChIP-qPCR analysis was carried out as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042690#s4" target="_blank">Materials and Methods</a> section using a polyclonal antibody raised against PARP-1 (Santa Cruz, H250; 2 µg/extract of one million cells) as precipitation agent. The treatment modalities and the ratio of PARP-1 bound promoter DNAs, isolated from differently treated cells (HeLa and HL60), and amplified by qPCR and calculated from the fluorescence of the Eva-Green complexes formed with the double-stranded PCR products, are shown. The c1/c2 values represent the ratio of the concentrations of PARP-1 bound <i>c-myc</i> promoter DNAs present in the ChIP products obtained from the treated (1) and from the non-treated (2) cell populations and calculated using the c1/c2 = 2^{n2 – n1} formula, where n1 and n2 are the number of PCR cycles needed to reach the same fluorescence value in the logarithmic phase of the PCR curves.</p

Influence of nuclear proteins on the kinetics of unfolding of the h <i>c-myc</i> GQ structure, assayed by measuring the decrease in FRET activities in the presence of the complementary DNA strand.

*<p>decrease in FRET activity during one minute (% of decrease in the maximal height of the FRET peak measured at 0 time).</p>‡<p>number of experiments.</p><p>F-<i>myc</i> GQ-R double labeled FRET oligonucleotide (25 pmol) was incubated either alone or with the tested proteins for one minute in a volume of 50 µl, than its FRET spectra was taken (0 min value). The complementary, antiparallel oligonucleotide to the GQ structure (in a tenfold molar excess) was admixed and FRET spectra was recorded at 1, 3 and 5 minutes after annealing has been initiated. The kinetics of the decrease of the FRET peak heights were calculated and shown as ΔFRET peak value during one minute of annealing as percentage of the zero minute FRET peak height values. Excitation was at 485 nm and emissions were recorded between 500–650 nms.</p

The expression of h PARP-1 <i>in vivo</i> increases the luciferase enzyme activities assayed in Del4 reporter plasmid transfected PARP −/− MEF cells.

<p>Logarithmically growing PARP −/− MEF cells were transfected with Del-4 plasmid together with the pcDNA3.1-<i>beta-galactosidase</i> expressing plasmid and in the absence or in the presence of h PARP-1 expression (pcDNA3.1-<i>parp-1</i> plasmid). After two days of incubation cells were splitted into six-well plates and grown for a day further. Than cells were harvested, lysed and their luciferase and beta-galactosidase enzyme activities were determined. The luciferase enzyme activities were normalized for beta-galactosidase activities and are shown in the figure. Asterisks show significant difference in the reporter enzyme activities between the two pools of sample (p<0.05).</p

Image_10_Integrated Systems Biology Approach Identifies Novel Maternal and Placental Pathways of Preeclampsia.pdf

<p>Preeclampsia is a disease of the mother, fetus, and placenta, and the gaps in our understanding of the complex interactions among their respective disease pathways preclude successful treatment and prevention. The placenta has a key role in the pathogenesis of the terminal pathway characterized by exaggerated maternal systemic inflammation, generalized endothelial damage, hypertension, and proteinuria. This sine qua non of preeclampsia may be triggered by distinct underlying mechanisms that occur at early stages of pregnancy and induce different phenotypes. To gain insights into these molecular pathways, we employed a systems biology approach and integrated different “omics,” clinical, placental, and functional data from patients with distinct phenotypes of preeclampsia. First trimester maternal blood proteomics uncovered an altered abundance of proteins of the renin-angiotensin and immune systems, complement, and coagulation cascades in patients with term or preterm preeclampsia. Moreover, first trimester maternal blood from preterm preeclamptic patients in vitro dysregulated trophoblastic gene expression. Placental transcriptomics of women with preterm preeclampsia identified distinct gene modules associated with maternal or fetal disease. Placental “virtual” liquid biopsy showed that the dysregulation of these disease gene modules originates during the first trimester. In vitro experiments on hub transcription factors of these gene modules demonstrated that DNA hypermethylation in the regulatory region of ZNF554 leads to gene down-regulation and impaired trophoblast invasion, while BCL6 and ARNT2 up-regulation sensitizes the trophoblast to ischemia, hallmarks of preterm preeclampsia. In summary, our data suggest that there are distinct maternal and placental disease pathways, and their interaction influences the clinical presentation of preeclampsia. The activation of maternal disease pathways can be detected in all phenotypes of preeclampsia earlier and upstream of placental dysfunction, not only downstream as described before, and distinct placental disease pathways are superimposed on these maternal pathways. This is a paradigm shift, which, in agreement with epidemiological studies, warrants for the central pathologic role of preexisting maternal diseases or perturbed maternal–fetal–placental immune interactions in preeclampsia. The description of these novel pathways in the “molecular phase” of preeclampsia and the identification of their hub molecules may enable timely molecular characterization of patients with distinct preeclampsia phenotypes.</p

Image_8_Integrated Systems Biology Approach Identifies Novel Maternal and Placental Pathways of Preeclampsia.pdf

<p>Preeclampsia is a disease of the mother, fetus, and placenta, and the gaps in our understanding of the complex interactions among their respective disease pathways preclude successful treatment and prevention. The placenta has a key role in the pathogenesis of the terminal pathway characterized by exaggerated maternal systemic inflammation, generalized endothelial damage, hypertension, and proteinuria. This sine qua non of preeclampsia may be triggered by distinct underlying mechanisms that occur at early stages of pregnancy and induce different phenotypes. To gain insights into these molecular pathways, we employed a systems biology approach and integrated different “omics,” clinical, placental, and functional data from patients with distinct phenotypes of preeclampsia. First trimester maternal blood proteomics uncovered an altered abundance of proteins of the renin-angiotensin and immune systems, complement, and coagulation cascades in patients with term or preterm preeclampsia. Moreover, first trimester maternal blood from preterm preeclamptic patients in vitro dysregulated trophoblastic gene expression. Placental transcriptomics of women with preterm preeclampsia identified distinct gene modules associated with maternal or fetal disease. Placental “virtual” liquid biopsy showed that the dysregulation of these disease gene modules originates during the first trimester. In vitro experiments on hub transcription factors of these gene modules demonstrated that DNA hypermethylation in the regulatory region of ZNF554 leads to gene down-regulation and impaired trophoblast invasion, while BCL6 and ARNT2 up-regulation sensitizes the trophoblast to ischemia, hallmarks of preterm preeclampsia. In summary, our data suggest that there are distinct maternal and placental disease pathways, and their interaction influences the clinical presentation of preeclampsia. The activation of maternal disease pathways can be detected in all phenotypes of preeclampsia earlier and upstream of placental dysfunction, not only downstream as described before, and distinct placental disease pathways are superimposed on these maternal pathways. This is a paradigm shift, which, in agreement with epidemiological studies, warrants for the central pathologic role of preexisting maternal diseases or perturbed maternal–fetal–placental immune interactions in preeclampsia. The description of these novel pathways in the “molecular phase” of preeclampsia and the identification of their hub molecules may enable timely molecular characterization of patients with distinct preeclampsia phenotypes.</p

Image_7_Integrated Systems Biology Approach Identifies Novel Maternal and Placental Pathways of Preeclampsia.pdf

<p>Preeclampsia is a disease of the mother, fetus, and placenta, and the gaps in our understanding of the complex interactions among their respective disease pathways preclude successful treatment and prevention. The placenta has a key role in the pathogenesis of the terminal pathway characterized by exaggerated maternal systemic inflammation, generalized endothelial damage, hypertension, and proteinuria. This sine qua non of preeclampsia may be triggered by distinct underlying mechanisms that occur at early stages of pregnancy and induce different phenotypes. To gain insights into these molecular pathways, we employed a systems biology approach and integrated different “omics,” clinical, placental, and functional data from patients with distinct phenotypes of preeclampsia. First trimester maternal blood proteomics uncovered an altered abundance of proteins of the renin-angiotensin and immune systems, complement, and coagulation cascades in patients with term or preterm preeclampsia. Moreover, first trimester maternal blood from preterm preeclamptic patients in vitro dysregulated trophoblastic gene expression. Placental transcriptomics of women with preterm preeclampsia identified distinct gene modules associated with maternal or fetal disease. Placental “virtual” liquid biopsy showed that the dysregulation of these disease gene modules originates during the first trimester. In vitro experiments on hub transcription factors of these gene modules demonstrated that DNA hypermethylation in the regulatory region of ZNF554 leads to gene down-regulation and impaired trophoblast invasion, while BCL6 and ARNT2 up-regulation sensitizes the trophoblast to ischemia, hallmarks of preterm preeclampsia. In summary, our data suggest that there are distinct maternal and placental disease pathways, and their interaction influences the clinical presentation of preeclampsia. The activation of maternal disease pathways can be detected in all phenotypes of preeclampsia earlier and upstream of placental dysfunction, not only downstream as described before, and distinct placental disease pathways are superimposed on these maternal pathways. This is a paradigm shift, which, in agreement with epidemiological studies, warrants for the central pathologic role of preexisting maternal diseases or perturbed maternal–fetal–placental immune interactions in preeclampsia. The description of these novel pathways in the “molecular phase” of preeclampsia and the identification of their hub molecules may enable timely molecular characterization of patients with distinct preeclampsia phenotypes.</p

Image_9_Integrated Systems Biology Approach Identifies Novel Maternal and Placental Pathways of Preeclampsia.pdf

<p>Preeclampsia is a disease of the mother, fetus, and placenta, and the gaps in our understanding of the complex interactions among their respective disease pathways preclude successful treatment and prevention. The placenta has a key role in the pathogenesis of the terminal pathway characterized by exaggerated maternal systemic inflammation, generalized endothelial damage, hypertension, and proteinuria. This sine qua non of preeclampsia may be triggered by distinct underlying mechanisms that occur at early stages of pregnancy and induce different phenotypes. To gain insights into these molecular pathways, we employed a systems biology approach and integrated different “omics,” clinical, placental, and functional data from patients with distinct phenotypes of preeclampsia. First trimester maternal blood proteomics uncovered an altered abundance of proteins of the renin-angiotensin and immune systems, complement, and coagulation cascades in patients with term or preterm preeclampsia. Moreover, first trimester maternal blood from preterm preeclamptic patients in vitro dysregulated trophoblastic gene expression. Placental transcriptomics of women with preterm preeclampsia identified distinct gene modules associated with maternal or fetal disease. Placental “virtual” liquid biopsy showed that the dysregulation of these disease gene modules originates during the first trimester. In vitro experiments on hub transcription factors of these gene modules demonstrated that DNA hypermethylation in the regulatory region of ZNF554 leads to gene down-regulation and impaired trophoblast invasion, while BCL6 and ARNT2 up-regulation sensitizes the trophoblast to ischemia, hallmarks of preterm preeclampsia. In summary, our data suggest that there are distinct maternal and placental disease pathways, and their interaction influences the clinical presentation of preeclampsia. The activation of maternal disease pathways can be detected in all phenotypes of preeclampsia earlier and upstream of placental dysfunction, not only downstream as described before, and distinct placental disease pathways are superimposed on these maternal pathways. This is a paradigm shift, which, in agreement with epidemiological studies, warrants for the central pathologic role of preexisting maternal diseases or perturbed maternal–fetal–placental immune interactions in preeclampsia. The description of these novel pathways in the “molecular phase” of preeclampsia and the identification of their hub molecules may enable timely molecular characterization of patients with distinct preeclampsia phenotypes.</p

Image_2_Integrated Systems Biology Approach Identifies Novel Maternal and Placental Pathways of Preeclampsia.pdf

<p>Preeclampsia is a disease of the mother, fetus, and placenta, and the gaps in our understanding of the complex interactions among their respective disease pathways preclude successful treatment and prevention. The placenta has a key role in the pathogenesis of the terminal pathway characterized by exaggerated maternal systemic inflammation, generalized endothelial damage, hypertension, and proteinuria. This sine qua non of preeclampsia may be triggered by distinct underlying mechanisms that occur at early stages of pregnancy and induce different phenotypes. To gain insights into these molecular pathways, we employed a systems biology approach and integrated different “omics,” clinical, placental, and functional data from patients with distinct phenotypes of preeclampsia. First trimester maternal blood proteomics uncovered an altered abundance of proteins of the renin-angiotensin and immune systems, complement, and coagulation cascades in patients with term or preterm preeclampsia. Moreover, first trimester maternal blood from preterm preeclamptic patients in vitro dysregulated trophoblastic gene expression. Placental transcriptomics of women with preterm preeclampsia identified distinct gene modules associated with maternal or fetal disease. Placental “virtual” liquid biopsy showed that the dysregulation of these disease gene modules originates during the first trimester. In vitro experiments on hub transcription factors of these gene modules demonstrated that DNA hypermethylation in the regulatory region of ZNF554 leads to gene down-regulation and impaired trophoblast invasion, while BCL6 and ARNT2 up-regulation sensitizes the trophoblast to ischemia, hallmarks of preterm preeclampsia. In summary, our data suggest that there are distinct maternal and placental disease pathways, and their interaction influences the clinical presentation of preeclampsia. The activation of maternal disease pathways can be detected in all phenotypes of preeclampsia earlier and upstream of placental dysfunction, not only downstream as described before, and distinct placental disease pathways are superimposed on these maternal pathways. This is a paradigm shift, which, in agreement with epidemiological studies, warrants for the central pathologic role of preexisting maternal diseases or perturbed maternal–fetal–placental immune interactions in preeclampsia. The description of these novel pathways in the “molecular phase” of preeclampsia and the identification of their hub molecules may enable timely molecular characterization of patients with distinct preeclampsia phenotypes.</p

Image_13_Integrated Systems Biology Approach Identifies Novel Maternal and Placental Pathways of Preeclampsia.pdf

<p>Preeclampsia is a disease of the mother, fetus, and placenta, and the gaps in our understanding of the complex interactions among their respective disease pathways preclude successful treatment and prevention. The placenta has a key role in the pathogenesis of the terminal pathway characterized by exaggerated maternal systemic inflammation, generalized endothelial damage, hypertension, and proteinuria. This sine qua non of preeclampsia may be triggered by distinct underlying mechanisms that occur at early stages of pregnancy and induce different phenotypes. To gain insights into these molecular pathways, we employed a systems biology approach and integrated different “omics,” clinical, placental, and functional data from patients with distinct phenotypes of preeclampsia. First trimester maternal blood proteomics uncovered an altered abundance of proteins of the renin-angiotensin and immune systems, complement, and coagulation cascades in patients with term or preterm preeclampsia. Moreover, first trimester maternal blood from preterm preeclamptic patients in vitro dysregulated trophoblastic gene expression. Placental transcriptomics of women with preterm preeclampsia identified distinct gene modules associated with maternal or fetal disease. Placental “virtual” liquid biopsy showed that the dysregulation of these disease gene modules originates during the first trimester. In vitro experiments on hub transcription factors of these gene modules demonstrated that DNA hypermethylation in the regulatory region of ZNF554 leads to gene down-regulation and impaired trophoblast invasion, while BCL6 and ARNT2 up-regulation sensitizes the trophoblast to ischemia, hallmarks of preterm preeclampsia. In summary, our data suggest that there are distinct maternal and placental disease pathways, and their interaction influences the clinical presentation of preeclampsia. The activation of maternal disease pathways can be detected in all phenotypes of preeclampsia earlier and upstream of placental dysfunction, not only downstream as described before, and distinct placental disease pathways are superimposed on these maternal pathways. This is a paradigm shift, which, in agreement with epidemiological studies, warrants for the central pathologic role of preexisting maternal diseases or perturbed maternal–fetal–placental immune interactions in preeclampsia. The description of these novel pathways in the “molecular phase” of preeclampsia and the identification of their hub molecules may enable timely molecular characterization of patients with distinct preeclampsia phenotypes.</p