Mobilization of putative high-proliferative-potential endothelial colony-forming cells during antihypertensive treatment in patients with essential hypertension

Recent studies have shown that in response to vascular damage or ischemia, bone marrow-derived endothelial progenitor cells (EPCs) are recruited into the circulation. To investigate whether antihypertensive treatment has an influence on the number of circulating EPCs, patients with essential hypertension were treated either with the angiotensin receptor antagonist telmisartan, the calcium channel blocker nisoldipine, or their combination for 6 weeks. At baseline and after 3 and 6 weeks of treatment, EPCs were identified and quantified by fluorescence-activated cell sorting (FACS) analysis and by their capacity to generate colony-forming units of the endothelial lineage (CFU-EC) in a methylcellulose-based assay. During treatment, patients in the nisoldipine groups, but not in the telmisartan group, showed a significant mobilization of EPCs, which in part had the capacity to generate large-sized colonies comprising more than 1,000 cells. Moreover, a remarkable correlation between the number of CFU-EC and the number of circulating CD133(+)/CD34(+)/CD146(+) cells was observed, thereby providing strong evidence that cells with this phenotype represent functional EPCs. No correlation was found between the numbers of CFU-EC and the blood pressure levels at any time point during the treatment. Hence, nisoldipine-induced mobilization of EPCs might represent a novel mechanism by which this antihypertensive compound independently of its blood pressure-lowering effect contributes to vasoprotection in patients with essential hypertension

Plasmodium falciparum Nucleosomes Exhibit Reduced Stability and Lost Sequence Dependent Nucleosome Positioning

The packaging and organization of genomic DNA into chromatin represents an additional regulatory layer of gene expression, with specific nucleosome positions that restrict the accessibility of regulatory DNA elements. The mechanisms that position nucleosomes in vivo are thought to depend on the biophysical properties of the histones, sequence patterns, like phased di-nucleotide repeats and the architecture of the histone octamer that folds DNA in 1.65 tight turns. Comparative studies of human and P. falciparum histones reveal that the latter have a strongly reduced ability to recognize internal sequence dependent nucleosome positioning signals. In contrast, the nucleosomes are positioned by AT-repeat sequences flanking nucleosomes in vivo and in vitro. Further, the strong sequence variations in the plasmodium histones, compared to other mammalian histones, do not present adaptations to its AT-rich genome. Human and parasite histones bind with higher affinity to GC-rich DNA and with lower affinity to AT-rich DNA. However, the plasmodium nucleosomes are overall less stable, with increased temperature induced mobility, decreased salt stability of the histones H2A and H2B and considerable reduced binding affinity to GC-rich DNA, as compared with the human nucleosomes. In addition, we show that plasmodium histone octamers form the shortest known nucleosome repeat length (155bp) in vitro and in vivo. Our data suggest that the biochemical properties of the parasite histones are distinct from the typical characteristics of other eukaryotic histones and these properties reflect the increased accessibility of the P. falciparum genome

Nucleosomes Stabilize ssRNA-dsDNA Triple Helices in Human Cells

Chromatin-associated non-coding RNAs modulate the epigenetic landscape and its associated gene expression program. The formation of triple helices is one mechanism of sequence-specific targeting of RNA to chromatin. With this study, we show an important role of the nucleosome and its relative positioning to the triplex targeting site (TTS) in stabilizing RNA-DNA triplexes in vitro and in vivo. Triplex stabilization depends on the histone H3 tail and the location of the TTS close to the nucleosomal DNA entry-exit site. Genome-wide analysis of TTS-nucleosome arrangements revealed a defined chromatin organization with an enrichment of arrangements that allow triplex formation at active regulatory sites and accessible chromatin. We further developed a method to monitor nucleosome-RNA triplexes in vivo (TRIP-seq), revealing RNA binding to TTS sites adjacent to nucleosomes. Our data strongly support an activating role for RNA triplex-nucleosome complexes, pinpointing triplex-mediated epigenetic regulation in vivo

EMBO J

Within the virion, adenovirus DNA associates with the virus-encoded, protamine-like structural protein pVII. Whether this association is organized, and how genome packaging changes during infection and subsequent transcriptional activation is currently unclear. Here, we combined RNA-seq, MNase-seq, ChIP-seq, and single genome imaging during early adenovirus infection to unveil the structure- and time-resolved dynamics of viral chromatin changes as well as their correlation with gene transcription. Our MNase mapping data indicates that the adenoviral genome is arranged in precisely positioned nucleoprotein particles with nucleosome-like characteristics, that we term adenosomes. We identified 238 adenosomes that are positioned by a DNA sequence code and protect about 60-70 bp of DNA. The incoming adenoviral genome is more accessible at early gene loci that undergo additional chromatin de-condensation upon infection. Histone H3.3 containing nucleosomes specifically replaces pVII at distinct genomic sites and at the transcription start sites of early genes. Acetylation of H3.3 is predominant at the transcription start sites and precedes transcriptional activation. Based on our results, we propose a central role for the viral pVII nucleoprotein architecture, which is required for the dynamic structural changes during early infection, including the regulation of nucleosome assembly prior to transcription initiation. Our study thus may aid the rational development of recombinant adenoviral vectors exhibiting sustained expression in gene therapy

Changes in adenoviral chromatin organization precede early gene activation upon infection

Within the virion, adenovirus DNA associates with the virus-encoded, protamine-like structural protein pVII. Whether this association is organized, and how genome packaging changes during infection and subsequent transcriptional activation is currently unclear. Here, we combined RNA-seq, MNase-seq, ChIP-seq, and single genome imaging during early adenovirus infection to unveil the structure- and time-resolved dynamics of viral chromatin changes as well as their correlation with gene transcription. Our MNase mapping data indicates that the adenoviral genome is arranged in precisely positioned nucleoprotein particles with nucleosome-like characteristics, that we term adenosomes. We identified 238 adenosomes that are positioned by a DNA sequence code and protect about 60–70 bp of DNA. The incoming adenoviral genome is more accessible at early gene loci that undergo additional chromatin de-condensation upon infection. Histone H3.3 containing nucleosomes specifically replaces pVII at distinct genomic sites and at the transcription start sites of early genes. Acetylation of H3.3 is predominant at the transcription start sites and precedes transcriptional activation. Based on our results, we propose a central role for the viral pVII nucleoprotein architecture, which is required for the dynamic structural changes during early infection, including the regulation of nucleosome assembly prior to transcription initiation. Our study thus may aid the rational development of recombinant adenoviral vectors exhibiting sustained expression in gene therapy

<i>P</i>. <i>falciparum</i> nucleosomes exhibit reduced heat and salt stabilities.

<p>(<i>A</i>) Preparation of histone octamers. Histone octamers were reconstituted from appropriate combinations of recombinant parasite and human histones (lanes 1–4), purified by gel filtration, separated by 15% SDS-PAGE and stained with Coomassie blue. The reconstitution H4hyb (lane 8) describes an octamer consisting of the parasite histones H2A, H2B, H3 and the human histone H4. (<i>B</i>) Temperature induced nucleosome sliding. <i>P</i>. <i>falciparum</i> nucleosomes and human nucleosomes were separately reconstituted on Cy3 and Cy5 labelled 601 DNA and then mixed in equimolar ratios. Nucleosomal mixtures were incubated 60 min at room temperature (lane 2) or at the indicated temperatures (lanes 3 to 8). Nucleosome positions were analyzed after the temperature incubation on a native 5% polyacrylamide gel and visualized by fluorescence scanning (upper panel: cy5 labelled human nucleosomes; middle panel: cy3 labelled <i>P</i>. <i>falciparum</i> nucleosomes) and ethidium bromide staining (lower panel: showing the mixture of human and parasite nucleosomes). The initial nucleosome position and free DNA are indicated and sliding products are marked by triangles. (<i>C</i>) Chloroquine stability assay. A 208 bp 601 (GC-rich) and the 210 bp KahrP (AT-rich) DNA fragment were reconstituted either with human (lower panel) or plasmodium octamers (upper panel) into nucleosomes and mixed at equimolar ratio. Nucleosomes were incubated with increasing concentrations of chloroquine (0, 0.05, 0.1, 0.3, 1, 3 and 9 mM), incubated for 10 min at room temperature and then analyzed by native polyacrylamide gel electrophoresis. The DNA and nucleoprotein complexes were visualized by fluorescence imaging staining. The positions of the free DNA and a scheme indicating the different nucleosome positions on the DNA template are given. (<i>D</i>) Stepwise salt elution of human and plasmodium histones from DNA. <i>P</i>. <i>falciparum</i> and human nucleosomes were reconstituted in parallel on linearized, biotinylated DNA and then coupled to magnetic beads. Magnetic beads were incubated stepwise with increasing salt concentrations as indicated and the eluted histones were collected and analyzed by SDS-PAGE and silver staining (lanes 1 to 9). The input fraction is shown in lane 10 and the positions of the histones are indicated. The upper gel shows the results for the human and the lower gel depicts the elution of the parasite histones.</p

Analyzing the sequence dependent binding preferences of plasmodium and human histone octamers.

<p>(<i>A</i>) <i>P</i>. <i>falciparum</i> genomic DNA (19,6% GC-content), human genomic DNA (40% GC-content) and the GC-rich human rDNA sequence (60% GC-content) were isolated and fragmented by sonication. The purified DNA was chemically labelled with Cy3 (<i>P</i>. <i>falciparum</i> DNA) or Cy5 (human genomic DNA and human rDNA). Plasmodium DNA was mixed at equimolar ratios with the human DNA and used for chromatin assembly by salt dialysis, with either plasmodium (lanes 2, 4, 7) or human histone octamers (lanes 1, 3, 6) at different histone to DNA ratios (0.9:1 and 1.1:1). Chromatin assemblies were analyzed by native polyacrylamide gels and fluorescence scanning. The upper panel shows the cy3 channel, revealing the assembly of the plasmodium DNA, the lower panel depicts the results of the human genomic DNA and the human rDNA as indicated (cy5 channel). The efficiency of nucleosome assembly on AT-rich and AT-poor DNA can be judged by the ratios of free and reconstituted DNA in the individual lanes of the competitive assembly reactions. The free and nucleosomal DNA is indicated. (<i>B</i>) Chloroquine stability assay. The 210 bp KahrP DNA (AT-rich, Cy3-labelled, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006080#ppat.1006080.s003" target="_blank">S3B Fig</a>), the Cy5 labeled 601 DNA (GC-rich, 160bp) and a Cy5 labelled KahrP DNA fragment (AT-rich, 150 bp) reconstituted either with human (hs nucleosomes, lanes 1–6) or plasmodium octamers (pf nucleosomes, lanes 7–12) into nucleosomes and then mixed at equimolar ratios. Nucleosomes were incubated with increasing concentrations of chloroquine (0 to 9 mM), incubated for 10 min at room temperature and then analyzed by native polyacrylamide gel electrophoresis. The free DNA (lane 13) and the nucleoprotein complexes were visualized by fluorescence scanning as indicated. The asterisk shows the position of contaminating single stranded DNA.</p

AT-repeat containing sequences in the linker region of the nucleosome determine the position of plasmodium histone octamers.

<p>(<i>A</i>) Nucleosome positioning at ATG (beginning of the protein coding region), ECR (end of protein coding region) and exon/intron boundaries of all annotated <i>P</i>. <i>falciparum</i> genes were aligned. Individual nucleosome positions were further characterized by a fuzziness score and the average fuzziness signal for all aligned genes was plotted as a heatmap (top). The color code is indicated on the right. The average frequency of identified nucleosome midpoints (middle) and of AT/TA (red) and AA/TT di-nucleotides (blue) over regulatory regions of all annotated genes is given. (<i>B</i>) Changes in AT/TA and AA/TT di-nucleotide frequencies within the nucleosome and its linker regions compared to the fuzziness score of nucleosome positioning. Five different fractions of nucleosomes, from well positioned (dark blue) to badly positioned (light blue) nucleosomes for <i>P</i>. <i>falciparum</i> (top panel) and <i>S</i>. <i>cerevisiae</i> (bottom panel) were analyzed. Vertical lines indicate nucleosome boundaries. The color code for the fuzziness score is indicated below. (<i>C</i>) A 150bp long HSP70 sequence was flanked by either two 15bp long AT-rich (AT), AA-homopolymers (AA) or GC-rich (CG) sequence linkers and amplified by PCR. The indicated PCR fragments were used for nucleosome assembly with plasmodium histones and analyzed on 5% native polyacrylamide gels. The templates used for assembly and the nucleosome positions are indicated. (<i>D</i>) Two genomic regions exhibiting positioned nucleosomes containing native AT-repeat flanking sequences (MAL and HSP) were cloned, amplified by PCR and used to analyze nucleosome positioning <i>in vitro</i>. MAL (lanes 1–3), HSP (lanes 9–11) and the MAL and HSP PCR fragments having the AT repeats replaced by GC-rich sequences (MAL-GC; HSP-GC; lanes 4–7) were used for nucleosome assembly with plasmodium (Pf) and human histones (Hs). Nucleosome positioning was analyzed on 5% native polyacrylamide gels. The free DNA, contaminating single stranded DNA (marked by an asterisk), positioned and delocalized nucleosomes are shown.</p

<i>P</i>. <i>falciparum</i> nucleosomes override nucleosome positioning signals.

<p>(<i>A</i>) Sequences encompassing the mouse rDNA promoter from position -190 to +90 were amplified by PCR and used as substrate for nucleosome assembly. Free DNA (lanes 1), nucleosome assembly reactions using the recombinant human histone octamers (lanes 2 and 3), octamers consisting of the plasmodium H2A, H2B, H3 and human H4 (H4hyb, lanes 4 and 5), the plasmodium octamers (lanes 6 and 7) and a size marker (M) were separated by native polyacrylamide gel electrophoresis and stained with ethidium bromide. Nucleosome positions and the respective histone to DNA ratios used for assembly are indicated. (<i>B</i>) Experimental setup like in (<i>A</i>) but a 370-bp fragment carrying the <i>D</i>. <i>melanogaster</i> HSP70 promoter was used for assembly. (<i>C</i>) Nucleosome assembly was performed on three different AT-rich sequences (A, B and C) amplified from the <i>P</i>. <i>falciparum</i> genome (Chromosome 11, position 1307500–1308399). The sequences were reconstituted into nucleosomes either with recombinant human or plasmodium histone octamers and analyzed by native polyacrylamide gel electrophoresis. (<i>D</i>) To analyze whether the full complement of histones assemble on DNA, the biotinylated HSP70-DNA (sequence used in <i>B</i>), was reconstituted into nucleosomes and separated on native polyacrylamide gels (lanes 2 and 3). (<i>E</i>) Plasmodium and human nucleosomes shown in (<i>D</i>) (I: Input—lanes 1 and 10) were coupled to magnetic beads and washed twice with 150 mM NaCl buffer (W: Wash—lanes 2 and 7). Histones were eluted with 2M salt (Elution–lanes 3 and 8), the individual fractions and proteins remaining bound to the magnetic beads (B: Beads–lanes 4 and 9) were analyzed by SDS-PAGE and Coomassie Blue staining.</p

Analysis of nucleosome remodeling and nucleosome spacing.

<p>(<i>A</i>) Competitive nucleosome remodeling reactions. In the same reaction Cy5-labelled plasmodium nucleosomes (upper panel) and Cy3-labelled human nucleosomes (middle panel) reconstituted on 601 DNA, positioned at the border of the 208bp long DNA fragment, were incubated with increasing concentrations of recombinant Chd3 (lanes 1 to 9) or Snf2H (lanes 10 to 18). Reactions were incubated for 60 min at 37°C in the presence or absence of ATP, as indicated. Nucleosome positions were analyzed by EMSA and imaged for the Cy5 and Cy3 channel, respectively. The lower panel shows the total nucleosomal reaction after ethidium bromide staining. The positions of the nucleosomes are indicated on the right side. (<i>B</i>) Recombinant histone octamers of the indicated type (human = Hs, plasmodium = Pf) were used for chromatin assembly with a 11kb plasmid by salt dialysis. Chromatin was subjected to partial MNase digestion (10 to 90 sec), stopping the reaction by the addition of SDS/EDTA. The DNA fragments were purified and visualized by agarose gel electrophoresis and ethidium bromide staining. The positions of the mono-, di- and tri-nucleosomal DNAs are indicated by black triangles marking the human and white triangles marking the plasmodium complexes. (<i>C</i>) Di-nucleosome density and box plots revealing the nucleosome repeat lengths <i>in vivo</i>. The size distribution of di-nucleosomal fragments derived from human (red) as well as plasmodium DNA (black) after MNase digestions (SRX885811-SRX885819)[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006080#ppat.1006080.ref016" target="_blank">16</a>]. MNase digestions were performed at different stages of the erythrocytic life cycle of <i>P</i>. <i>falciparum</i>. Each stage of the life cycle is plotted individually together with the corresponding human DNA fraction.</p