DNA Methylation

<p><b>A</b>. X Chromosome DNA Methylation and XIST Expression. Methylation levels of genes in the X-chromosome (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118307#pone.0118307.s009" target="_blank">S6A Table</a>) are shown on the heatmap. Hierarchical clustering was performed on the samples, as indicated by the dendrogram. The genes are ordered according to their location (from the beginning to the end of the chromosome). Samples that show loss of DNA methylation for the “Enz” cluster are highlighted in blue, those that show DNA methylation for the “Ecm” cluster are highlighted in pink, and for both clusters in mauve. Genes located in the regions of loss of DNA methylation are listed to the right of the heatmap. XIST expression is shown on the line graph, with the detection limit for the microarray indicated by the red line. <b>B</b>. DNA methylation at imprinted loci. Methylation levels for imprinted probes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118307#pone.0118307.s009" target="_blank">S6B Table</a>) are shown on the heatmap. Hierarchical clustering was performed on the samples, as indicated by the dendrogram. The genes are ordered according to chromosome location; genes are listed to the left. The inset at the right shows a detail of the NESP/GNAS complex locus, indicating the positions of the CpG sites that were hypermethylated (red triangle) vs. hypomethylated (green triangle) in the late passage samples relative to the NESP/GNAS and NESPAS exons. <b>C, D, E</b>. Heatmaps showing differential DNA methylation genes for early vs. late passage <b>(C)</b>, mechanical vs. enzymatic passage <b>(D)</b>, and Mef vs. Ecm substrate <b>(E)</b>. In heatmap <b>(C)</b>, the black boxes indicate genes for which the DNA methylation levels in the late passage MefMech (P103) samples was more similar to those in the early passage samples. Probes were selected by multivariate regression. Functional enrichments identified by GREAT analysis are shown to the right of the heatmaps, visualized using REVIGO [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118307#pone.0118307.ref013" target="_blank">13</a>]. Samples were arranged according to passage and culture method, and hierarchical clustering was performed on the genes only. In the functional enrichment results, the size of the node indicated the number of contributing GO terms, and color of the nodes indicates the FDR (darker color for lower FDR), and the edge length indicates the similarity between GO terms (shorter edge for more similar terms).</p

Eukaryotic transcriptomes and prokaryotic metatranscriptomes of three marine sponges.

Files Cym_Euk_165645.fasta, Sco_Euk_90948.fasta, Ted_Euk_113832.fasta are the eukaryotic transcriptomes for Cymbastella concentrica, Scopalina sp., Tedania anhelens respectively. Files Meta_Cym_RZ_228060.fasta, Meta_Sco_RZ_64649.fasta, Meta_Ted_RZ_56167.fasta are the prokaryotic metatranscriptomes of those same sponges. The numbers in the file names represent the number of transcripts within the file. See supporting table S1 C and D of the manuscript for details

Low Permeability Zone Remediation via Oxidant Delivered by Electrokinetics and Activated by Electrical Resistance Heating: Proof of Concept

This study proposes and proves (in concept) a novel approach of combining electrokinetic (EK)-assisted delivery of an oxidant, persulfate (PS), and low temperature electrical resistivity heating (ERH), to activate PS, to achieve remediation of contaminated, low permeability soil. This unique combination is able to overcome existing challenges in remediating low permeability materials, particularly associated with delivering remediants. A further benefit of the approach is the use of the same electrodes for both EK and ERH phases. Experiments were conducted in a laboratory-scale sand tank packed with silt and aqueous tetrachloroethene (PCE) and bracketed on each side by an electrode. EK first delivered unactivated PS throughout the silt. ERH then generated and sustained the target temperature to activate the PS. As a result, PCE concentrations decreased to below detection limit in the silt in a few weeks. Moreover, it was found that activating PS at ∼36 °C eliminated more PCE than activating it at >41 °C. It is expected this results from the reactive SO₄^•– radical being generated more slowly, which ensures more complete reaction with the contaminant. The novel application of EK-assisted PS delivery followed by low temperature ERH appears to be a viable strategy for low permeability contaminated soil remediation

Histone H3 Variant Regulates RNA Polymerase II Transcription Termination and Dual Strand Transcription of siRNA Loci in <i>Trypanosoma brucei</i>

<div><p>Base J, β-D-glucosyl-hydroxymethyluracil, is a chromatin modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. In <i>Trypanosoma brucei</i>, J is enriched, along with histone H3 variant (H3.V), at sites involved in RNA Polymerase (RNAP) II termination and telomeric sites involved in regulating variant surface glycoprotein gene (<i>VSG</i>) transcription by RNAP I. Reduction of J in <i>T</i>. <i>brucei</i> indicated a role of J in the regulation of RNAP II termination, where the loss of J at specific sites within polycistronic gene clusters led to read-through transcription and increased expression of downstream genes. We now demonstrate that the loss of H3.V leads to similar defects in RNAP II termination within gene clusters and increased expression of downstream genes. Gene derepression is intensified upon the subsequent loss of J in the <i>H3</i>.<i>V</i> knockout. mRNA-seq indicates gene derepression includes <i>VSG</i> genes within the silent RNAP I transcribed telomeric gene clusters, suggesting an important role for H3.V in telomeric gene repression and antigenic variation. Furthermore, the loss of H3.V at regions of overlapping transcription at the end of convergent gene clusters leads to increased nascent RNA and siRNA production. Our results suggest base J and H3.V can act independently as well as synergistically to regulate transcription termination and expression of coding and non-coding RNAs in <i>T</i>. <i>brucei</i>, depending on chromatin context (and transcribing polymerase). As such these studies provide the first direct evidence for histone H3.V negatively influencing transcription elongation to promote termination.</p></div

Increased production of nascent RNA in cSSR following the loss of H3.V.

<p>A region on chromosome 2 (950–975 kb) representing cSSR 2.5 is shown where H3.V regulates transcription. (A) Base J and H3.V co-localize at sites of RNAP II termination within cSSRs [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.ref013" target="_blank">13</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.ref014" target="_blank">14</a>]. H3.V ChIP-seq reads and base J IP-seq reads are plotted as reads per million reads (RPM), as previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.ref014" target="_blank">14</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.ref035" target="_blank">35</a>]. (B) ORFs are shown with the top strand in blue and the bottom strand in red. (C) mRNA-seq reads from wild type <i>T</i>. <i>brucei</i> are plotted as RPM. Reads that mapped to the top strand are shown in blue and reads that mapped to the bottom strand in red. (D) Small RNA-seq reads from WT and <i>H3</i>.<i>V KO</i> are mapped as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.g001" target="_blank">Fig 1A</a>. (E-G) Strand-specific RT-PCR analysis of nascent RNA in cSSRs. (E) Schematic representation (not to scale) of primer location and direction at cSSR 2.5 (primers shown as green arrows in this and all subsequent figures). The arrowhead below the line indicates the poly(A) processing site for the final gene in the PTU. (F) Strand-specific RT-PCR analysis. cDNA was synthesized using the reverse primer. PCR was performed using the same reverse primer to make the cDNA plus the forward primer, as indicated. Data is also presented for an additional cSSR on chromosome 1 (cSSR 1.4, 635–637 kb). Wild type: WT; Wild type+DMOG: -J; <i>H3</i>.<i>V</i> KO: KO; <i>H3</i>.<i>V</i> KO+DMOG: KO-J. 40S ribosomal protein S11 provides a positive control and minus RT (-RT) negative control is shown. (G) Nested qPCR. Primers were designed within the PCR reaction in F to use in subsequent qPCR analysis. White bars: Wild type; grey bars: Wild type+DMOG; dark grey bars: <i>H3</i>.<i>V</i> KO; black bars: <i>H3</i>.<i>V</i> KO+DMOG. All products were normalized to 40S ribosomal protein S11. The average of three independent strand-specific RT-PCR nested qPCR experiments is plotted. Error bars represent the standard deviation. P values were calculated using Student’s t test. *, p value ≤ 0.05; **, p value ≤ 0.01.</p

H3.V regulates <i>VSG</i> gene expression from silent telomeric bloodstream expression sites.

<p>(A) A schematic diagram of the silent ES15 (not to scale). The box with stripes represents the 70 bp repeats. Numbers indicate <i>ESAG</i> genes. Grey box represents the <i>VSG</i> pseudogene 11 (Tb427.BES126.13). (B-C) mRNA-seq and RT-qPCR analysis of the indicated <i>VSG</i> genes in silent expression sites. As described in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.g003" target="_blank">3D</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.g005" target="_blank">5E</a>, white bars: Wild type; grey bars: Wild type+DMOG; dark grey bars: <i>H3</i>.<i>V</i> KO; black bars: <i>H3</i>.<i>V</i> KO+DMOG. For mRNA-seq analysis, p values determined by Cuffdiff are indicated by asterisks: *, p value ≤ 0.05; **, p value ≤ 0.01. For RT-qPCR analysis, p values were calculated using Student’s t test. *, p value ≤ 0.05; **, p value ≤ 0.01.</p

Loss of H3.V stimulates the production of siRNAs in <i>T</i>. <i>brucei</i>.

<p>(A) Small RNA-sequencing reads for three representative cSSRs are shown (11.9, 11.4, and 10.9; where cSSR 11.9 refers to the ninth termination site on chromosome 11) where H3.V loss does not lead to read-through transcription, but does lead to increased siRNAs. Small RNA reads are plotted as reads per million reads mapped (RPM). ORFs and the genomic location (kb) are shown above the graphs. WT: wild type; KO: <i>H3</i>.<i>V</i> KO; KO+DMOG: <i>H3</i>.<i>V</i> KO + DMOG. Blue: top strand; red: bottom strand. (B and C) Length distribution of small RNAs. (B) Length distribution of small RNAs from cSSR 11.9. Shown is the RPM for each RNA length observed in cSSR 11.9, a site with a statistically significant increase in 21-27nt RNAs in the <i>H3</i>.<i>V</i> KO (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.s012" target="_blank">S2 Table</a>). The average of three independent small RNA-seq experiments is plotted. White bars, WT; black bars, <i>H3</i>.<i>V</i> KO. Error bars represent the standard deviation. (C) Length distribution of small RNAs genome-wide. The RPM for each RNA length observed in the entire small RNA-seq data set is shown. Data are plotted as in B.</p

Increased production of nascent RNA in cSSR following the loss of H3.V.

<p>A region on chromosome 2 (950–975 kb) representing cSSR 2.5 is shown where H3.V regulates transcription. (A) Base J and H3.V co-localize at sites of RNAP II termination within cSSRs [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.ref013" target="_blank">13</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.ref014" target="_blank">14</a>]. H3.V ChIP-seq reads and base J IP-seq reads are plotted as reads per million reads (RPM), as previously described [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.ref014" target="_blank">14</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.ref035" target="_blank">35</a>]. (B) ORFs are shown with the top strand in blue and the bottom strand in red. (C) mRNA-seq reads from wild type <i>T</i>. <i>brucei</i> are plotted as RPM. Reads that mapped to the top strand are shown in blue and reads that mapped to the bottom strand in red. (D) Small RNA-seq reads from WT and <i>H3</i>.<i>V KO</i> are mapped as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.g001" target="_blank">Fig 1A</a>. (E-G) Strand-specific RT-PCR analysis of nascent RNA in cSSRs. (E) Schematic representation (not to scale) of primer location and direction at cSSR 2.5 (primers shown as green arrows in this and all subsequent figures). The arrowhead below the line indicates the poly(A) processing site for the final gene in the PTU. (F) Strand-specific RT-PCR analysis. cDNA was synthesized using the reverse primer. PCR was performed using the same reverse primer to make the cDNA plus the forward primer, as indicated. Data is also presented for an additional cSSR on chromosome 1 (cSSR 1.4, 635–637 kb). Wild type: WT; Wild type+DMOG: -J; <i>H3</i>.<i>V</i> KO: KO; <i>H3</i>.<i>V</i> KO+DMOG: KO-J. 40S ribosomal protein S11 provides a positive control and minus RT (-RT) negative control is shown. (G) Nested qPCR. Primers were designed within the PCR reaction in F to use in subsequent qPCR analysis. White bars: Wild type; grey bars: Wild type+DMOG; dark grey bars: <i>H3</i>.<i>V</i> KO; black bars: <i>H3</i>.<i>V</i> KO+DMOG. All products were normalized to 40S ribosomal protein S11. The average of three independent strand-specific RT-PCR nested qPCR experiments is plotted. Error bars represent the standard deviation. P values were calculated using Student’s t test. *, p value ≤ 0.05; **, p value ≤ 0.01.</p

Decreased efficiency of RNAP II termination and increased gene expression following the loss of histone H3.V.

<p>A region on chromosome 3 (617–670 kb) representing cSSR 3.3 and chromosome 7 (453–525 kb) representing cSSR 7.3 is shown where H3.V regulates transcription of a cluster of genes. (A-C) Base J and H3.V co-localize at sites of RNAP II termination within a PTU. H3.V ChIP-seq reads and base J IP-seq reads, ORFs, and mRNA-seq reads from wild type <i>T</i>. <i>brucei</i> are plotted for cSSR 3.3 (left) and cSSR 7.3 (right) as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.g002" target="_blank">Fig 2</a>. (D) RT-qPCR analysis of genes numbered according to the ORF maps above in panel B. As described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.g002" target="_blank">Fig 2G</a>, white bars: Wild type; grey bars: Wild type+DMOG; dark grey bars: <i>H3</i>.<i>V</i> KO; black bars: <i>H3</i>.<i>V</i> KO+DMOG. Transcripts were normalized against 40S ribosomal protein S11, and are plotted as the average and standard deviation of three replicates. P values were calculated using Student’s t test. *, p value ≤ 0.05; **, p value ≤ 0.01. The silent gene cluster at cSSR 7.3 consists of nine highly similar retrotransposon hot spot protein genes, therefore the primers used to analyze gene 2 also amplify the additional upstream genes. (E and F) Strand-specific RT-PCR analysis of read-through transcription of the two cSSRs analyzed in A-D. Above each panel is a schematic representation (not to scale) of primer location and direction at a transcription termination site (TTS). The vertical arrow indicates the proposed TTS as described in the text [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.ref035" target="_blank">35</a>]. The long solid arrow indicates the direction of transcription and the dashed arrow indicates read-through transcription past the TTS. cDNA was synthesized using the reverse primer (relative to transcription). PCR was performed using the same reverse primer to make the cDNA plus the forward primer, as indicated. 40S ribosomal protein S11 provides a positive control and a minus RT (-RT) negative control is shown.</p

H3.V and base J have independent yet additive roles in regulating termination and gene expression.

<p>(A) Heatmap of genes upregulated in the <i>H3</i>.<i>V</i> KO. For the list of genes represented on the heatmap see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.s011" target="_blank">S1 Table</a>. Clustering of genes at the top indicate those that are further upregulated upon loss of base J in the <i>H3</i>.<i>V</i> KO. J and H3.V columns indicate whether each gene is located within 10 kb of the modification (filled black box), as described in the Materials and Methods section. (B-D) H3.V/J localization, gene map, and mRNA-seq reads plotted for a gene cluster on chromosome 7 at cSSR 7.7 (position 1750–1800 kb shown) is illustrated as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.g003" target="_blank">Fig 3</a>. (E) Plot of the mRNA-seq data for the genes indicated (numbered) in the ORF map. The average RPKM of triplicate mRNA-seq libraries was used to determine fold changes, with wild type set to 1. Error bars indicate the standard deviation between mRNA-seq replicates and p values, determined in Cuffdiff, are indicated by asterisks: *, p value ≤ 0.05; **, p value ≤ 0.01. (F) RT-qPCR analysis of gene expression for the indicated genes (according to the ORF map) as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005758#pgen.1005758.g003" target="_blank">Fig 3D</a>. White bars: Wild type; grey bars: Wild type+DMOG; dark grey bars: <i>H3</i>.<i>V</i> KO; black bars: <i>H3</i>.<i>V</i> KO+DMOG. P values were calculated using Student’s t test. *, p value ≤ 0.05; **, p value ≤ 0.01.</p