The <em>S. pombe</em> Histone H2A Dioxygenase Ofd2 Regulates Gene Expression during Hypoxia

<div><p>Post-translational modification of histone proteins are known to play an important role in regulating chromatin structure. In an effort to find additional histone modifications we set out to screen enzymes of the 2-oxoglutarate and Fe(II)-dependent (2-OG-Fe(II)) dioxygenase family for activity towards histones. Here we show that the <em>Schizosaccharomyces pombe</em> 2-OG-Fe(II) dioxygenase domain containing protein-2 (Ofd2) is a histone H2A dioxygenase enzyme. Using a combination of peptide screening and alanine scanning substitution analysis, we identify an HxxLR motif in H2A as a substrate for Ofd2 activity. Transcriptional profiling indicates that Ofd2 regulates the repression of oxidative phosphorylation genes during hypoxic stress. We show that Ofd2 is recruited to the 5′ end of oxidative phosphorylation genes specifically during hypoxia and that it uses its dioxygenase activity to regulate their transcription. Together, these data uncover a novel histone H2A modifying activity involved in the regulation of gene expression during hypoxia.</p> </div

Significantly^a hypoxia repressed genes in Ofd2Δ.

a<p>Threshold of 1.5 fold (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029765#pone.0029765.s001" target="_blank">Figure S1</a>).</p>b<p>Presented as average change in gene expression in hypoxia over expression in normal oxygen conditions.</p

Calculation of 3D genome structures for comparison of chromosome conformation capture experiments with microscopy: An evaluation of single-cell Hi-C protocols

<p>Single-cell chromosome conformation capture approaches are revealing the extent of cell-to-cell variability in the organization and packaging of genomes. These single-cell methods, unlike their multi-cell counterparts, allow straightforward computation of realistic chromosome conformations that may be compared and combined with other, independent, techniques to study 3D structure. Here we discuss how single-cell Hi-C and subsequent 3D genome structure determination allows comparison with data from microscopy. We then carry out a systematic evaluation of recently published single-cell Hi-C datasets to establish a computational approach for the evaluation of single-cell Hi-C protocols. We show that the calculation of genome structures provides a useful tool for assessing the quality of single-cell Hi-C data because it requires a self-consistent network of interactions, relating to the underlying 3D conformation, with few errors, as well as sufficient longer-range <i>cis</i>- and <i>trans-</i>chromosomal contacts.</p

Role for Ofd2 dioxygenase domain in oxygen sensing.

<p>A) Three yeast strains (972, SP13 & h90) that were wt and deleted for Ofd2 (Ofd2<b>Δ</b>) were cultured for 90 min at both normal oxygen and hypoxia conditions. Total RNA samples were then prepared and mRNA levels of 3 repressed genes (<i>cyc1, qcr8 & SPAC3A11.07</i>) and two induced genes (<i>erg3 & hem13</i>) were quantitated by RT-qPCR. Expression levels were then normalised to <i>act1</i> and the fold change in expression levels of hypoxia over normal oxygen conditions were plotted as relative hypoxia mRNA. Data are the mean from 3 replicate qPCR. Error bars equal 1 standard deviation. B) Hypoxia time course analysis of mRNA levels for 3 repressed and 1 induced gene in wild type (wt), Ofd2<b>Δ</b>, Ofd2<b>Δ</b> rescued (Ofd2<b>Δ</b>+NT81 Ofd2) and iron binding mutant rescued (Ofd2<b>Δ</b>+NT81 Ofd2H132A) strains. At 0, 30, 60 and 90 min after hypoxia treatment total RNA samples were prepared and mRNA levels were quantitated by RT-qPCR and normalised to <i>act1</i>. Expression levels at each time point were then plotted relative to the levels at time 0. Data are the mean from 2 replicate qPCR. Error bars equal 1 standard deviation. B) Western blot analysis of 25 µg of whole cell extracts of NT81 Ofd2 and NT81 Ofd2H132A strains at various time points after hypoxia treatment with anti-flag and anti actin antibodies.</p

Ofd2 localises to repressed genes during hypoxia.

<p>A strain containing flag tagged Ofd2 was grown under hypoxic or normal oxygen conditions for 60 min. Chromatin immunoperciitation was then performed using anti-flag or anti mouse IgG as indicated. Binding to various amplicons in 3 repressed genes. (A–C) and 1 induced gene (D) was assayed using qPCR. Bound DNA is plotted relative to input DNA. Data are the mean from 2 replicate qPCRs. Error bars equal 1 standard deviation. Location of amplicons in each gene is depicted above each graph and arrows indicate 5′ end of gene.</p

Histone H2A dioxygenase activity.

<p>A) General reaction schematic for 2-OG-Fe(II) hydroxylases. R represents an amino acid sidechain. B) ClustalW sequence alignmant of AlkB, Ofd2 and ALKBH1. The secondary structure B-strands for AlkB are dipicted as arrows. Residues in AlkB involved in iron binding are boxed in red. Dark and light shading denote conserved and similar residues respectively. Species abbreviations: Ec <i>Escherichia.coli</i>; Sp <i>Schizosaccharomyces pombe</i>; Hs <i>Homo sapiens</i>. C) Dioxygenase assay of Ofd2 and Ofd2 H132A iron binding mutant. Calf thymus bulk histone (cBH) and individual histones H3, H2A, H2B and H4 and recombinant H2A (rH2A). Dioxygenase activity was evaluated with the CO₂ capture assay using 1ug of purified Ofd2 with 25ug cBH or 5ug of individual histones, except for ++ where 25ug was used. – indicate control reactions containing no substrates. Data is presented as mean from 2 replicates. Error bars equal 1 standard deviation.</p

Structural imaging mode.

<p>Fixed <i>S</i>. <i>pombe</i> expressing cytoplasmic Cdc22-mEos proteins were imaged during a PALM experiment. 5,000 frames were analysed with Peak Fit and the resulting list of localisations was used to produce a super-resolved picture directly after fitting <b>(A)</b>, or after applying the vlsPALM thresholds defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125438#pone.0125438.g002" target="_blank">Fig 2B</a><b>(B)</b>. The corresponding diffraction-limited image of the two cells is shown as an inset in <b>(A)</b>. Close-ups of the white rectangles in <b>(A-B)</b> are shown in <b>(C-D)</b>. The contrast of the large intracellular vesicles of the yeast is increased after vlsPALM filtering (white arrows in <b>(C-D)</b>).</p

Variation of the PSF in three dimensions.

<p>A vls (green plane, <b>A</b>) is defined as a volume above and below the focal plane of the microscope from which an emitter is imaged as a diffraction-limited spot on the detector (green optical path, <b>A</b>). A fluorophore emitting from outside the vls (red volume above and below the vls, <b>A</b>), is blurred on the image plane of the detector (red optical path, <b>A</b>). The z-stacks of 28 sub-diffraction beads were superposed to image the axial variation of the PSF of the instrument. The contrast-adjusted rendered volume <b>(B)</b> of the z-stack shows the axial variation of the width of the PSF. Three examples of (xy) planes of the z-stack in, above and below the vls are shown in <b>(C-E)</b>. For each plane, a contrast-adjusted image (left column) and an intensity surface plot (right column) of the plane underlines the axial variation of the width (orange arrows) and the amplitude (blue arrows) of the PSF.</p

Confidence mode.

<p>Embryonic stem cells expressing Cenp-A-mEos proteins were fixed and imaged. The corresponding movie (summed in <b>A</b>) was analysed with Peak Fit and the resulting list of localisations was separated between in vls (green) and out of vls (red) localisations using the vlsPALM thresholds defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125438#pone.0125438.g002" target="_blank">Fig 2C</a>. vlsPALM allows the identification of the in-focus localisations <b>(B)</b>. All localisations were plotted either as fitted <b>(C)</b> or in a super-resolved picture <b>(D)</b>, but coloured according to the vlsPALM filtering. Three categories of Cenp-A clusters were observed: some were almost entirely within the vls (<b>D-F</b>, 1); others were spanning one extremity of the vls, partly in the vls (<b>D-F</b>, 2); the last ones were entirely out of the vls (<b>D-F</b>, 3). <b>(E)</b> shows the diffraction-limited and super-resolved close-ups of the Cenp-A clusters defined in <b>(D)</b>. <b>(F)</b> displays the number of localisations in (green) and out of (red) the vls for each cluster. Such classification allows selecting in-focus clusters for further quantification and preventing under-counting due to undetectable out-of-focus emitters.</p