Formation of oligomer complexes by secreted HtrA1 protein in cell culture.

<p>(<b>A</b>) Serum rich (10%) conditioned medium from HeLa cells contained a higher level of HtrA1 complexes than low serum (0.2%) conditioned medium. Medium was collected at the indicated time points and subjected to non-reducing (- β-ME) or reducing (+ β-ME) SDS-PAGE. Immunoblotting with polyclonal anti-HtrA1 antibody detected full length HtrA1 (arrow) and HtrA1-containing complexes (arrowheads) under non-reducing conditions. Full length HtrA1 could be detected under non-reducing conditions. (<b>B</b>) Serum rich and low serum conditioned media subjected to native PAGE revealed two prominent bands when probed with monoclonal anti-HtrA1 antibody (bracket). A dilution series is shown for clarity. (<b>C</b>) A schematic diagram of the human HtrA1 expressed from the full-length construct, tagged with an HA epitope (red) at the N-terminus and V5/hexa-His tag (blue) at the C-terminus. S denotes the signal peptide. (<b>D</b>) Exogenous HtrA1 is stably expressed in HEK293 cells. HtrA1 could be detected in conditioned medium from HeLa cells and stably transfected HEK-HtrA1 cells, but not that from HEK293 cells or medium that has not been exposed to cells, when probed with monoclonal anti-HtrA1 antibody (middle panel). Probing with anti-V5 antibody (right panel) detected specific bands (arrows, arrowheads) only in HEK-HtrA1 conditioned medium, although a non-specific band was detected in all samples (asterisk). Coomassie blue staining is included as a loading control (left panel). (<b>E</b>) Serum rich (10%) conditioned medium contained a higher level of exogenous HtrA1 complexes than serum free conditioned medium. Exogenous HtrA1 was captured from conditioned medium via the hexa-His tag, using Ni-NTA columns. Full-length HtrA1 (arrow) could be detected in the input conditioned medium (control) and column-captured fraction (bound) under both serum conditions. HtrA1 complexes (arrowheads) were only detected under serum rich conditions. CM: conditioned medium.</p

MPPs and HtrA1 appear to interact directly.

<p>(<b>A</b>) MPPs displayed differential activity in affecting The pattern of extracellular HtrA1 complexes (arrowheads) from HeLa cell low-serum conditioned medium is differentially altered by treatment with MPPs (25 mM, 37°C, 1 hr), as demonstrated when probed with polyclonal anti-HtrA1 antibody. (<b>B</b>) MPPs may induce conformational changes upon binding to HtrA1. The accessibility of N-terminal and C-terminal epitopes in the presence of MPPs was determined by ELISA and compared to DMSO controls. Error bars indicate the standard deviation. (<b>C</b>) Extracellular HtrA1 (arrows) could be precipitated from HEK-HtrA1 conditioned medium using HEMIN-conjugated agarose beads but not control, unconjugated agarose beads. (<b>D</b>) Competitive binding experiments using conditioned medium from HEK-HtrA1 or HeLa cells pre-incubated with MPPs revealed reduced recovery of HtrA1 (arrows) in the presence of competitor compounds when probed with polyclonal anti-HtrA1 antibody. (<b>E</b>) Degradation of Fibulin 5 in fixed HeLa cells treated with HtrA1 conditioned medium was enhanced in the presence of TPP, ZPP and PPP-IX. RMA: Rosmarinic acid, TPP: tin protoporphyrin IX, ZPP: zinc protoporphyrin IX, PPP-IX: protoporphyrin IX, HEM: HEMIN, CPP: cobalt protoporphyrin IX, CM: conditioned medium.</p

PPP-IX binds to the protease domain of HtrA1 and disease-associated mutations eliminate binding.

<p>(<b>A</b>) PPP-IX competed with HEMIN for endogenous HtrA1 binding. HeLa cell lysate was subjected to HEMIN pull down. Less HtrA1 was recovered in the presence of PPP-IX. Akt was used as a control and was not purified from the same pull down. (<b>B</b>) The HtrA1 protease domain was sufficient for interaction with MPPs. A schematic diagram of the deletion constructs tagged with HA and V5/hexa-His epitopes is shown (top). Cell lysates from HEK293 expressing variant HtrA1 proteins were subjected to HEMIN pull down and eluates (H) were examined for tagged protein with the anti-HA antibody (bottom left). The amount of recovered protein compared to input (I) is shown (bottom right). (<b>C</b>) PPP-IX competed with HEMIN for binding to the HtrA1 protease domain in a dose-dependent manner, with less HtrA1 protein recovery as PPP-IX concentration increased, revealed by immunoblotting for anti-HA. Akt was not similarly purified. The amount of recovered protein compared to input is shown (bottom). (<b>D</b>) Disease associated mutations reduced the interaction between HtrA1 and MPPs. The position of engineered single amino acid mutations in variant HtrA1 constructs is shown in the schematic diagram (top). Variant HtrA1 protein pulled down from transfected cell lysates with HEMIN agarose (H) was probed for HtrA1 (left panel) and the amount of recovered protein compared to input (I) was calculated (right panel). R274Q and V297 M significantly reduced the binding of HtrA1 to HEMIN (*: p<0.01). HtrA family member HtrA2 is not purified, demonstrating that the interaction is specific to HtrA1.</p

The formation of HtrA1 extracellular complexes can be affected by small molecules.

<p>(<b>A</b>) A schematic of the chemical intervention strategy for selecting molecules capable of affecting extracellular HtrA1 complex formation. “Hit compounds” were identified when the HtrA1 complex pattern was altered in comparison to DMSO treatment. (<b>B</b>) Conditioned serum from HEK-HtrA1 and HeLa cells was probed with anti-HA and anti-HtrA1 antibody respectively after treatment with the seven hit compounds. All compounds increased HtrA1 complex abundance (arrowheads) compared to the DMSO treated control. DMSO treatment did not alter complex formation. CBD: (S)-(-)-Carbidopa, RMA: Rosmarinic acid, ZPP: zinc protoporphyrin IX, TPP: tin protoporphyrin IX, ACTD: Actinomycin D, YM: YM 90709, AZ: AZ 10417808. (<b>C</b>) Schematic diagrams of the chemical structure of the non-MPP hit compounds. Dashed lines indicate a conserved chemical moiety in Carbidopa and Rosmarinic acid. (<b>D</b>) Schematic diagram of the chemical structures of the protoporphyrin IX-based metalloporphyrins used.</p

Stage and Gene Specific Signatures Defined by Histones H3K4me2 and H3K27me3 Accompany Mammalian Retina Maturation In Vivo

<div><p>The epigenetic contribution to neurogenesis is largely unknown. There is, however, growing evidence that posttranslational modification of histones is a dynamic process that shows many correlations with gene expression. Here we have followed the genome-wide distribution of two important histone H3 modifications, H3K4me2 and H3K27me3 during late mouse retina development. The retina provides an ideal model for these studies because of its well-characterized structure and development and also the extensive studies of the retinal transcriptome and its development. We found that a group of genes expressed only in mature rod photoreceptors have a unique signature consisting of de-novo accumulation of H3K4me2, both at the transcription start site (TSS) and over the whole gene, that correlates with the increase in transcription, but no accumulation of H3K27me3 at any stage. By <em>in silico</em> analysis of this unique signature we have identified a larger group of genes that may be selectively expressed in mature rod photoreceptors. We also found that the distribution of H3K4me2 and H3K27me3 on the genes widely expressed is not always associated with their transcriptional levels. Different histone signatures for retinal genes with the same gene expression pattern suggest the diversities of epigenetic regulation. Genes without H3K4me2 and H3K27me3 accumulation at any stage represent a large group of transcripts never expressed in retina. The epigenetic signatures defined by H3K4me2 and H3K27me3 can distinguish cell-type specific genes from widespread transcripts and may be reflective of cell specificity during retina maturation. In addition to the developmental patterns seen in wild type retina, the dramatic changes of histone modification in the retinas of mutant animals lacking rod photoreceptors provide a tool to study the epigenetic changes in other cell types and thus describe a broad range of epigenetic events in a solid tissue <em>in vivo</em>.</p> </div

Genes with the same expression patterns show different histone signatures in retina.

<p>(<b>A</b>) H3K4me2 and H3K27 accumulation in examples of genes up-regulated during retina maturation. (<b>B</b>) H3K4me2 and H3K27 accumulation in examples of genes down-regulated during retina maturation. Closed arrowheads show TSS of each gene (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s018" target="_blank">Text S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s006" target="_blank">Tables S4</a> &S5). (<b>C</b>) Cluster analysis of H3K4me2 and H3K27me3 occupancy around TSS (+/−2.5 Kb) at all developmental stages for the genes upregulated in mature retina. Tree-view shows 4 clusters (C1–C4) with distinct epigenetic patterns for H3K4me2 (upper panel) and H3K27me3 (middle panel) but with same expression patterns (lower panel). (<b>D</b>) Same analysis as in (<b>C</b>) for the genes downregulated in mature retina. Cluster analysis of H3K4me2 and Tree-view shows 3 clusters (C1′–C3′) with distinct epigenetic patterns for H3K4me2 (upper panel) and H3K27me3 (middle panel) but with same expression patterns (lower panel). For <b>A</b> and <b>B</b> y-axis is the number of reads in a 100 bp interval. For <b>C</b> and <b>D</b>, (upper panels) y-axis is the normalized occupancy, or the number of reads for a given histone modification in an interval +/−2.5 kb around the TSS of given gene, normalized for the total number of mapped reads in given experiment.</p

Gene-wide coverage by H3K4me2 and H3K27me3 as a landscape for maintenance of gene activities.

<p>(<b>A</b>) Comparison of average normalized occupancy per 1 Kb of H3K4me2 (upper panels) and H3K27me3 (lower panels) between promoter region (from −2.5 Kb to TSS) and whole gene body (from TSS to TES) for the clusters from Fig. 4c, C1 to C4 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s014" target="_blank">Table S12</a>). (<b>B</b>) Same comparison as in (<b>A</b>) for the clusters from Fig. 4d, C1′ to C3′ (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s015" target="_blank">Table S13</a>). (<b>C</b>) Same comparison as in (<b>A</b>) for genes specific expression in retina but not photoreceptors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s016" target="_blank">Table S14</a>). Y-axis is average occupancies for genes in given cluster, where occupancy is (number of reads in an interval×5,000,000 reads×1 kb)/(total number of reads in experiment × length of genome interval in kb).t-Test for two-sample assuming equal variances was done to compare occupancy on promoter and gene body for each stage and * marks those with statistically significant differences: (*) 0.05><i>P</i>>0.01, (**) 0.01><i>P</i>>0.001, (***) <i>P</i><0.001 (see details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s018" target="_blank">Text S1</a>).</p

Changes in histone modifications during retina maturation.

<p>(<b>A</b>) Immunofluorescence microscopic images of sagittal sections of developmental mouse retina tissue array stained with anti-H3K4me2 (<b>A</b>, green, upper panels) and anti-Rhodopsin (<b>A</b>, red, upper panels), anti-H3K27me3 (<b>A</b>, green, lower panels) and anti-SVP38 (<b>A</b>, red, lower panels), and counterstained with Hoechst in cell nuclei (<b>A,</b> blue). ONBL, outer neuroblast layer; INBL, inner neuroblast layer; GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer. Scale bar represents 25 µm. (<b>B</b>) H3K4me2 labeling with high magnification for ONL from adult retina (Scale bar = 6 µm). (<b>C</b>) H3K27me3 labeling with high magnification for ONL from adult retina (Scale bar = 6 µm). (<b>D</b>) Averaged and normalized intensity profiles for fluorescence of each specific antibody or Hoechst staining across the nuclear centers (e.g. white bars in <b>B</b> and <b>C</b>) of rod photoreceptor nuclei (n = 5). (<b>E</b>) Adult retina outer nuclear layer labeled with an antibody recognizing Crx. (<b>F</b>) Control labeling of adult ONL showing lack of staining with secondary antibody alone. (<b>G</b>) Averaged and normalized intensity profile for Crx labeling (green) and Hoechst (blue) across rod nuclear centers (bar in <b>E</b>). (<b>H, I</b>) Confocal images with high magnification for RGCL from adult retina (Scale bar = 15 µm). Cells marked * or # show distinct cellular distribution with H3K4me2 antibody (<b>H</b>, green) and a′ or b′ show distinct cellular distribution with H3K27me3 antibody (<b>I</b>, green).</p

Genes with the same expression patterns show different histone signatures in retina.

<p>(<b>A</b>) H3K4me2 and H3K27 accumulation in examples of genes up-regulated during retina maturation. (<b>B</b>) H3K4me2 and H3K27 accumulation in examples of genes down-regulated during retina maturation. Closed arrowheads show TSS of each gene (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s018" target="_blank">Text S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s006" target="_blank">Tables S4</a> &S5). (<b>C</b>) Cluster analysis of H3K4me2 and H3K27me3 occupancy around TSS (+/−2.5 Kb) at all developmental stages for the genes upregulated in mature retina. Tree-view shows 4 clusters (C1–C4) with distinct epigenetic patterns for H3K4me2 (upper panel) and H3K27me3 (middle panel) but with same expression patterns (lower panel). (<b>D</b>) Same analysis as in (<b>C</b>) for the genes downregulated in mature retina. Cluster analysis of H3K4me2 and Tree-view shows 3 clusters (C1′–C3′) with distinct epigenetic patterns for H3K4me2 (upper panel) and H3K27me3 (middle panel) but with same expression patterns (lower panel). For <b>A</b> and <b>B</b> y-axis is the number of reads in a 100 bp interval. For <b>C</b> and <b>D</b>, (upper panels) y-axis is the normalized occupancy, or the number of reads for a given histone modification in an interval +/−2.5 kb around the TSS of given gene, normalized for the total number of mapped reads in given experiment.</p

Epigenetic signatures correlate with gene expression patterns.

<p>(<b>A</b>) H3K4me2 and H3K27 accumulation in examples of genes specifically expressed in other non-retinal tissues. (<b>B</b>) Percentage of the genes lacking both H3K4me2 and H3K27me3 accumulation (18%, 4,527 genes) at TSS regions (+/−2.5 Kb) through all developmental stages from all RefSeq genes (totally 20,626 genes) tested by an analysis of Euclidean raw distances (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s018" target="_blank">Text S1</a>). (<b>C</b>) Categorizes genes (4.527 genes in <b>c</b>) lacking both H3K4me2 and H3K27me3 accumulation at TSS by their biological function and tissue specificity. Top 10 categories of genes are listed and “others” (48%) are genes with GO annotations and not listed in the pie chart; “unknown” (19%) are genes without GO annotations and not listed in the pie chart (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046867#pone.0046867.s004" target="_blank">Table S2</a>). (<b>D</b>) H3K4me2 and H3K27 accumulation in examples of genes ubiquitously expressed in most tissues. Y-axis represents the number of reads in a 100 bp interval.</p