13 research outputs found
Pipeline for detection and validation of Hh-responsive enhancers.
<p>Ci/Gli cluster identification and background genome generation were performed as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145225#pone.0145225.s002" target="_blank">S2 Fig</a>. The cluster (CC) for a given genomic region was calculated as the total number of sites observed in the Dm or Dp genome (observed) divided by the average number of sites per background genome for that species (expected). Clusters of Ci/Gli sites with a (CC) ≥ 4 were further filtered as follows: a) Clusters were required to contain at least one Ci/Gli site of ≥0.81 MSS; b) Dm Clusters were required to overlap in position (but not sequence) with a cluster in Dp; c) Clusters in exon or repeat regions were excluded. The entire table of selected clusters, sorted by chromosomal location, is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145225#pone.0145225.s007" target="_blank">S4 Table</a>. The list of clusters was then ranked by average MSS of the predicted Ci/Gli sites and the top 17 were examined functionally (these included 16 novel hits and one known enhancer, <i>ptc</i><sup><i>-0</i>.<i>6</i></sup>). The Hh-responsive enhancer activity of genomic regions containing selected clusters was functionally evaluated by means of a transgenic fly assay as well as by chicken neural tube electroporation. For genomic regions that showed apparent Hh responsiveness, Ci/Gli sites were mutated and re-assayed to confirm direct Ci/Gli regulation.</p
Mapping six Hh regulated enhancers in four genetic loci.
<p>(A-D) Genomic landscape of the <i>ptc</i>, <i>inv</i>, <i>rdx</i> and <i>gsb</i> loci with fragments tested marked by green bars. All predicted Ci/Gli binding sites are highlighted (red/orange tick marks, annotated according to MSS, as noted at top of Fig). The sequence conservation track (gray bars) marks conservation among the 12 sequenced <i>Drosophila</i> species, whereas the dark and light blue bars represent clusters of predicted Ci/Gli binding sites in Dm and Dp, respectively. Black brackets at right indicate 5Kb.</p
Novel enhancers directly respond to Hh signaling in the wing imaginal disc and embryo.
<p>(A-K) β-galactosidase or GFP marks the expression of enhancers in the pouch of the wing imaginal disc. A diagram of the fragments tested and location and MSS for all Ci/Gli sites is shown for each candidate (yellow rectangles). Each wild type enhancer responds to Hh signaling along the anterior-posterior compartment boundary of the wing disc, with the exception of <i>inv</i><sup><i>+16</i>.<i>8</i></sup>(G). Active enhancers lose Hh responsiveness in the wing imaginal disc when predicted Ci/Gli binding sites are mutated, as shown in the right of each panel. (L-O) GFP marks the expression of the noted enhancers in the embryo. <i>En</i> expression (red) marks cells producing Hh ligand. When the predicted Ci/Gli binding sites in these enhancers are mutated (M-O), activity in Hh-responsive cells is severely reduced.</p
Expression of a complex <i>inv</i> enhancer in the chicken neural tube and <i>Drosophila</i> wing imaginal disc.
<p>(A) Genomic landscape of the <i>inv</i> locus depicting the <i>inv</i><sup><i>long</i></sup>, <i>inv</i><sup><i>+16</i>.<i>8</i></sup> and <i>inv</i><sup><i>+18</i>.<i>6</i></sup> constructs. Ci/Gli binding sites are shown as red/orange bars; the intensity of red coloration indicates the MSS. Sequence conservation is indicated by the track at bottom of the panel. (B) Transverse sections of Hamburger-Hamilton stage 21–22 chicken embryos are shown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145225#pone.0145225.g005" target="_blank">Fig 5</a>. DAPI (gray, far left column) depicts nuclei. tdTOMATO (red, middle left column) marks cells electroporated with pCIT or <i>SmoM2</i>. GFP (green, middle right column) reports enhancer activation. Anti-NKX6.1 antibody staining (magenta, far right column) marks Hh-responsive cells. The <i>inv</i><sup><i>long</i></sup> (top row) enhancer demonstrates GFP expression in the ventral neural tube (white arrowhead). The expression of <i>inv</i><sup><i>long</i></sup> is strengthened and broadened with co-electroporation of <i>SmoM2</i> (middle row). Mutagenesis of Ci/Gli binding sites demonstrates that enhancer activity is Ci/Gli dependent (bottom row). (C) Tabulation of activity in the chicken neural tube of <i>inv</i><sup><i>long</i></sup> constructs containing different Ci/Gli site compositions. Green boxes indicate wild type Ci/Gli sequences; purple boxes indicate mutated Ci/Gli sites. Constructs that have functional Ci/Gli sites that correspond to <i>inv</i><sup><i>+18</i>.<i>6</i></sup> (Construct A) or <i>inv</i><sup><i>+16</i>.<i>8</i></sup> (Construct B and C) exhibit GFP expression in the neural tube. However, the central Ci/Gli binding sites are insufficient to drive enhancer activity alone (construct D).</p
Identification and Validation of Novel Hedgehog-Responsive Enhancers Predicted by Computational Analysis of Ci/Gli Binding Site Density
<div><p>The Hedgehog (Hh) signaling pathway directs a multitude of cellular responses during embryogenesis and adult tissue homeostasis. Stimulation of the pathway results in activation of Hh target genes by the transcription factor Ci/Gli, which binds to specific motifs in genomic enhancers. In <i>Drosophila</i>, only a few enhancers (<i>patched</i>, <i>decapentaplegic</i>, <i>wingless</i>, <i>stripe</i>, <i>knot</i>, <i>hairy</i>, <i>orthodenticle</i>) have been shown by <i>in vivo</i> functional assays to depend on direct Ci/Gli regulation. All but one (<i>orthodenticle</i>) contain more than one Ci/Gli site, prompting us to directly test whether homotypic clustering of Ci/Gli binding sites is sufficient to define a Hh-regulated enhancer. We therefore developed a computational algorithm to identify Ci/Gli clusters that are enriched over random expectation, within a given region of the genome. Candidate genomic regions containing Ci/Gli clusters were functionally tested in chicken neural tube electroporation assays and in transgenic flies. Of the 22 Ci/Gli clusters tested, seven novel enhancers (and the previously known <i>patched</i> enhancer) were identified as Hh-responsive and Ci/Gli-dependent in one or both of these assays, including: <i>Cuticular protein 100A</i> (<i>Cpr100A</i>); <i>invected</i> (<i>inv</i>), which encodes an <i>engrailed</i>-related transcription factor expressed at the anterior/posterior wing disc boundary; <i>roadkill (rdx)</i>, the fly homolog of vertebrate <i>Spop</i>; the segment polarity gene <i>gooseberry (gsb)</i>; and two previously untested regions of the Hh receptor-encoding <i>patched (ptc)</i> gene. We conclude that homotypic Ci/Gli clustering is not sufficient information to ensure Hh-responsiveness; however, it can provide a clue for enhancer recognition within putative Hedgehog target gene loci.</p></div
Validation of predicted Hh-responsive enhancers in the chicken neural tube.
<p>Transverse sections of Hamburger-Hamilton stage 21–22 chicken embryos are shown. DAPI (grayscale, far left column) depicts nuclei. tdTOMATO (red, middle left column) marks cells electroporated with pCIT or <i>SmoM2</i>. GFP (green, middle right column) reports enhancer activation. Anti-NKX6.1 antibody staining (magenta, far right column) denotes Hh-responsive cells. (A) Chicken embryos co-electroporated with an enhancerless pGanesh construct (containing only an Hsp70 minimal promoter) and either pCIT or a constitutively active <i>SmoM2</i>. An arrowhead (middle right column; bottom row) depicts a few GFP positive cells in pGanesh electroporated embryos. Note the ectopic NKX6.1 expression (far right column) indicative of overactive Hh signaling in electroporated cells (white arrow). (B-E) Candidate Hh-responsive <i>inv</i><sup><i>+16</i>.<i>8</i></sup> (B top row), <i>inv</i><sup><i>+18</i>.<i>6</i></sup> (C top row), <i>Cpr100A</i> (D top row), and <i>Plc21C</i> (E top row) constructs all exhibit GFP expression in cells in which Hh is activated by co-electroporation of <i>SmoM2</i>. However, chicken embryos co-electroporated with <i>SmoM2</i> in combination with a Ci/Gli-binding deficient mutant (CiKO) of each candidate (bottom rows) show a complete absence of GFP expression in the case of <i>inv</i><sup><i>+16</i>.<i>8</i></sup><i>-CiKO</i> (B) and <i>inv</i><sup><i>+18</i>.<i>6</i></sup><i>-CiKO</i> (C), despite ectopic NKX6.1 expression in both conditions (far right column). <i>Cpr100A-CiKO</i> (D) has a greatly diminished expression pattern with only a few GFP positive cells (white arrowhead) remaining (middle right column; bottom row). <i>Plc21C-CiKO</i> (E) does not show loss of GFP expression, indicating that it is not a direct Hh target, since its response to Hh signaling is not Ci/Gli dependent. <i>Rdx</i> (F top row) GFP expression corresponds to Hh expressing cells and shows no expression once Ci/Gli sites are mutated (<i>rdx-CiKO</i> bottom row).</p
Identification of the domains of PSTPIP1 that are required for filament formation and filament binding.
<p>(A) A schematic view of the PSTPIP1 molecule (416 amino acids) is shown at top. The F-BAR domain (∼300 amino acids) includes the FCH domain, an uncharacterized intervening region (X) and a coiled coil domain (CC). A short helical region (helix 5) of the F-BAR structure is contained within the 5′ end of an uncharacterized region (Y) downstream of the CC domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006147#pone.0006147-Henne1" target="_blank">[17]</a>. The SH3 domain includes amino acid residues 364–416. The location of three mutations associated with PAPA syndrome (at amino acids 230, 250 and 266) is shown; a mutation at position 232 abolishes PSTPIP1 binding to pyrin and to PTP HSCF, a PEST-type protein tyrosine phosphatase (PTP) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006147#pone.0006147-Shoham1" target="_blank">[5]</a>. The specific deletion constructs tested are outlined in the lower schematic. Results of these studies are tabulated at right. “Forms filaments” indicates that the protein forms filamentous structures when transfected alone. “Binds filaments” means that the protein binds to formed full length filaments of co-transfected PSTPIP1. (B–J) Truncated versions of myc-tagged PSTPIP1 shown in (A) were transfected alone or in combination with full-length PSTPIP1-FLAG. B–E) The FCH and coiled-coil portion of PSTPIP1 bound filaments formed by full-length PSTPIP1 (B–D), but was not able to form filaments when transfected alone (E). (F,G) A PSTPIP protein containing the coiled-coil and SH3 region of PSTPIP1 was not able to form filaments (not shown), nor was it able to bind to filaments formed by full-length PSTPIP1 (G). (H) PSTPIP1 lacking the SH3 domain forms filaments when transfected alone. Thus, the SH3 domain is not required for filament formation. (I,J) The two PAPA-associated mutants, A230T (I) and E250Q (J) form long straight filaments similar to those of wildtype PSTPIP1.</p
Pyrin recruits PSTPIP1 to ASC specks.
<p>All images are from transfected COS cells. Representative images are shown. (A) The apoptotic speck protein, ASC (red), is normally diffusely distributed throughout the cell in cytoplasm and nucleus. (B) ASC (in this case, green) can coalesce into a small perinuclear aggregate, the speck. (C–D) Pyrin (red) is recruited to ASC specks via its PyD as previously shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006147#pone.0006147-Richards1" target="_blank">[32]</a>. (E–G) PSTPIP1 is not detected in ASC specks in the absence of co-transfected pyrin. (H–J) In 70% of cells transfected with untagged ASC, PSTPIP1-FLAG and pyrin-myc, PSTPIP1 is recruited to the speck (arrow, H). (K–P) In 30% of cases, transfection of the three proteins results in localization of pyrin in both PSTPIP1 filaments and in the speck (K–M), or exclusively in the speck (N–P). (Q–S) FLAG-tagged W232A PSTPIP1 does not interact with pyrin, and is not recruited to specks. (T–Y) Recruitment of PAPA mutants by myc-pyrin to the ASC speck. (T–V) A230T-FLAG. (W–Y) E250Q-FLAG. Pyrin recruits these mutant forms to ASC specks in 95% of transfected cells.</p
Patterns of pyrin and PSTPIP1 expression in native and transfected cells.
<p>(A) Immunostaining of native PSTPIP1 (green) in human monocytes reveals a finely branched pattern of filaments. (B) In human neutrophils, the PSTPIP1 distribution is filamentous and concentrated at the edge of the cell. (C) In transfected COS cells, PSTPIP1 forms long straight filamentous structures. (D) In human monocytes, pyrin (red) is distributed in a filamentous reticular pattern that extends throughout the cytoplasm and encircles the nucleus. (E) When epitope tagged pyrin (green) is transfected into COS cells, no reticular network is seen; pyrin is diffusely cytoplasmic.</p
Co-expression of pyrin alters the distribution of PSTPIP1 in transfected cells.
<p>(A–E) In cells co-transfected with myc-tagged pyrin and FLAG-tagged PSTPIP1, PSTPIP1 filaments are branched and reticulated, and pyrin co-localizes with these filaments. (A) and (C) illustrate the original images, while (B) and (D) are processed images that have been deconvoluted to remove background and enhance the signal of the filaments. A branched network of filaments surrounding the nucleus is evident. (E) Overlay of pyrin and PSTPIP1 staining pattern after deconvolution; pyrin appears to be concentrated at the nodes of branch points. (F–H) FLAG-tagged PSTPIP1 lacking the SH3 domain, PSTPIP1(-SH3), can recruit myc-tagged pyrin to filaments. Pyrin binding causes the filaments to be highly branched or reticular (compare the PSTPIP1 pattern in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006147#pone-0006147-g004" target="_blank">Figure 4G</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006147#pone-0006147-g002" target="_blank">Figure 2H</a>). (I–K) The W232A mutation of PSTPIP1, which cannot bind pyrin, forms straight filaments (green, J), and pyrin (red, I) does not decorate or reticularize these filaments. The overlay is shown in (K). Note that though filaments appear yellow, there is no filamentous pattern of pyrin (I); rather, pyrin is uniformly distributed.</p