Heterochromatin modulation of the <i>Or59b</i> cluster.

<p>(A) GFP expression (green) driven by the <i>Or59b</i> reporter or <i>Or59b</i> cluster in <i>su(var)3–9</i>^<i>06</i> heterozygote flies. Synaptic neuropil regions are labeled with the presynaptic marker nc82 (magenta). Control flies were crossed to <i>w</i>^<i>1118</i>. (B-E) GFP expression (green) driven by mutated <i>Or59b</i> cluster versions in <i>su(var)3–9</i>^<i>06</i> heterozygote flies. Note that the loss of GFP expression driven by the <i>Or59b</i> cluster with a mutated Pou motif or with a distant E-box is rescued in <i>su(var)3–9</i>^<i>06</i> heterozygote flies. (F) GFP expression (green) driven by an <i>Or59b</i> cluster with an additional E-box in <i>su(var)3–9</i>^<i>06</i> heterozygote flies. Note that the E-box rescues the produced <i>su(var)3–9</i>^<i>06</i> expression phenotypes. (G) Model depicting the function of the cluster in the regulation of <i>Or59b</i> expression. Our results propose that the Hox/Pou motif regulates the heterochromatin state and allows bHLH proteins to bind the E-box, which induces expression. The E-box and Pou motif sequences overlap to generate unstable binding, and a steady state is generated that drive expression in the Ab2a and Ab7b OSN classes. Cooperative interactions between E-boxes stabilize expression in the face of environmental perturbations. Schematic models of the results are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005051#pgen.1005051.s005" target="_blank">S5</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005051#pgen.1005051.s006" target="_blank">S6</a> Figs.</p

Temperature stress or starvation hampers <i>Or59b</i> cluster function.

<p>(A) Following 3 days of starvation, the mRNA levels of indicated OR genes were measured by qPCR and compared with control flies (* p < 0.05; ** p < 0.01; error bars represent SEM). (B) GFP expression (green) driven by the <i>Or59b</i> reporter or <i>Or59b</i> cluster following 3 days of starvation. Synaptic neuropil regions are labeled with the presynaptic marker nc82 (magenta). Phenotype penetrance is marked as a percentage at the top-right corner of the image. (C) Following 3 days at 14°C, the mRNA levels of the indicated ORs were measured by qPCR and compared with flies maintained at 24°C (* p < 0.05; ** p < 0.01; *** p < 0.001; error bars represent SEM). (D) GFP expression produced by the <i>Or59b</i> reporter and the <i>Or59b</i> cluster at 14°C. Note that the same insertion of the <i>Or59b</i> cluster construct produces both loss- and gain-of-expression phenotypes at 14°C. Schematic interpretations of the results are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005051#pgen.1005051.s006" target="_blank">S6 Fig.</a></p

Spatial expression pattern is dictated by the structure of the <i>Or59b</i> cluster.

<p>GFP expression (green) produced by (A) the <i>Or59b</i> cluster, (B) the cluster with the E-box displaced 125bps, (C) with the E-box 10bps upstream the cluster, (D-E) the Ebox 5 or 10 bp downstream the cluster. Synaptic neuropil regions are labeled with the presynaptic marker nc82 (magenta). A schematic representation of different rearrangements is shown under each figure. Schematic interpretations of the results are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005051#pgen.1005051.s005" target="_blank">S5 Fig.</a></p

One motif cluster combines the TF regulation of <i>Or59b</i> gene expression.

<p>(A) Diagram of the 1000-bp upstream region of the <i>Or59b</i> gene showing the locations of the Pou (blue), Acj6^Hox (orange), Pdm3^Hox (red) and E-box (green) motifs. The gray box marks the cluster of Hox/pou/E-box motifs. Below, the 36-bp <i>Or59b</i> cluster sequence is presented. (B-D) A whole-mount brain shows GFP expression driven by the <i>Or59b</i> cluster (green) and the synaptic neuropil marked by nc82 (magenta). The marked region defines the whole brain and the antennal lobes. (C) GFP expression from the <i>Or59b</i> reporter and <i>Or59b</i> cluster in the antenna and in the antennal lobe, where it marks axonal projections to the DM4 and VC4 glomeruli. (D) Loss of expression produced by the <i>Or59b</i> cluster is observed in the <i>Acj6-</i>, <i>Fer1-</i> and <i>Pdm3-IRs</i> but not in <i>Atro-IR</i> or <i>UAS-Atro</i> overexpression lines. Control flies were crossed to <i>Peb-Gal4</i>.</p

Cooperative regulation between the cluster and surrounding motifs produces robust class-specific expression.

<p>(A) GFP expression (green) driven by the <i>2×Or59b</i> cluster at 14°C and 24°C. Note the three expression phenotypes produced by the <i>2×Or59b</i> cluster at 14°C. Synaptic neuropil regions are labeled with the presynaptic marker nc82 (magenta). (B) GFP expression (green) produced by the <i>Or85a</i> cluster at 14°C, 24°C or following 3 days of starvation. Note that GFP expression is equally strong in different lines at 14°C. (C) The <i>Or59b</i> cluster with an additional E-box produces robust expression at 14°C and 24°C. (D) The fractions of the brains showing stable or bimodal expression of GFP according to the genotype and temperature. Note that only the <i>Or59b</i> cluster and 2×<i>Or59b</i> cluster are unstable at 14°C. (E) Quantification of GFP positive cells in the antenna. <i>Or59b</i> cluster shows a varied number of GFP positive cells at 14°C compared to 24°C. <i>Or59b</i> and <i>Or59b</i> cluster with an additional E-box show stable expression in both temperatures. Schematic interpretations of the results are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005051#pgen.1005051.s006" target="_blank">S6 Fig.</a></p

The Acj6 to Pdm3 ratio dictates the <i>Or59b</i> cluster-driven expression.

<p>(A) GFP (green) expression driven by <i>Or59b</i> cluster constructs with mutated Acj6^Hox, Pdm3^Hox, Pou and E-box motifs. Synaptic neuropil regions are labeled with the presynaptic marker nc82 (magenta). (B) GFP expression driven by the <i>Or59b</i> cluster in different backgrounds. Loss of expression of the <i>Or59b</i> cluster is observed in <i>acj6</i>^<i>6</i> +/− flies and is rescued in <i>acj6⁶/pdm3^MIO3202</i> flies. Control flies were crossed to <i>w</i>^<i>1118</i>. Schematic interpretations of the results are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005051#pgen.1005051.s005" target="_blank">S5 Fig.</a></p

A regulatory matrix for <i>Drosophila</i> OR expression.

<p>(A) The regulatory matrix represents in situ hybridizations for 32 ORs/TF-IR, indicated as wild-type levels (gray dots) and lost (black dots) OR expression. Trichoid ORs marked in orange. (B) Bar diagram, representing number of ORs that required each TF for expression. Trichoid ORs marked as orange insets in each bar. (C) Number of ORs regulated by 0–7 TFs depicted as bar graphs. (See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001280#pbio.1001280.s006" target="_blank">Table S2</a> for statistics and see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001280#pbio.1001280.s003" target="_blank">Figure S3</a> for domain and sensilla arranged matrix).</p

The location of the binding site upstream of the OR dictates Xbp1 function.

<p>(A) Motif density plot, showing motifs found upstream of OR genes that did not require the matching TF (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001280#pbio.1001280.s007" target="_blank">Table S3</a> for statistics). (B) Bar graph depicting the total number of motifs located upstream or downstream the TATA box for ORs that either require the TF (“essential”) or not (“nonessential”) for expression. (C) Double in situ labeling of <i>Or98a</i> and <i>Or56a</i> in wild type (Wt) and <i>xbp1-IR</i> antennae revealed ectopic <i>Or98a</i> expression next to <i>Or56a</i>. The RNAi phenotypes are summarized as a matrix (grey, wild-type expression; red, ectopic; and black, loss of expression). (D) One Xbp1 motif (purple) was found next to the TATA box (green) of <i>Or98a</i>. The <i>Or98a</i> promoter construct produced expression in a single domain (light blue oval, black expression). Whereas, the same <i>Or98a</i> promoter construct with a mutated Xbp1 motif (red) produced a distal expansion of the expression.</p

Expression of OR gene regulators in the adult <i>Drosophila</i> antenna.

<p>(A) The identified TFs belong to different protein families as indicated by their protein domain organization. (B) In situ hybridizations and immunohistology on wild-type antenna sections showing the expression pattern of each TF (red) counterstained with the nuclear marker DAPI (blue). (C,D) RNAi-mediated reduction of Acj6 (C) and Xbp1(D) does not affect the overall expression pattern of the other six TFs. (E) Expression of the TFs (magenta) in either <i>Or47b-CD8::GFP</i> or <i>Or92a-CD8::GFP</i> (green) expressing OSNs. Note, that the Or47b expressing OSNs lack expression of onecut and zf30c (arrows).</p

An RNAi screen identifies seven TFs required for OR expression.

<p>(A) Whole mount preparations of antenna from the two screening rounds (GFP in black). In the first round, expression of <i>Or98a-CD8::GFP</i> and <i>Or23a-CD8::GFP</i> in two mid-antennal domains (light blue and orange oval) were analyzed. In the second round, <i>Or92a-CD8::GFP</i> expression in the most proximal (dark blue oval) antenna domain and <i>Or47b-CD8::GFP</i> expression in the most distal (red oval) antenna domain were analyzed. (B) Statistics from the screen is depicted as a graph, summarizing the number of IR lines that did not affect OR expression (Wt, white), led to lethality (Lethal, grey) or lost OR expression (Loss of OR expression, Green). (C) Phenotype summary for the seven TF-IRs and the analyzed OSN classes, wild-type OR expression (grey dots) and loss of OR expression (black dots). (D) Antenna from each TF-IR with representative OR expression phenotypes. (E) Whole mount antennal lobe with the Or92a-CD8::GFP OSN projections shown in green and the synaptic marker, nc82, delineating the glomeruli of the antennal lobe, in magenta. The boxed region indicates the antennal lobe area in the right panel, which compares the RNAi and mutant phenotypes of <i>acj6</i>, <i>sim</i>, <i>xbp1</i>, <i>zf30c</i>. Note the loss of <i>Or92a</i> in both the mutant and RNAi lines.</p