Role of Architecture in the Function and Specificity of Two Notch-Regulated Transcriptional Enhancer Modules

<div><p>In <em>Drosophila melanogaster</em>, <em>cis</em>-regulatory modules that are activated by the Notch cell–cell signaling pathway all contain two types of transcription factor binding sites: those for the pathway's transducing factor Suppressor of Hairless [Su(H)] and those for one or more tissue- or cell type–specific factors called “local activators.” The use of different “Su(H) plus local activator” motif combinations, or codes, is critical to ensure that only the correct subset of the broadly utilized Notch pathway's target genes are activated in each developmental context. However, much less is known about the role of enhancer “architecture”—the number, order, spacing, and orientation of its component transcription factor binding motifs—in determining the module's specificity. Here we investigate the relationship between architecture and function for two Notch-regulated enhancers with spatially distinct activities, each of which includes five high-affinity Su(H) sites. We find that the first, which is active specifically in the socket cells of external sensory organs, is largely resistant to perturbations of its architecture. By contrast, the second enhancer, active in the “non-SOP” cells of the proneural clusters from which neural precursors arise, is sensitive to even simple rearrangements of its transcription factor binding sites, responding with both loss of normal specificity and striking ectopic activity. Thus, diverse cryptic specificities can be inherent in an enhancer's particular combination of transcription factor binding motifs. We propose that for certain types of enhancer, architecture plays an essential role in determining specificity, not only by permitting factor–factor synergies necessary to generate the desired activity, but also by preventing other activator synergies that would otherwise lead to unwanted specificities.</p> </div

ASE5 and the mα enhancer are active in distinct cell types in development.

<p>(A) Diagram showing the relationship between the expression specificities of the mα enhancer and ASE5. Drawing at left represents a late third-instar wing imaginal disc; expression territories of the mα enhancer are shown in green. This enhancer is active primarily in proneural clusters (PNCs), each of which gives rise to a sensory organ precursor (SOP) for one of the external sensory organs of the adult fly. One PNC is shown in expanded form in the middle of the panel, to illustrate that the mα enhancer is active specifically in the “non-SOP” cells of each cluster (green), and not in the SOP (white circle) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002796#pgen.1002796-Castro1" target="_blank">[11]</a>. The right part of the panel illustrates the cell lineage by which the SOP generates the four cells that make up an external mechanosensory organ. ASE5 is active specifically in one of these post-mitotic progeny cells, the socket cell (green), which is also marked by high-level expression of Su(H) (red) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002796#pgen.1002796-Barolo2" target="_blank">[10]</a>. (B) Diagrams illustrating the architecture of the two transcriptional enhancer modules analyzed in this study. ASE5 is defined by a 0.4-kb genomic DNA fragment <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002796#pgen.1002796-Barolo2" target="_blank">[10]</a> (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002796#pgen.1002796.s010" target="_blank">Text S1</a>), while the mα enhancer is contained within a 1.0-kb fragment <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002796#pgen.1002796-Castro1" target="_blank">[11]</a>. Known transcription factor binding sites within each module are shown. Essential motifs within ASE5 include five high-affinity Su(H) sites (green S), four strong Vvl sites (blue V1, V2), and a single 11-bp sequence (AACGCGAAGCT) designated the A motif (red A). Functional motifs within the mα enhancer include five high-affinity Su(H) sites, two strong Vvl sites, and a proneural protein “E box” site (red E). Motifs are defined as follows: S, YGTGDGAA (TGTGTGAA omitted); V1, RYRYAAAT; V2, AATTAA; E, RCAGSTG. (C–P) Distinct specificities of ASE5 and the mα enhancer are demonstrated by the patterns of GFP reporter expression (green) they drive in transgenic flies at three different developmental stages. Shown are wing imaginal discs of late third-instar larvae (C, J), pupal nota at 24 hours APF (D–F, K–M), and dorsal epithelium of adult abdomen (G–I, N–P). Socket cells of external sensory organs are marked by anti-Su(H) antibody stain (red). Note that <i>ASE5-GFP</i> is active specifically in both pupal (D–F) and adult (G–I) socket cells [as marked by Su(H) immunoreactivity], but is inactive in the PNCs of both the third-instar wing disc (compare C to J) and the pupal notum (compare D to K). By contrast, <i>mα-GFP</i> is specifically active in PNCs at both stages (J, K) and also exhibits expression in the wing margin territory (J), but is inactive in both pupal — note lack of overlap between green (<i>mα-GFP</i>) and red [Su(H)] signals in M — and adult socket cells (N–P).</p

Rearrangement of required sequence elements has little effect on the activity of ASE5.

<p>(A) Diagrams of ASE5-GFP reporter gene constructs in the “shuffle” series. The five Su(H) binding sites are marked in green; box A (see text) is in red; box B is in blue. Other wild-type (wt) sequences are shown in black, while mutant (mt) sequence (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002796#s4" target="_blank">Materials and Methods</a>) is marked in gray. All constructs are of the same size as wild-type ASE5; the positions of the box A and box B elements are exchanged with those of similar-sized segments elsewhere in the module. ASE5-shuffle1–4 retain wild-type sequences of ASE5, while ASE5-shuffle5–8 bear mutated sequences between the Su(H) sites, box A, and box B. Observed levels of GFP expression in socket cells are summarized at right. Wild-type ASE5 is scored as very strong (+++++); other constructs vary from very strong to moderate (+++) to very weak (+). Constructs that fail to drive detectable GFP expression are indicated as negative (−). (B–K, B′–K′) Effects of motif rearrangements on the activity of ASE5 are examined in nascent socket cells of notum microchaetes at 24 hours APF (B–K; see arrowheads in B), and in mature socket cells in the anterior proximal wing in adults (B′–K′); results are summarized in (A).</p

Mutation of bHLH repressor binding motifs in the 4D and 1B enhancer segments within a <i>neur</i> rescue construct causes ectopic expression of <i>neur</i>.

<p>(A) Diagram of the region surrounding the <i>neur</i> locus. Shown are the boundaries of neur4D and neur1B, the extent of the <i>neur</i> rescue constructs, the locations of bHLH-R binding motifs (those mutated in the rescue constructs are indicated by X’s), and the location of the GFP coding sequence in the tagged rescue constructs. (B-G) Comparison of <i>neur</i> transcript accumulation in wing imaginal discs from neurRC-WT (B and D) and neurRC-4D,1BRm (C and E) larvae. Boxes in B and C surround the developing chordotonal organ of the tegula, shown under higher magnification in D and E. (F) Quantification of the area of <i>neur</i> probe <i>in situ</i> hybridization signal over the chordotonal organ of the tegula [17340±2888 SEM (n = 9) vs. 24040±1575 SEM (n = 21)]. (G) Quantification of the white intensity over the same region, which is inversely proportional to the darkness of staining [112±6.68 SEM vs. 89.2±2.8 SEM]. (H-Q) Comparison of GFP signal in wing imaginal discs from neurRC-WT-GFP (H, J, L, N, and P) and neurRC-4D,1BRm-GFP (I, K, M, O, and Q) larvae. Boxes in H and I denote regions shown at higher magnification in the indicated panels. J and K show GFP signal alone; L and M shown Sens protein signal alone; N and O show the merged signals (GFP in green, Sens in magenta). P and Q are likewise merged images. aDC, pDC: anterior and posterior dorsocentral macrochaetes; aSC, pSC: anterior and posterior scutellar macrochaetes; Ch. Or.: chordotonal organ.</p

neurRC bristle counts.

<p>neurRC bristle counts.</p

Mutation of bHLH repressor binding motifs in the neur4D and neur1B enhancers causes proneural motif-dependent ectopic reporter gene expression in non-SOPs.

<p>(A-B’, D, and E) Comparison of neur4DRm and neur4DP_SRm GFP reporter activities in third-instar wing imaginal discs (A-B’) and 12 h APF nota (D and E). (C) Quantification of ectopic GFP-expressing cells in the scutellar and dorsocentral macrochaete clusters in wing discs from larvae carrying the indicated reporter constructs. The proportion of discs exhibiting ectopic GFP is indicated for each genotype, and the graph reflects the average number of ectopic GFP cells over all discs. (F and G) Comparison of neur1BRm and neur1BP_SRm GFP reporter activities in wing discs. (H) Quantification of ectopic GFP-expressing cells adjacent to the posterior dorsocentral (pDC) macrochaete SOP cell in wing discs from larvae carrying the indicated reporter constructs. Graph presented as described for C. A’ and B’ show the scutellar and dorsocentral regions of the wing disc (see boxes in A and B); insets in F and G show only the region surrounding the pDC SOP. F and G show only the GFP signal; in the remaining images, GFP is in green and Sens protein is in magenta. Caret in A’ points to GFP-positive, Sens-negative cells; see text for details. Error bars in C and H represent standard error of the mean (SEM).</p

Two <i>neuralized</i> SOP enhancers contain conserved binding sites for both proneural proteins and bHLH repressors.

<p>(A) Diagram of the <i>neur</i> locus and flanking genes, showing the locations of the neur4D and neur1B SOP enhancers [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007528#pgen.1007528.ref013" target="_blank">13</a>]. (B) Expanded diagram of the neur4D enhancer, marking the positions of proneural and bHLH-R binding motifs, along with other conserved sequences. (C-H) GFP expression (green) driven by a wild-type (WT) neur4D reporter construct (C, C’, E, and G) or by a proneural motif mutant (P_Sm) version (D, D’, F, and H) in representative third-instar wing imaginal discs (C-D’), 12 h APF nota (E and F), and 24 h APF nota (G and H); C’ and D’ show the scutellar and dorsocentral regions of the wing disc (see boxes in C and D). SOPs are marked by Sens protein (magenta). Caret (<) in (E) identifies two small, adjacent GFP-positive, Sens-negative nuclei. (I) Expanded diagram of the neur1B enhancer, showing the positions of proneural and bHLH-R binding motifs, along with other conserved sequence blocks; refer to (B) for symbol definitions. (J-Q) GFP expression driven a wild-type (WT) neur1B reporter construct (J, L, N, and P), a construct in which the the single P_S-type proneural motif is mutated (P_Sm; K and M), and a construct in which both the P_S- and P_A-type proneural motifs are mutated (P_S+Am; O and Q) in third-instar wing imaginal discs (J, K, N, and O), 24 h APF nota (L and M), and third-instar leg imaginal discs (P and Q). In panels L and M, GFP is in green, Sens protein in magenta. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007528#pgen.1007528.s002" target="_blank">S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007528#pgen.1007528.s003" target="_blank">S2</a> Figs.</p

Forcing persistent non-SOP expression of <i>neur</i> causes both loss and gain of SOPs.

<p>(A) Quantification of macrochaete gain and loss on the dorsal head and thorax of flies of the genotypes indicated at right. Error bars represent SEM. (B and C) Scutellar bristle positions in 24 hr APF nota of the indicated genotypes, stained with anti-Cut antibody, show loss of the SOP with <i>neur</i> misexpression. (D-I) Uniform expression of <i>UAS-neur</i> driven by <i>tub-GAL4</i> in <i>neur</i> mutant clones using the MARCM system in either a wing imaginal disc (D-F) or a 12 hr APF notum (G-I). GFP (green in F and I) marks the territories of <i>tub>neur</i> expression; anti-Sens antibody signal (magenta in F and I) marks SOPs. Brackets in D and F mark SOP loss at the region of overlap between <i>tub>neur</i> activity and the wing margin. Sens-positive cells boxed in D are in a different focal plane from the GFP-expressing cells.</p

Through N signaling, proneural-dependent SOP expression of Neur promotes the inhibition of both <i>neur</i> transcription and Neur function in non-SOP cells.

<p>Proneural proteins activate <i>neur</i> transcription both directly, via binding sites in the neur4D and neur1B enhancers, and indirectly by activating expression of other positive regulators of <i>neur</i> in the SOP. <i>neur</i>-dependent N signaling, combined with proneural factor activity, non-autonomously promotes expression of both <i>E(spl)</i> bHLH-Rs and BFMs in non-SOP cells. The bHLH-Rs repress further transcription of <i>neur</i> directly, through binding motifs in neur4D and neur1B, and similarly inhibit the expression of other SOP-specific targets. The BFMs bind Neur and block its interaction with Dl, preventing non-SOP cells from sending an effective N signal back to the SOP.</p

Lateral inhibition: Two modes of non-autonomous negative autoregulation by <i>neuralized</i>

<div><p>Developmental patterning involves the progressive subdivision of tissue into different cell types by invoking different genetic programs. In particular, cell-cell signaling is a universally deployed means of specifying distinct cell fates in adjacent cells. For this mechanism to be effective, it is essential that an asymmetry be established in the signaling and responding capacities of the participating cells. Here we focus on the regulatory mechanisms underlying the role of the <i>neuralized</i> gene and its protein product in establishing and maintaining asymmetry of signaling through the Notch pathway. The context is the classical process of “lateral inhibition” within <i>Drosophila</i> proneural clusters, which is responsible for distinguishing the sensory organ precursor (SOP) and non-SOP fates among adjacent cells. We find that <i>neur</i> is directly regulated in proneural clusters by both proneural transcriptional activators and <i>Enhancer of split</i> basic helix-loop-helix repressors (bHLH-Rs), via two separate cis-regulatory modules within the <i>neur</i> locus. We show that this bHLH-R regulation is required to prevent the early, pre-SOP expression of <i>neur</i> from being maintained in a subset of non-SOPs following SOP specification. Lastly, we demonstrate that Neur activity in the SOP is required to inhibit, in a cell non-autonomous manner, both <i>neur</i> expression and Neur function in non-SOPs, thus helping to secure the robust establishment of distinct cell identities within the developing proneural cluster.</p></div