Gain of Function Notch Phenotypes Associated with Ectopic Expression of the Su(H) C-Terminal Domain Illustrate Separability of Notch and Hairless-Mediated Activities

<div><p>The Notch signaling pathway is instrumental for cell fate decisions. Signals from the Notch receptor are transduced by CSL-type DNA-binding proteins. In <i>Drosophila</i>, this protein is named Suppressor of Hairless [Su(H)]. Together with the intracellular domain of the activated Notch receptor ICN, Su(H) assembles a transcriptional activator complex on Notch target genes. Hairless acts as the major antagonist of the Notch signaling pathway in <i>Drosophila</i> by means of the formation of a repressor complex together with Su(H) and several co-repressors. Su(H) is characterized by three domains, the N-terminal domain NTD, the beta-trefoil domain BTD and the C-terminal domain CTD. NTD and BTD bind to the DNA, whereas BTD and CTD bind to ICN. Hairless binds to the CTD, however, to sites different from ICN. In this work, we have addressed the question of competition and availability of Su(H) for ICN and Hairless binding in vivo. To this end, we overexpressed the CTD during fly development. We observed a strong activation of Notch signaling processes in various tissues, which may be explained by an interference of CTD with Hairless corepressor activity. Accordingly, a combined overexpression of CTD together with Hairless ameliorated the effects, unlike Su(H) which strongly enhances repression when overexpressed concomitantly with Hairless. Interestingly, in the combined overexpression CTD accumulated in the nucleus together with Hairless, whereas it is predominantly cytoplasmic on its own. </p> </div

Scheme of bristle development and the myc-CTD construct.

<div><p>A) During bristle development, a sensory organ precursor cell (SOP) is singled out by lateral inhibition from a cluster of equipotential, proneural cells. By activating the Notch pathway, the SOP forces the surrounding cells into a secondary fate (labeled dark grey). The SOP divides asymmetrically, unequally activating the Notch pathway in the daughter cells: the pIIa cell receives a Notch signal and gives rise to the outer cell lineage, whereas inner cell fate is derived from pIIb. The pIIa daughter that receives a Notch signal will form the socket, the other daughter cell will form the bristle shaft (according to [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081578#B36" target="_blank">36</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081578#B38" target="_blank">38</a>]).</p> <p>B) The Su(H) protein consists of three highly conserved domains, the N-terminal domain (NTD, blue), the β-trefoil domain (BTD, green) and the C-terminal domain (CTD, orange). The N-terminal helix (orange) is in the proximate neighborhood of the CTD in the three-dimensional structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081578#B7" target="_blank">7</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081578#B8" target="_blank">8</a>]. The numbers represent the amino acids of the protein. In the CTD construct, codons 417 to 528 where fused to a myc coding sequence providing the start methionine and the myc-tag for antibody staining (black).</p></div

Modell of a Su(H) cytoplasmic pool.

<p>A) The large cytoplasmic pool of Su(H) is not available for complex formation in the nucleus, however, is used to replenish nuclear Su(H). This explains the apparent limitation of Su(H) when ICN is overexpressed. The export mechanism is currently unknown and may represent a further level of Su(H) regulation. B) Overexpression of Su(H) increases the cytoplasmic protein pool, thereby raising the level of available nuclear Su(H). This may cause Notch gain of function phenotypes in tissues, where Notch signalling takes place, or in the presence of exogenous ICN. The darker colour represents mutant Su(H) protein to exemplify the model; wild type Su(H) would behave identical. C) Combined overexpression of Su(H) and Hairless allows formation of repressor complex, as long as endogenous wild type Su(H) is available from the cytoplasmic pool. Lack of binding of Hairless to mutant Su(H) predicts an enrichment of activator complexes containing the mutant Su(H) and ICN.</p

Quantification of the CTD effect in S2 cell culture.

<div><p>A) Transfection of S2 cells with a luciferase reporter gene containing several Su(H) binding sites was performed and activity measured in the presence of the given constructs. The activation by ICN was taken as 100%; this is enhanced about 3.5 fold by addition of Su(H). Addition of myc-CTD activates as well, although to a lesser degree, despite its lack of DNA-binding. The activation by exogenous Su(H) is effectively repressed by exogenous full length Hairless (H). Interestingly, Hairless is less able to suppress activation by myc-CTD. Control is empty pMT-vector. Three independent experiments were sampled, standard deviation is indicated.</p> <p>B) Co-immunoprecipitation was performed on protein extracts from fly heads overexpressing myc-CTD and Hairless in the developing eye (genotype: gmr-Gal4 / +; UAS-HFL UAS-myc-CTD/ +). Anti-myc antibodies were used for precipitation (IP), detection was with anti-Hairless A (α-H; upper blot 7.5% PAGE) and anti-myc antisera (α-myc; lower blot 12% PAGE), respectively. PE, protein extract as input control; M, mock control; S, protein ladder (sizes in kilodaltons, kD). Myc-CTD has an expected size of 13.8 kDa (arrowheads), the two Hairless isoforms are of approximately 150 and 120 kDa (arrows). The smaller bands in the protein extract detected by anti-H antibodies are most likely degradation products; * light chain immunoglobulins. </p></div

Overexpression of CTD causes Notch gain of function phenotypes.

<div><p>A) Overexpression of GFP results in wild type looking flies and was used as control. The enlargement highlights the microchaetae (open arrow), macrochaetae (closed arrow), bristle shafts and sockets (asterisk). The cartoon depicts the relevant molecules: Su(H) in blue, green and orange reflecting the NTD, BTD and CTD, respectively is cytoplasmic and nuclear (dashed line), where it binds to the DNA (grey). Hairless (red) is bound to Su(H) in either compartment. ICN (green) is bound to nuclear Su(H) on the DNA. Hairless and ICN are balanced in the wild type: activation and repression take place on the DNA (red/green double-headed arrow). </p> <p>B) Overexpression of the activated Notch intracellular domain (UAS-RICN) causes a transformation of inner into outer fate and shaft into socket fate, giving rise to double and quadruple sockets (see four arrows in enlargement). Nearly all bristles are affected. As Notch overexpression affects multiple tissues, the flies die as pharate adults. In the cartoon, ectopic ICN is shown as enlarged circle that directly outcompetes endogenous Hairless, thereby shifting signaling into the activation mode. </p> <p>C) Overexpression of Su(H) is shown for comparison. It causes a transformation of shaft into socket cells affecting the majority of micro- and macrochaetae (open and closed arrows). The cartoon depicts the ectopic Su(H) molecules in both, the nuclear and the cytoplasmic compartments, where they may form additional activator complexes or curb Hairless activity, respectively, shifting the balance in the active mode.</p> <p>D) The myc-CTD transgene likewise enforces a shaft to socket cell transformation, affecting macro- and microchaetae alike (open and closed arrows). As highlighted in the cartoon, CTD cannot bind to the DNA, hence its impact on Notch target gene activity must be indirect. CTD may trap Hairless in the cytoplasm, reducing its availability in the nucleus and shifting the balance into an active mode. PHOTOs on the left were taken with the ES120 camera (colored).</p></div

Complex genetic interactions of novel <i>Suppressor of Hairless</i> alleles deficient in co-repressor binding

<div><p>Throughout the animal kingdom, the Notch signalling pathway allows cells to acquire diversified cell fates. Notch signals are translated into activation of Notch target genes by CSL transcription factors. In the absence of Notch signals, CSL together with co-repressors functions as a transcriptional repressor. In <i>Drosophila</i>, repression of Notch target genes involves the CSL homologue Suppressor of Hairless (Su(H)) and the Notch (N) antagonist Hairless (H) that together form a repressor complex. Guided by crystal structure, three mutations <i>Su(H)</i>^<i>LL</i>, <i>Su(H)</i>^<i>LLF</i> and <i>Su(H)</i>^<i>LLL</i> were generated that specifically affect interactions with the repressor H, and were introduced into the endogenous <i>Su(H)</i> locus by gene engineering. In contrast to the wild type isoform, these <i>Su(H)</i> mutants are incapable of repressor complex formation. Accordingly, Notch signalling activity is dramatically elevated in the homozygotes, resembling complete absence of <i>H</i> activity. It was noted, however, that heterozygotes do not display a dominant <i>H</i> loss of function phenotype. In this work we addressed genetic interactions the three H-binding deficient <i>Su(H)</i> mutants display in combination with <i>H</i> and <i>N</i> null alleles. We included a null mutant of <i>Delta (Dl)</i>, encoding the ligand of the Notch receptor, as well as of <i>Su(H)</i> itself in our genetic analyses. <i>H</i>, <i>N</i> or <i>Dl</i> mutations cause dominant wing phenotypes that are sensitive to gene dose of the others. Moreover, <i>H</i> heterozygotes lack bristle organs and develop bristle sockets instead of shafts. The latter phenotype is suppressed by <i>Su(H)</i> null alleles but not by H-binding deficient <i>Su(H)</i> alleles which we attribute to the socket cell specific activity of Su(H). Modification of the dominant wing phenotypes of either <i>H</i>, <i>N</i> or <i>Dl</i>, however, suggested some lack of repressor activity in the <i>Su(H)</i> null allele and likewise in the H-binding deficient <i>Su(H)</i> alleles. Overall, <i>Su(H)</i> mutants are recessive perhaps reflecting self-adjusting availability of Su(H) protein.</p></div

DNA binding is not altered in Su(H) mutants.

<p>Electromobility shift assay for binding of Su(H) protein variants to the radiolabelled <i>E(spl)</i>m8-S1 oligo <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027986#pone.0027986-Bailey1" target="_blank">[44]</a>. Control, no protein added (lane 1). The binding of the mutant Su(H)^WA, Su(H)^RE, Su(H)^LEWA, or Su(H)^WARE proteins (lane 3–6) to DNA was similar as the wild-type Su(H) protein (lane 2).</p

Expression analysis of a vg^BE lacZ reporter.

<div><p>Influence on the vestigial boundary enhancer lacZ reporter line (vg^BE lacZ) by the ectopic expression of UAS-constructs in the <i>omb</i> expression pattern using omb-Gal4. The vg^BE lacZ expression is shown in red (A-G’, and A’’’-G’’’), the GFP control is shown in green (A,A’’), as is Hairless (D,D’’, E,E’’), myc-CTD (C,C’’,G,G’’) and Su(H) (B-B’’, F-F’’). A-B’’’ and D-F’’’ were used as reference. A’’’-G’’’ show enlargements boxed in A-G; the dashed line marks the overexpression domain.</p> <p>Compared to control (UAS-GFP) (A-A’’’), overexpression of UAS-Su(H) not only causes overproliferation but also induction of vg^BE lacZ expression within the entire overexpression domain (asterisks) (B-B’’’). UAS-myc-CTD overexpression resulted in a mild activation of the vg^BE lacZ reporter activity within the omb-expression domain (CTD) (C-C’’’), similar to the downregulation of Hairless by RNA-interference (UAS-dsH) (D-D’’’) (small arrows in C’,C’’’,D’,D’’’). Interestingly, myc-CTD induced tissue overproliferation typical of Notch gain of function (asterisk in C,C’’). Hairless H (UAS-HFL) overexpression repressed the vg^BE lacZ reporter (E,E’,E’’’; arrowhead), which was normalized in the presence of UAS-myc-CTD (H / CTD) (G-G’’’). (F-F’’’) The combined ectopic expression of UAS-Su(H) together with Hairless H (UAS-HFL) gives a strong super-repressor phenotype: the vg^BE lacZ activity is eliminated in the expression domain (arrowhead in F’, F’’’) and loss of tissue is observed (arrow in F). Discs are oriented with anterior to the left and ventral downwards. Size bar in A (for A-G’’) represents 100 µm; and in A’’’ (for A’’’-G’’’) 20 µm.</p></div

CTD cannot form a super-repressor.

<div><p>A) The consequences of an overexpression of Hairless (H), shown as a reference, are double shafts mostly affecting microchaetae (open arrow) and more rarely macrochaetae (closed arrow). Note also partial transformation of microchaetae (arrowhead). Bristle loss is also observed (asterisk) and can be explained by a transformation of outer into inner cell fates (pIIa to pIIb, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081578#pone-0081578-g001" target="_blank">Fig. 1A</a>). The cartoon depicts ectopic Hairless enlarged. By replacing activator with repressor complexes Hairless enforces a repressive mode.</p> <p>B) Combined overexpression of Su(H) and Hairless (H) is shown as a reference. It causes a super-repressor phenotype which reflects a strong loss of Notch activity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081578#B11" target="_blank">11</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081578#B15" target="_blank">15</a>]. In addition to double shafts (open arrow) and bristle loss (asterisk), bushes of bristles are observed (arrowhead) that reflect a collapse of the lateral inhibition process. Note that the flies die before eclosion as pharate adults. In the cartoon, the super-repressors are shown enlarged, as is the shift in the repression mode. </p> <p>C) Combined overexpression of myc-CTD with Hairless (H) subtly disturbs bristle development: a double socket points to a Notch gain of function (arrow), whereas bristle loss to a Hairless gain of function (asterisk), as does the slightly higher bristle density. The cartoon depicts the model that ectopic CTD is able to quench the effects of ectopic Hairless, as expected if the two bound already in the cytosol. PHOTOs on the left were taken with the ES120 camera (colored).</p></div

Genetic interactions of <i>Su(H)</i> alleles with the deficiency <i>N</i>^<i>5419</i>.

<p><b>(A)</b> Typical examples of wings from female flies of the given genotype are shown. In the upper two rows, wings from controls and <i>Su(H)</i> mutant alleles are depicted. The lower two rows show combinations with the deficiency <i>N</i>^<i>5419</i>. <i>N</i> mutants are characterized by wing incisions (asterisk) and thickened L3 and L5 longitudinal veins (arrowhead points to L5). The doubly heterozygotes of <i>N</i>^<i>5419</i> and any <i>Su(H)</i> mutant allele results in an amelioration of the wing phenotypes. <b>(B)</b> Quantitative analysis summarizing percentage of notched wings derived from females of the given genotype (n, total number of wings is given in each column). Standard deviation is given from 2–4 independent experiments. Statistical significance was determined on the total by ANOVA two-tailed Tukey-Kramer approach for multiple comparisons; significant differences are color coded correspondingly (highly significant ***, p<0.001; very significant **, p<0.01; significant *, p<0.05). Note high variations in the three control crosses <i>N</i>^<i>5419</i>/+ (derived from <i>N</i>^<i>5419</i>/FM7c x Oregon R), <i>N</i>^<i>5419</i>/<i>y</i>^<i>1</i> <i>w</i>^<i>67c23</i>, and <i>N</i>^<i>5419</i>/+; <i>Su(H)</i>^<i>gwt</i>/+. The two null alleles <i>Su(H)</i>^<i>Δ47</i> and <i>Su(H)</i>^<i>attP</i> show drastically different genetic interactions–the former rescuing <i>N</i> wing phenotypes, the latter behaving rather like a wild type allele. Moreover, <i>N</i>^<i>5419</i>/+; <i>Su(H)</i>^<i>LLF</i>/+ does not differ from the controls.</p