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. Author manuscript; available in PMC: 2015 Aug 15.
Published in final edited form as: Dev Biol. 2014 Jun 2;392(2):256–265. doi: 10.1016/j.ydbio.2014.05.019

Daughterless homodimer synergizes with Eyeless to induce Atonal expression and retinal neuron differentiation

Miho Tanaka-Matakatsu 1, John Miller 1, Daniel Borger 1, Wei-Jen Tang 1, Wei Du 1,*
PMCID: PMC4106475  NIHMSID: NIHMS602443  PMID: 24886829

Abstract

Class I Basic Helix-Loop-Helix (bHLH) transcription factors form homodimers or heterodimers with class II bHLH proteins. While bHLH heterodimers are known to have diverse roles, little is known about the role of class I homodimers. In this manuscript, we show that linked a dimer of Daughterless (Da), the only Drosophila class I bHLH protein, activates Atonal (Ato) expression and retinal neuron differentiation synergistically with the retinal determination factor Eyeless (Ey). The HLH protein Extramacrocheate (Emc), which forms heterodimer with Da, antagonizes the synergistic activation from Da but not the Da-Da linked dimer with Ey. We show that Da directly interacts with Ey and promotes Ey binding to the Ey binding site in the Ato 3′ enhancer. Interestingly, the Ey binding site in the Ato 3′ enhancer contains an embedded E-box that is also required for the synergistic activation by Ey and Da. Finally we show that mammalian homologs of Ey and Da can functionally replace their Drosophila counterparts to synergistically activate the Ato enhancer, suggesting that the observed function is evolutionary conserved.

Keywords: Atonal, Eyeless, Pax6, Daughterless, bHLH proteins, Drosophila eye development

Introduction

Da is the only class I bHLH transcription factor in Drosophila and is known to form heterodimers with spatially and temporally specific class II bHLH proteins. These heterodimers bind to E-box DNA sequences (CANNTG) (Jarman et al., 1993b; Murre et al., 1989) to regulate the proliferation and differentiation of diverse cell types. In addition, Da can also form homodimers, which bind to E-box sequences weakly (Cabrera and Alonso, 1991; Jarman et al., 1993a). However little is known if Da homodimers play any role in development and differentiation.

During Drosophila eye development, photoreceptor differentiation initiates in the morphogenetic furrow (MF) and requires the activities of the class II bHLH protein Atonal (Ato) (Jarman et al., 1994). Ato expression is regulated by two distinct enhancers: the Ato 3′ enhancer controls the initial upregulation of Ato in the MF while the Ato 5′ enhancer controls the later refinement of Ato expression in R8 equivalence groups and eventually in single R8 cells (Sun et al., 1998). Therefore the Ato 3′ enhancer contains the information that integrates developmental control mechanisms for Ato upregulation and retinal neuron differentiation.

The Ato 3′ enhancer was shown to be directly activated by the Retinal Determination (RD) factors Ey, Sine oculis (So), and Eyes absent (Eya) through adjacent Ey and So binding sites (Tanaka-Matakatsu and Du, 2008; Zhang et al., 2006). Ey is expressed in the anterior eye disc with reduced levels in the MF, while Eya and So are expressed from the pre-proneural (PPN) domain to the posterior eye disc (Bessa et al., 2002; Lopes and Casares, 2010) (Fig. S1A–B, S1E). On the other hand, expression of the WT Ato 3′ enhancer, as well as Ato protein, initiates as a uniform band in cells slightly anterior to the MF marker Dpp-lacZ but posterior to the initiation of Eya expression (Fig. S1B–E) (Tanaka-Matakatsu and Du, 2008). These observations suggest that additional mechanisms in the PPN domain control the proper onset of Ato 3′ enhancer activation.

Removing the HLH protein Emc, overexpressing the Notch ligand Dl, or removing the Notch signaling component Su(H) has been found to induce precocious photoreceptor differentiation in the PPN domain (Baonza and Freeman, 2001; Bhattacharya and Baker, 2011; Brown et al., 1995; Fu and Baker, 2003; Li and Baker, 2001). The observed effects of Su(H) and Dl are likely mediated by regulating the levels of Emc and Hairy (Baonza and Freeman, 2001; Bhattacharya and Baker, 2009). Emc functions by antagonizing the activities of Da (Bhattacharya and Baker, 2011; Van Doren et al., 1991). Da has been shown to activate its own expression as well as that of Emc, which is expressed in all cells anterior and posterior to the MF (Bhattacharya and Baker, 2011; Brown et al., 1995). Interestingly the expression of Emc is downregulated in the MF by Hh and Dpp signaling, which leads to increased Da levels in the MF (Bhattacharya and Baker, 2011; Lim et al., 2008) and correlates with Ato upregulation and initiation of retinal neuron differentiation. Therefore the HLH proteins Emc and Da potentially provide additional control of the Ato 3′ enhancer activity.

In this report, we used a Da-Da linked dimer to characterize the role of the Da homodimer in Ato 3′ enhancer activation and the interactions between Da and Ey. Our results suggest an evolutionarily conserved mechanism of regulating Ato expression and retinal differentiation via the bHLH protein Da/E12 and Ey/Pax6 proteins.

Materials and Methods

Fly Strains, Misexpression and Mosaic analysis

Fly culture and crosses were performed according to standard procedure at 25°C. To generate mitotic clones using hs-Flipase, larvae were heat shocked for 1hr at 37°C during the second instar stage. To induce flip-on clones, embryos were collected every 2hrs, larvae were heat shocked for 15min at 34°C during the second instar stage. Fly strains used in this study are: UAS-ey (BL6294), UAS-Pax6 (Halder et al., 1995), UAS-E12-da (Tapanes-Castillo and Baylies, 2004), UAS-emc (Baonza et al., 2000), UAS-da52.2 (Cadigan et al., 2002), UAS-daRNAi (BL26319), UAS-ato RNAi(BLs26316, 34929, 35017), 30A-GAL4 (BL37534) (Brand and Perrimon, 1993), Dpp-lacZ BS3.0 (BL5528) (Blackman et al., 1991), h22 emc1 FRT80B (recombinant of h22 (BL108280) and emc1 FRT80B (BL5532) (Brown et al., 1995), emcAP6 FRT80B (Bhattacharya and Baker, 2011), so1/CyO (BL401) (Cheyette et al., 1994), yw, hsFLP; arm-lacZ M(2) FRT40A, da10 FRT40A (BL5531) (Caudy et al., 1988), yw, hsFLP; pπMyc FRT80B, yw, hsFLP; RpS3 FRT80B arm-lacZ, yw, hsFLP; AyGAL4, UAS-lacZ (BL4410), yw, hsFLP; AyGAL4, UAS-GFP.

Histochemistry

Imaginal disc immunohistochemistry and in situ hybridization were performed as previously described (Tanaka-Matakatsu and Du, 2008). Primary antibodies were used at the following dilutions: rabbit α-Ey 1:1000 (Halder et al., 1998) (gift from Uwe Walldorf), rabbit α-Da DAP7555 1:50 (Cronmiller and Cummings, 1993), mouse α-Eya 1: 40 (DSHB), rabbit α-Ato 1:2000 (Jarman et al., 1995), mouse α-β gal JIE7 Concentrated 1:500 (DSHB), mouse α-Glass 1:20 (DSHB), mouse α-Elav 1:50 (DSHB), mouse α-Myc 1:40 (9E10, DSHB), mouse α-GFP 1:500 (BD Bioscience). Dye conjugated secondary antibodies were from Jackson ImmunoResearch and used at 1:500 dilution: donkey α-mouse Cy3, donkey α-rabbit Cy3, goat α-mouse Cy2, goat α-rabbit-FITC, donkey anti-rat Cy5. DAPI was used at 1:100 (5ug/ml) for DNA staining. Images were taken using a Zeiss AxioImager microscope with an ApoTome.

Transgenic flies

PCR amplified and sequenced Da-Da or Ato-Da tethered dimers were subcloned into the pUAST vector. See the supplementary materials for primer sequences. The resulting constructs were used to established transgenic fly lines as described previously (Tanaka-Matakatsu and Du, 2008).

Molecular Modeling of Da bHLH and Ey Paired domain binding DNA

The structural models of Da bHLH and Ey PD were generated using the I-TASSER molecular modeling server (Roy et al., 2010), based on the bHLH domain of heterodimer E47/neuroD1 (pdb=2ql2) and the human Pax-6 PD-DNA complex (pdb=6pax). The DNA binding domains of Da and Ey were then docked onto the same DNA according to the models and avoiding steric clashes.

Statistical analysis

Wing discs from all genotypes were stained with the photoreceptor marker Glass to detect photoreceptor differentiation. Wing discs were imaged under Zeiss AxioImager using a x10 objective. Twenty wing discs were counted for each genotype unless otherwise indicated. Images were taken at same image acquisition settings for GFP luminosity and GFP area determination. The number of pixels in the Glass positive area was obtained in Photoshop CS3. The adult hinges were imaged under a Leica MZFLIII stereomicroscope at zoom 3.2. Fifty hinges were counted for each genotype. The count data were analyzed and box-and-whisker plots were created in Excel.

GST-pull down Assay

DH5α that carried the respective GST-fusion plasmids were grown to OD600=0.6 and were induced with 0.1mM IPTG for 2hr at 37°C. GST or GST fusion proteins were immobilized with Glutathione sepharose beads (Sigma). Bound proteins were blocked with 1mg/ml BSA in 1xPBS supplemented with 1mM DTT, 1mM PMSF and 1% Triton X-100 for 30min at 4°C before the binding reaction. In vitro transcribed/translated proteins were labeled with 35S-Met using TNT T7 coupled Reticulocyte lysate System (Promega). The binding reaction was performed in 0.1mg/ml BSA in 1xPBS for 2hrs at 4°C. Samples were washed with 1xPBS supplemented with 0.2% NP-40, 1mM DTT and 1mM PMSF for 10min, 3 times. Samples were resolved by SDS-PAGE and autoradiographed using a STORM 860 Phosphorimager (Molecular Dynamics).

Chromatin Immunoprecipitation

ChIP was performed as described before (Tanaka-Matakatsu et al., 2009) with the following modifications. Third instar imaginal discs were dissected and fixed for 25min on ice. Protein A beads were blocked O/N with 100μg/ml BSA and 500μg/ml ssDNA at 4°C before used in experiments. Chromatin samples were incubated with Normal Rabbit Serum, Rabbit α-Ey, Rabbit α-Da-CC (DAP 7555) (Cronmiller and Cummings, 1993) or Rabbit α-Da-BP (Wei et al., 2000) for 8hr at 4 °C. Reverse crosslinking was performed at 65°C for 6hrs. See the primer list for PCR primers.

Results

Emc blocks while Da promotes precocious activation of the Ato 3′ enhancer in the PPN region of the developing eye disc

Deletion mapping of the Ato 3′ enhancer revealed that the 6.4BB, 5.0SB and 3.6BB enhancers showed a maximal level of Ato 3′ enhancer activity, while the previously reported enhancers (Tanaka-Matakatsu and Du, 2008; Zhang et al., 2006) were significantly weaker (2.8PB and 2.0BE, Fig. S2). Therefore the 6.4BB and the 3.6BB Ato 3′ enhancers were used in this study.

To determine if Hairy and Emc regulate initial Ato upregulation, we examined the effect of h22 emc1 double mutant clones on the Ato 3′ enhancer activity. As shown in Fig. 1A–B″, inactivation of both Hairy and Emc alleles led to precocious Ato 3′ enhancer activation (Fig. 1B–B″) and neuronal differentiation (Fig. 1A–A″). In addition, mutant clones of emcAP6, a null allele for emc, also exhibited precocious Ato 3′ enhancer activation (Fig. 1E–E″). These results indicate that EMC prevents precocious activation of the Ato 3′ enhancer in the PPN region, which is consistent with the previously observed function of Emc in preventing precocious photoreceptor differentiation in the PPN region (Bhattacharya and Baker, 2011; Brown et al., 1995).

Figure 1. Da promote photoreceptor differentiation and the Ato 3′ enhancer activation in the PPN region.

Figure 1

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(A–B″) h emc double mutant clones induced precocious retinal cell differentiation in the PPN region (Elav staining in green in A or gray in A′) and precocious activation of the Ato 6.4BB-GFP enhancer (GFP shown in green in B or gray in B′). Mutant clones (pointed by white arrows) were marked by the absence of GFP or anti-Myc (magenta) in A and B respectively. Da (C–C″) or Da-Da (D–D″) overexpressing clones, marked by anti-β-gal (magenta in C, D or gray in C″, D″), induced precocious 6.4BB-GFP activation (green in C, D or gray in C′, D′, white arrows point to clones) in the PPN region. (E–E″) emcAP6 null mutant clones precociously activated the Ato 6.4BB-GFP (white arrows). (F–G″) Flip-out clones of Da-Da linked dimers induced precocious Elav expression (F–F″) and Ato protein upregulation (G–G″) in PPN region of 3rd instar eye disc. Anterior of the eye disc is to the left and dorsal is up. The arrowheads point to the MF. Scale bars are 20μm in A–G.

Emc functions by blocking the activity of Da (Bhattacharya and Baker, 2011). To determine if Da regulates Ato induction in the developing eye, we tested the effect of Da overexpression in eye discs. Da overexpression clones in the PPN region induced precocious Ato 3′ reporter expression (Fig. 1C–C″), similar to the effect of removing the Da negative regulators EMC and Hairy (Fig. 1B–B″ and 1E–E″). These results suggest that Emc blocks Ato induction in the PPN at least in part by inhibiting Da function.

Da and Emc regulate Ey-induced Ato 3′ enhancer activation and ectopic eye formation

Ectopic expression of Ey using the 30A-Gal4 driver (Brand and Perrimon, 1993), which drives expression in a ring-like domain surrounding the wing pouch (Fig. S3N), was able to activate the 3.6BB-GFP reporter in a subset of cells near the anterior-posterior (AP) boundary and induce ectopic eye formation in the hinge region (Fig. 2A, 2N). The RD factor So was shown to be required for Ey-dependent ectopic eye induction (Pignoni et al., 1997). Indeed, introducing one copy of a so mutation decreased 30A>Ey induced Ato 3′ enhancer activation (Fig. 2J and 2M, p=2.2 × 10−4) and decreased the size of induced ectopic eyes (Fig. S3H, S3K). Therefore, 30A>Ey flies provide a sensitized genetic background that can be used to identify additional factors that regulate Ato 3′ enhancer activation.

Figure 2. Ey and Da dimer synergistically activate Ato 3′ enhancer and induce larger ectopic eyes.

Figure 2

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(A–I) The level of Ato 3.6BB-GFP reporter activation in the third instar wing discs. (A–C) Expression of Ey (A), Ey and Da (B), Ey and Da-Da linked dimer (C) using the 30A>Gal4 driver induce an expanded area of the GFP reporter expression (show in green) and increased the area of Glass expression (A′–C′, magenta, white brackets). Knockdown of Da using daRNAi decreased the 3.6BB-GFP positive area in the discs with Ey (D) as well as Ey and Da (E) expression. Reducing the gene dosage of hairy22 emc1 (G), emcAP6 (H) or h22(I) increased the 3.6BB-GFP expression area, while reducing the gene dosage of so1 (J) reduced the 3.6BB-GFP positive area. (K–L′) effects of Da (K) or Da-Da (L) on 3.6BB-GFP Ato 3′ eye enhancer activation and photoreceptor marker Glass expression. Wing discs were counterstained with DAPI (blue). White arrows point to the areas of GFP expression with photoreceptor marker Glass expression. (M) Box-whisker plots display the distribution of ratio values of relative Glass+ GFP positive area over the whole wing disc. N=20 for each genotype. The bottom and top edges of the gray box indicate the range of values between the 25th and 75th percentiles (IQR). The line inside the gray box indicates the median value. Positive or negative value whiskers extended to the greatest or least values from the box, respectively. The values exceeding 1.5xIQR are shown with outlier dots (black filled circle). Each p value to Ey is p<0.0001 as determined by Student’s t test. (N–P) Misexpression of UAS-Ey under the 30A-Gal4 driver induces ectopic eye formation on the adult hinge (black arrows). Ectopic expression genotypes are listed at the bottom of each panel. (Q) Box-whisker plots for ectopic eye sizes. N=50 for each genotype. Each p value to Ey is p<8.6E-09. In this and all the subsequent figures, all the scale bars for wing discs and adult hinge are 50μm and 200μm, respectively.

To determine if Emc and Da affect ectopic Ey-induced Ato 3′ enhancer activation in the wing disc, we examined the effect of altering the activity of Da or Emc in the 30A>Ey wing discs. As shown in Fig. 2, reducing the gene dosage of the da negative regulators h and emc in the 30A>Ey background caused significant expansion of the 3.6BB-GFP positive area (Fig. 2G–I, 2M, p<4.3 × 10−7) and increased ectopic eye sizes (Fig. S3E–G, S3K). Conversely, knockdown of Da by daRNAi in the 30A>Ey background significantly reduced the GFP positive area (Fig. 2D and 2M, p=0.1 × 10−3) and reduced the size of Ey-induced ectopic eyes (Fig. S3B–D, S3K). These data suggest that endogenous Da collaborates with Ey to activate the Ato 3′ enhancer and induce ectopic eye formation.

We further determined the effect of increasing the expression of Da in the 30A>Ey flies. While expression of Da alone had no significant effect, coexpression of Da with Ey increased the 3.6BB reporter expression domain (Fig. 2B, 2K and 2M). Furthermore, increased activation of the Ato 3′ enhancer was correlated with both an increased number of cells expressing the photoreceptor specific marker Glass and increased sizes of ectopic eye induced in the wing hinge region (Fig. 2A–B′, 2N–O, 2Q). Finally, daRNAi decreased the 3.6BB-GFP positive area and ectopic eye sizes in the context of Ey and Da coexpression (Fig. 2D–F, 2M, p< 4 × 10−5, Fig. S3A–D, S3K). Therefore, Da can synergize with Ey to activate the Ato 3′ enhancer and induce ectopic eye formation.

The Da homodimer but not the Ato-Da heterodimer activates the Ato 3′ enhancer synergistically with Ey

We generated Ato-Da and Da-Da linked dimers (Neuhold and Wold, 1993) to test if the Da-Da homodimer and Ato-Da heterodimer can synergize with Ey to activate the Ato 3′ enhancer. Interestingly, coexpression of Da-Da linked dimer and Ey induced a larger 3.6BB-GFP positive area than coexpression of Da and Ey (Fig. 2A–C, 2M). Furthermore, increased Ato 3′ enhancer activation by Ey and Da-Da was correlated with increased expression of Glass (Fig. 2A′–C′) and increased size of induced ectopic eyes (Fig. 2N–Q). The relative ratio of the median value of ectopic eye sizes was: Ey: Ey, Da: Ey, Da-Da= 1:3.1:5.1 respectively (Fig. 2Q). On the other hand, even though the Ato-Da linked dimer showed stronger activation of the Ato target, Dap-HB (Sukhanova et al., 2007), than the Da-Da linked dimer did (Fig. 3A–C), the Ato-Da linked dimer failed to synergize with Ey to induce increased the Ato 3′ reporter expression (Fig 3E–H) and slightly decreased Ey-induced ectopic eye size (Fig. 3I–L). In addition, the Ato-Da linked dimer failed to induce precocious Ato 3′ enhancer activation or precocious photoreceptor differentiation in the PPN region of developing eye disc (Fig. 3M–M″). On the other hand, Da-Da flip-out clones induced precocious Ato expression and photoreceptor differentiation in the PPN region of developing eye disc (Fig. 1F–G″). We conclude that the Da-Da homodimer but not the Ato-Da heterodimer can synergize with Ey to activate the Ato 3′ enhancer and induce ectopic eye formation.

Figure 3. The Ato-Da linked dimer does not promote the Ato 3′ enhancer activation or ectopic eye induction.

Figure 3

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(A–D) Expression of Ato-Da or Da-Da linked dimers using the GMR-Gal4 driver activated the previously characterized Ato target Dap-HB reporter in the eye discs. (E–H) Expression of the Ato-Da linked dimer did not synergize with Ey to activate 3.6BB-GFP expression (white arrows in E and G). The Ey, Ato-Da p value to Ey is p=0.57 (H). Genotypes are listed at the bottom of the each panel. (I–L) Coexpression of Ato-Da linked dimer and Ey using the 30A>Gal4 driver moderately decreased Ey-dependent ectopic eye formation (compare I to K, black arrows). Wing discs and adult hinges median rational values are shown. Each p value to Ey is p<4.56×10−8 (L). (M–M″) Linked dimer Ato-Da flip-out clones were insufficient to induce precocious Ato 3′ enhancer activation or photoreceptor differentiation in the PPN region of the developing eye disc. White arrows indicate the Ato-Da expression clones. Clones were labeled with anti-β-Gal (red) and photoreceptor cells marked by Elav (Blue). Scale bars are 50μm in A–G, 200μm in I-K, and 20μm in M. White arrowheads indicate the MF.

We further compared the effect of inactivating Da or Ato by RNAi in developing eye discs. While Ato RNAi clones spanning the MF induced increased expression of the Ato 3′ reporter (Fig. S4C), Da RNAi caused initial delay in Ato 3′ enhancer activation followed by increased enhancer activation (Fig. S4A). These observations are consistent with the idea that the Ato/Da heterodimer is involved in the downregulation of Ato 3′ enhancer in the MF, while Da has an additional function in the normal onset of Ato expression. Interestingly, expression of Ey in Da RNAi clones blocks the delay in Ato 3′ enhancer activation (Fig. S4B), suggesting that increased levels of Ey can compensate for the loss of Da in the initial activation of the Ato 3′ enhancer.

Emc antagonizes the ability of Da but not Da-Da dimer to activate the Ato 3′ enhancer with Ey

Emc functions at least in part by forming an inactive complex with Da (Bhattacharya and Baker, 2011). We tested the ability of Emc to inhibit synergistic Ato 3′ enhancer activation by Ey and Da or the Da-Da linked dimer in vivo. Indeed, coexpression of Emc with Ey and Da significantly reduced the 3.6BB-GFP reporter expression (Fig. 4A–C, 4I, the p value Ey, Da to Ey, Da, Emc is p= 5.38 × 10−11). In contrast, Emc was unable to reduce the 3.6BB-GFP expression induced by Ey and the Da-Da dimer (Fig. 4E–G, 4I, the p value of Ey, Da-Da to Ey, Da-Da, Emc is p= 0.94) or inhibit Ato-Da linked dimer induced Dap-HB reporter expression (Fig. 3B and 3D). In addition, Emc could not reduce the expression of the photoreceptor specific marker Glass which is induced by Ey and the Da-Da dimer (Fig. 4E′ and 4F′). Therefore Emc can antagonize the activation of the Ato 3′ enhancer by Da with Ey, but it cannot antagonize activation by the Da-Da linked dimer with Ey.

Figure 4. Emc inhibited Da but not Da-Da linked dimer to activate the Ato 3.6BB-GFP reporter in conjunction with Ey.

Figure 4

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(A– H) Ectopic expression genotypes are listed at the bottom of each panel. Discs were counterstained with DAPI (blue). White arrows indicate the area with GFP expression. Wing discs with median ratio values are shown. (I) Statistical analysis of ratio change of GFP area in each genotype is shown in box-whisker plot. p=8.64×10−10 (between Ey, Da, Emc and Ey, Da) and p= 0.94 (between Ey, Da-Da, Emc and Ey, Da-Da).

The Ato Ey2 binding site contains an internal E-box motif that is required for full enhancer activity

Ey contains two distinct DNA binding domains, a Paired domain (PD) and a Homeodomain (HD) (Callaerts et al., 1997). The PD but not the HD is required for eye induction (Punzo et al., 2001; Punzo et al., 2004). The crystal structure of the PD of Pax6, the mammalian homolog of Ey (Quiring et al., 1994), revealed that the Pax6 PD is composed of two subdomains and a linker region (Fig. 5A). Interestingly, the two subdomains make major groove contacts while the central linker makes minor groove contacts over an 8-bp central region of the 20 bp Pax6 binding site (Xu et al., 1999). Alignment of the Ato Ey2 binding site with the Pax6 binding site in the crystal structure revealed the presence of an E-box located in the central region not contacted by the N- or C-terminal part of the PD (Fig. 5A, E-box shown in green). The CATTTG type of E-box was reported to bind to Da-Da homodimers but not to Da-Ac or Da-Sc heterodimers (Jarman et al., 1993b; Kunisch et al., 1994; Yang et al., 2001) and molecular modeling suggested that the major groove of the E-box in the Ey binding site remain accessible for binding by the Da-Da homodimer when the Ey PD binds to the Ey2 site (Fig. 5A) (Ellenberger et al., 1994; Ma et al., 1994). Therefore the Ey2 binding site in the Ato 3′ enhancer is potentially a composite site that allows the binding of both the Da-Da homodimer and Ey.

Figure 5. Synergistic activation of the Ato 3′ enhancer by Ey and Da requires the E-Box sequence within the Ey2 binding site.

Figure 5

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(A) A structure model of Ey PD and Da bHLH binding simultaneously to Ey2 of Ato 3′ enhancer. PAI and RED subdomains (yellow) linked with the linker region (light yellow). Da bHLH dimer (pink) binds to the E-box (green) through the major groove. Ato Ey2 and Math5 T sequences are shown below. E-box sequence and its location are highlighted in green. (B) ChIP assay using 3rd instar imaginal discs from larva expressing Ey and Da-Da. Rabbit anti-Ey and Da antibodies were used. Normal Rabbit Serum (NRS) were used as a control. Diluted input chromatin samples are on left lanes 2–4. A 100bp molecular weight marker (MW) is in lane 1. (C–J) Mutations of Ey binding site or the E-box sequences reduced the Ato 3.6BB-GFP activation. Imaginal discs with median ratio values are shown. The attP/attB mediated site specific recombination systems were used to generate the WT, Ey2MUT and Ey2-So3 reporter transgenic lines. (C–E) Reporter GFP expressions in the third instar eye discs. Levels of luminosity are shown in (K). N=26 in each genotype. P values to WT is p<1.6 × 10−9. (F–H) Mutant reporters reduced the GFP area size in the 30A>Ey ectopic expression wing discs. GFP areas are marked by white arrows. Change in GFP area in the wing disc is shown in (L). N=13 in each genotype. P values are p<7.6×10−8. (I and J, M and N) Da and Ey failed to synergistically activate the Ato 3′ enhancer with either the Ey2 mutation (Ey2 Mut, I) or replacement of the Ey2 with the So3 Ey binding site that does not have an E-box (Ey2-So3, J). N=15 and p=0.501(M). N=15 and p=0.578(N).

A gel shift experiment was carried out to determine the effect of Da on the binding of Ey to the Ey2 site in the Ato 3′ enhancer (Fig. S5D). Interestingly, SL2+ cell extracts expressing WT Ey alone showed relatively weak Ey DNA binding activity (Fig. S5A, lane 1). Addition of the Da bHLH linked dimer to WT Ey extracts significantly elevated Ey2 binding activity (Fig. S5A, lanes 2 and 3), which was specifically competed away by oligonucleotides containing the Ey binding site (Fig. S5A, lanes 4 and 5). These results support the idea that the Da dimer promotes the DNA binding activity of the Ey PD. It should be noted that we did not observe an obviously slower migrating complex corresponding Da-bHLH dimer/Ey/DNA. It is possible that such a trimolecular complex is not stable enough in vitro under our gel shift conditions and may require additional proteins to form stable complexes in vivo. Alternatively, it is possible that the Ey/Da-Da/DNA and Ey/DNA complexes run at a similar location in our gel shift experiments. Experiments using antibodies against Ey and Da demonstrate the presence of Ey in the gel shift complex (Fig. S5B, lanes 6 and 10). However the effects of anti Da antibody were not very clear (Fig. S5B, lanes 7 and 8, Anti Da CC seems to slightly reduced Ey gel shift while the anti Da antibody BP appears to give a slight super shift). It is likely that better antibodies suitable for gel shift experiments will be needed to clarify this issue. ChIP experiments were carried out to further test the binding of Ey and Da to the Ato 3′ enhancer in vivo. Both Ey and the Da-Da linked dimer were found to bind to the Ato 3′ enhancer in vivo (Fig. 5B).

To determine the functional significance of this E-box binding site for Ato 3′ enhancer function, we used the attP-attB mediated site directed integration system (Fig. S6A–C) (Bateman et al., 2006; Fujioka et al., 2008) to generate matched 3.6BB-GFP transgenic fly lines carrying WT or mutant Ey2 sites. The Ey2-Mut has mutations in both the Ey and E box motif while the Ey2-So3 replaces the Ey2 with a previously characterized Ey site that does not contain an E-box (Ostrin et al., 2006; Punzo et al., 2002) (sequences are shown in the supplementary primer list). As shown in Figure 5, both the Ey2-Mut (Fig. 5D) and Ey2-So3 eye discs (Fig. 5E) exhibited reduced GFP expression levels compared to the WT eye discs (Fig. 5C, 5K). In addition, due to the presence of an additional Ey binding site, Ey3, in the 3.6BB Ato 3′ enhancer (Fig. S5C–D), ectopic Ey can still induce low levels of GFP reporter expression in the Ey2-Mut and Ey2-So3 wing discs (Fig. 5F–H, 5L). However, coexpression of Ey and Da failed to induce synergistic activation of the mutant Ato 3′ enhancers (Fig. 5G–J, 5M–N), even though Ey+Da induced synergistic activation of the WT Ato 3′ enhancer (Fig. 2A–B, 2J). Therefore, both the Ey and E-box binding sites of the Ey2 are required for full Ato 3′ enhancer activity in vivo.

The RED subdomain of Ey PD interacts with the bHLH domain of the Da dimer

We next tested the possibility that Ey and Da can directly interact in vitro. Specific interactions between Ey and Da were detected by using 35S labeled Ey proteins and GST-Da proteins (Fig. 6B, Ey deletion constructs shown in Fig. S6). Importantly, Ey interacted specifically with the C-terminus of Da (Da-C1) that includes the bHLH domain but not the N-terminal part of Da (Da-N, Fig. 6B). Furthermore, deletion of the PD significantly decreased the binding between Da and Ey (Fig. 6B). Therefore the C-terminus of Da and the PD of Ey contribute to the interaction between Ey and Da.

Figure 6. PD of Ey physically interacts with bHLH domain of Da.

Figure 6

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(A) Diagram of Da constructs. The pink areas indicate the bHLH domain and the dark grey areas indicate the repression domain. See also Fig. S6 for the diagram of Ey WT and deletion constructs. (B–C, E–F) GST-pull down assays to detect interaction between 35S-labelled proteins (marked with *) to GST-fusion proteins. IVT in each GST-pull down assay panel represented 50% input. (B) Binding of 35S-labeled Ey and Ey-ΔPD to GST Da constructs indicated. Deletion of PD of the Ey decreased Da binding to Ey. (C) Ey protein contains PD domain interact with 35S-labeled Da bHLH linked dimer. (D) Diagram of PD deletion constructs. The PD consists of the N-terminal PAI subdomain, C-terminal RED subdomain, and a linker. Both PAI and RED contain three alpha helical structures (α1 to α6, in orange). Amino acid substitution of residues KR to AA in the PD are indicated with * in PD construct 8. (E) 35S-labeled Da bHLH linked dimer pulled down by GST-PD (lane 2), GST-α3 to RED (lane 5), and to a lesser degree GST-RED (lane 6) proteins. The lane number corresponds to the PD deletion proteins in D. (F) Amino acids substitutions of KR to AA change in RED subdomain of the PD (lane 8) decreased interaction between the PD and Da full length protein.

Since the Da bHLH linked dimer was able in enhance binding of Ey to the Ey2 binding site (Fig. S4), we further tested the interactions between the Da bHLH linked dimer and Ey. The Da bHLH dimer only specifically interacted with GST-Ey proteins that contain the PD (EyN and PD) but not GST fusions that lack the PD (EyC and HD, Fig. 6C). Therefore the PD of Ey can directly interact with the bHLH domain of Da.

The Ey PD consists of an N-terminal PAI subdomain, a C-terminal RED subdomain, and a linker between the two (Fig. 6D) (Jun and Desplan, 1996; Xu et al., 1999). We further defined the subdomains of the PD that interact with Da bHLH. We found that the GST-fusion protein that contained only the RED subdomain was able to interact with Da bHLH dimer weakly (Fig. 6E lane 6, Fig. 6D #6 deletion protein) and the GST-fusion that contain the α3 helix of PAI, linker, and RED domain showed strong interaction (Fig. 6E lane 5, Fig. 6D #5 deletion protein). In addition, deletions of any part of the RED subdomain significantly decreased the interaction between Ey PD and Da (Fig. S7A–B) and mutation of two amino acids in the RED subdomain also decreased interaction (Fig. 6F lane 8, Fig. 6D #8 mutant protein). Therefore most of the sequences in the RED subdomain are required for interaction with the Da bHLH linked dimer and sequences in the linker and C-terminal PAI subdomain contribute to maximal interaction.

Synergistic function of Ey/Pax6 and Da/E12 proteins are evolutionary conserved

Pax6 is the mammalian homolog of Ey and can induce ectopic eye formation (Callaerts et al., 1997; Halder et al., 1995). We tested if Pax6 can replace Ey to synergize with Da and induce ectopic eyes. Pax6 expression alone can only induce a small amount of ectopic Ato 3′ activation and coexpression of Da significantly increased ectopically induced GFP area (Fig. 7A–B, Fig. 7H), which is correlated with the synergistic induction of ectopic retinae by Da and Pax6 (Fig. 7E–F, Fig. 7I). Therefore mammalian Pax6 can functionally replace Ey to synergistically induce ectopic eye formation with Da. Furthermore, the mouse class I bHLH protein E12 and fly Da linked dimer (E12-Da) (Tapanes-Castillo and Baylies, 2004) can function similarly to the Da-Da linked dimer and induce more ectopic retinae and ectopic Ato 3′ enhancer activation in conjunction with Pax6 than Pax6 with Da (Fig. 7D, 7G). The ratio of the median relative sizes of ectopic eye area was: Pax6: Pax6, Da: Pax6, E12-Da = 1: 5.1 : 11.1 respectively (Fig. 7I). Therefore the mammalian Pax6 and E12 proteins can functionally replace fly Ey and Da in the synergistic activation of the Ato 3′ enhancer and induction of ectopic eyes, indicating that the synergistic function between Ey and Da is evolutionary conserved.

Figure 7. Synergistic effects of Ey and Da are evolutionary conserved.

Figure 7

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(A–D and E–G) Ectopic eye sizes and the 3.6BB-GFP positive areas induced by Pax6 coexpression with fly Da or mammalian E12-fly Da chimeric protein. (A–D, H) Third instar wing discs that expressed Pax6 (A), Pax6 and Da (B), Pax6 and Da-Da linked dimer (C), or Pax6 and E12-Da linked dimer (D) expanded the area of the 3.6BB-GFP reporter expression (green, white arrowheads). (H) Relative increase of the 3.6BB-GFP area in the wing disc. P values are p< 0.0001 to the Pax6. N=20. (E–G, I) Effects on ectopic eye induction on adult hinge area. Ectopic eyes pointed by black arrows. Coexpression of E12-Da chimeric protein and Pax6 enhanced the ectopic eye size. (I) Ectopic eye size on adult hinge measured by pixel number. P<0.00001 to the Pax6. N=50

Discussion

Our results show that the Da homodimer and Ey synergistically initiate Ato expression and induce ectopic eyes. We suggest that the synergistic effects of Da and Ey are mediated by the protein-protein interactions between the Da-Da homodimer and Ey and by the presence of an embedded E-box motif within the Ey2 site. Molecular modeling of the Ey/Ey2 binding site complex based on the Pax6/DNA crystal structure (Fig. 5A), revealed that the major groove of the E-box within Ey2 is available for binding by the Da-Da homodimer. Placement of the Da-Da bHLH domain onto the E-box of Ey/Ey2 site structure revealed its proximity to the RED domain and helix α3 of the PAI domain, which mediates the interaction between Ey PD and Da bHLH (Fig. 6D–E). The requirement of the Da-Da homodimer also explains the inhibitory effect of Emc, which forms a heterodimer with Da, inhibits Da-Da homodimer formation, and prevents strong Ato 3′ enhancer activation. As EMC and Da regulate each other’s expression and EMC is downregulated in the MF by Hh and Dpp (Bhattacharya and Baker, 2011), decreased EMC expression in the MF lead to increased Da expression, which promotes Da homodimerization and induce synergistic Ato 3′ enhancer activation with Ey. On the other hand, we found that Ato-Da linked dimers failed to induce precocious photoreceptor differentiation. This is in contrast to the observed effect of precocious photoreceptor differentiation induced by Ato and Da expression (Bhattacharya and Baker, 2011). Since Ato and Da expression can potentially increase the levels of both the Ato-Da heterodimer as well as the Da-Da homodimer, it is possible that the Da-Da homodimer contributes to the observed precocious photoreceptor induction by Ato+ Da expression. Alternatively, our Ato-Da linked dimer may not be very active, unable to activate some critical targets of the Ato Da heterodimer required for inducing precocious photoreceptor differentiation.

Interestingly, the functional interactions between Da and Ey are evolutionarily conserved. The Ey homolog Pax6 and Da homolog E12 can functionally replace their respective counterparts to synergistically activate the Ato 3′ enhancer and induce ectopic eyes. The observed functional conservation is consistent with the fact that interactions between Ey and Da are mediated by conserved domains: the PD and bHLH. Furthermore, Math5, the mouse Ato homolog required for the differentiation of the retinal ganglion cell (Wang et al., 2001), is also regulated by Pax6. Additionally, an Ey2 type Ey/Pax6 binding site with embedded E-box motif is also observed in the enhancer that regulates Math5 (Fig. 5A, Math5 T) (Hufnagel et al., 2007), raising the possibility that the function of Ey/Pax6 and Da/E12/E47 in synergistically inducing Ato/Math5 expression and retinal neuron differentiation is also conserved.

Mammalian E2A is alternatively spliced to produce E12 and E47 proteins, with a key difference being the presence or absence of the DED inhibitory domain, which inhibits the DNA binding and homodimerization of the E12 protein (Shirakata and Paterson, 1995; Sun and Baltimore, 1991). The published Da sequence has the DED inhibitory domain, similar to the E12 protein. Consistent with this, homodimerization of Da is not very strong. It is possible that the reduced ability of Da to homodimerize in conjunction with its self-activation (Bhattacharya and Baker, 2011) makes the levels of the Da homodimer very sensitive to changes in the level of its dimerization partners such as Emc and Ato.

Supplementary Material

01

Highlights.

  • Ey and Da synergistically activate Ato 3′ enhancer and induce ectopic eye

  • The Da homodimer but not the Ato-Da heterodimer synergizes with Ey

  • Ey Paired domain and Da bHLH domain physically interact

  • Mammalian Ey and Da homologs synergistically activate Ato and induce ectopic eye

Acknowledgments

We thank Drs. Nick Baker, Yuh-Nung Jan, Walter Gehring, Nadean Brown, Uwe Walldorf, Jason Clements, Stephen Cohen, Antonio Baonza, Claire Cronmiller, Bruce Patterson, Ilaria Rebay, James Posakony, Francesca Pignoni, Miki Fujioka, Mary Baylies, Justin Kumer and the fly community that supplied us fly stocks and reagents in the course of this work. We also thank the Bloomington stock center (Indiana University), the Drosophila Genomics Resource Center, and the Developmental Studies Hybridoma Bank (University of Iowa) for fly stocks and reagents. This study was supported by grants from National Institutes of Health GM074197 and CA149275.

Footnotes

Author Contributions

M.T.-M. and W.D. conceived the experiments, analyzed the data and wrote the manuscript. M.T.-M. generated all the constructs and was involved in all aspects of the experiments. JM and DB contributed to the quantitative data generation and manuscript preparation. W.J.T. generated structural models based on molecular modeling and contributed to the manuscript preparation.

Competing interests statement

The authors declare that they have no competing financial interests.

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