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1 Graduate School of Frontier Biosciences, Osaka University, Yamadaoka 1-3, Suita, Osaka 565-0871, Japan
2 Dynamic NanoMachine Project, International Cooperative Research Project, Japan Science and Technology Agency, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
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Abstract |
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-crystallin enhancer previously demonstrated to be cooperatively bound by SOX2 and PAX6. The N-3 core sequence was also bound by these factors in a cooperative fashion, but with a higher threshold of these factors’ levels than DC5, and the enhancer effect of the tetrameric sequence activated by exogenous SOX2 and PAX6 was less pronounced than that of DC5. The observations suggest that gene activation mechanisms that depend on the cooperative interaction of SOX2 and PAX6 but with different thresholds of the factor levels are crucial for the regulation of visual system development. |
Introduction |
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-crystallin is activated by the cooperative binding of PAX6 and SOX2 to its lens-specific enhancer element (Kamachi et al. 2001). These lines of evidence indicate the major contribution of the co-regulatory loops formed between Pax6 and Sox2 genes in the regulation of lens development (Kondoh et al. 2004) and suggest the involvement of analogous regulatory loops in retinal development.
The cooperative binding of PAX6 and SOX2 to the lens-specific enhancer element (DC5) of the -crystallin gene has remarkable features (Kamachi et al. 2001). (i) The PAX6 Paired domain binding site sequence juxtaposed with the SOX binding site deviates considerably from the "consensus" PAX6 binding sequence determined in vitro using the PAX6 Paired domain (Epstein et al. 1994; Czerny & Busslinger 1995), and this non-canonical PAX6 site binds PAX6 poorly in the absence of SOX2. (ii) However, in the presence of SOX2, PAX6 binds to the DC5 sequence very efficiently through direct molecular interaction between these two proteins, and strongly activates the enhancer, which is not active without the simultaneous binding of the two factors. (iii) This cooperative PAX6 binding with SOX2 and strong activation of the enhancer depend on the deviation of the PAX6 binding site sequence of DC5 from the consensus sequence, as replacement of the PAX6 binding site of DC5 with the consensus sequence destroys both their cooperative binding and the strong synergistic activation. This synergistic action exerted by PAX6 and SOX2 is presumably not unique to this lens-specific DC5 element, but may be employed as a general molecular mechanism underlying the ocular tissue development that depends on the PAX6 and SOX2 transcription factors (Kamachi et al. 2001).
The expression of Sox2 covers the entire neural primordium (neural plate and neural tube) and the placodal area of the surface ectoderm in the early stage embryos (Uwanogho et al. 1995; Rex et al. 1997; Uchikawa et al. 2003; Matsumata et al. 2005). However, our recent studies indicated that the continuity of the expression domains of Sox2 is brought about by the overlap of many different expression domains which are individually regulated by various specific enhancers responding to local regulatory cues (Uchikawa et al. 2003, 2004; Takemoto et al. 2006). These embryonic enhancers of the Sox2 gene are significantly conserved among the genomes of various vertebrate species (Uchikawa et al. 2003, 2004; Okuda et al. 2006), indicating that common mechanisms underlie the regulation of Sox2 expression in the embryonic CNS and placodes.
Among these early stage Sox2 enhancers, the enhancer N-3 is highly relevant to the process of visual system development. (i) The embryonic domains showing the activity of enhancer N-3, that is, the diencephalon, optic vesicle and lens placode, are associated with the Pax6 expression. (ii) Most notably, the lens placodal activity of enhancer N-3 is dependent on the contact of the optic vesicle with the surface ectoderm, in the absence of which the cells in the placodal region of the surface ectoderm fail to activate the enhancer N-3. These observations strongly suggest that the molecular mechanisms of the regulation of enhancer N-3 have a central role in embryonic visual system development.
We therefore undertook detailed analysis of enhancer N-3 first by identifying a minimal essential regulatory element of enhancer N-3 as a 36-bp core element, and second by investigating the transcription factors interacting with and regulating the core element. This study demonstrated that the 36-bp core element of enhancer N-3 consists of SOX binding sequence and a non-canonical PAX6 binding sequence, and is activated by the cooperative action of the transcription factors PAX6 and SOX2, but with a higher threshold of the levels of these transcription factors than is required in the case of the DC5 element. This suggests that visual system development is regulated through genetic elements that differentially respond to the PAX6/SOX2 levels.
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Results |
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The activity of enhancer N-3 was visualized by EGFP expression in chicken embryos electroporated at stage (st.) 4 with ptk-EGFP carrying enhancer N-3 and cultured using New's technique (Uchikawa et al. 2003, 2004).
As briefly reported previously (Uchikawa et al. 2003), the enhancer N-3 is activated around st. 8 in the di- and mesencephalon (Fig. 1A), and this activity is also maintained in the optic vesicle that evaginates from the diencephalon after st. 10 (Fig. 1B "ov"). After st. 12 the enhancer N-3 is also activated in the area of the pre-placodal surface ectoderm contacted by the optic vesicle (Fig. 1Bb "le"), which soon forms the lens placode.
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It was also noted that the domains of an embryo exhibiting enhancer N-3 activity roughly corresponded to the Pax6-expressing domains at st. 11, suggesting a close association between the two (Fig. 1Bc).
Identification of the essential core sequence of the Sox2 enhancer N-3
Many of the enhancers involved in developmental regulation comprise a core element that primarily defines the spatio-temporal specificity of the enhancer and non-core elements that are responsible for generating a strong enhancer effect (e.g. Goto et al. 1990; Takemoto et al. 2006). The core element is essential for the overall enhancer activity, and its removal from the enhancer sequence totally inactivates the enhancer, while removal of a non-core element at most reduces the strength of the enhancer effect. The core element alone is usually not sufficient for eliciting an enhancer effect, but multiplication of the core element exponentially augments the enhancer activity while maintaining the specificity, resulting in mimicry of the original enhancer with respect to the specificity and strength. Thus, the core element of an enhancer is operationally defined as a minimal essential sequence, which by multimerization generates an enhancer effect that largely recapitulates the specificity of the original enhancer. We searched for the possible core element sequence of enhancer N-3.
Using a functional assay, enhancer N-3 was originally defined in the chicken genomic sequence as the 582-bp sequence from –15 133 to –14 552 bp relative to the Sox2 translational initiation site (Uchikawa et al. 2003). Overlapping fragments C2-1 to C2-5 of a 639-bp genomic sequence including enhancer N-3 and short surplus sequences were prepared and examined for the enhancer activity to activate tk-EGFP in electroporated chicken embryos (Fig. 2A). Among these five fragments of the enhancer N-3, only fragment C2-3 covering the 121-bp region from –14 968 to –14 848 showed an enhancer activity with specificity analogous to that of the original enhancer N-3 (Fig. 3Ab, showing an enhanced activity of C2-3 as a dimeric form). Thus, this 121-bp fragment was subjected to further analysis.
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Involvement of the putative SOX binding sites S1 (5') and S2 (3') in the enhancer activity of the 50-bp sequence was examined and also the requirement of the intervening sequence by introduction of mutations, and the enhancer activity of these mutated versions of the 50-bp sequence was tested by embryo electroporation. As shown in Fig. 3Ba, a mutation in the S1 SOX site (Mut-S1) did not inactivate the enhancer activity of the 50-bp sequence, indicating the dispensability of this site. By contrast, mutations of either the S2 SOX binding site (Mut-S2) or the intervening sequence (Mut-P) abrogated the enhancer activity of this 50-bp sequence (Fig. 3Bb,c). This observation allowed us to delimit the minimal essential sequence required for the enhancer activity to the 36-bp sequence indicated in Fig. 2B.
The tetramerized 36-bp sequence not only showed enhancer activity but also closely resembled the activity of the original enhancer N-3 (Fig. 3C). In addition, when the 36-bp sequence was removed from the full-length enhancer N-3 sequence (639-bp), the enhancer was completely inactivated (Fig. 3D). These results indicate that the 36-bp sequence containing the putative SOX site plus additional 5'-extending sequence satisfies the criteria of the enhancer N-3 core sequence.
Requirement of two sequence motifs bound by SOX2 and PAX6 for the N-3 core enhancer activity
The DNA sequence 5' of the S2 SOX binding motif includes a motif which may be assignable as a PAX6 Paired domain binding motif (Fig. 2B, and to be detailed in Fig. 5A), although deviating considerably from the consensus PAX6 binding sequences determined in vitro (Epstein et al. 1994; Czerny & Busslinger 1995). The involvement of PAX6 and SOX2 in the regulation of this enhancer was suggested by the similarity of the Pax6 expression domain and the enhancer N-3-active domain, as indicated above, and by the fact that Sox2 is the major Sox gene expressed in the CNS of these early developmental stages (Uwanogho et al. 1995; Rex et al. 1997; Okuda et al. 2006). Thus, the 36-bp core sequence of enhancer N-3 contains two DNA sequence motifs provisionally assigned as SOX and PAX6 binding sequences. To confirm this assignment, we carried out electrophoretic mobility shift assays (EMSA) using full-length SOX2 and Pax6 proteins synthesized in vitro (Fig. 4A). Wild-type and mutated versions of the 36-bp core sequence flanked by polylinker sequences were used as probe DNAs. As shown in Fig. 4A, SOX2 bound strongly to the wild-type sequence, and this binding was abolished by the mutation disrupting the SOX-binding sequence. PAX6 also bound to the sequence despite the deviation of the sequence from the PAX6 binding consensus, and this binding was abolished by the mutation altering the PAX6 binding motif, confirming the specificity of the binding of these proteins to the 36-bp N-3 core.
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Cooperative binding of SOX2 and PAX6 to the N-3 core and DC5 sequences
Alignment of the enhancer N-3 core sequence and the DC5 sequence of the -crystallin enhancer reveals interesting similarities (Fig. 5A): (i) Both sequences can be aligned with the PAX6 Paired domain binding consensus sequence (Epstein et al. 1994), although there are mismatches at many positions, and these positions differ between the N-3 core and DC5 sequences. (ii) Nevertheless, the spacing between the binding sequences for SOX and PAX6 is perfectly conserved between the DC5 and N-3 core sequences. As the DC5 sequence is a target of cooperative binding by SOX2 and PAX6, we examined whether SOX2 and PAX6 bind to the N-3 core sequence in an analogously cooperative fashion, and also whether a significant difference exists in the affinity to these transcription factors between DC5 and the N-3 core.
The DNA binding domain-containing fragments of SOX2 and PAX6 were expressed in Escherichia coli and purified, and their binding to the DC5 and N-3 core sequences was compared. The HMG-domain-containing fragment of SOX2 (SOX2/HMG+) also contained the C-proximally extended Group B1 homology sequence that has been implicated in the cooperative interaction with PAX6 (Kamachi et al. 1999, 2001), while PAX6/PD contained the Paired domain of PAX6 plus a short C-proximal extension (Fig. 5B).
A systematic EMSA analysis was carried out using varying amounts of SOX2/HMG+ and PAX6/PD proteins alone and in combination (Fig. 5C–E). The capacity of the N-3 core sequence to bind PAX6/PD protein was first compared with that of DC5 (Fig. 5C). The N-3 core sequence showed a moderate intensity of band of complex at 2.5 ng of PAX6/PD, whereas the DC5 sequence demonstrated a weak but detectable band at 0.5 ng of PAX6/PD, and a strong band at 2.5 ng. This indicates that the N-3 core sequence requires a several-fold higher level of PAX6/PD protein for effective binding than DC5.
The SOX2/HMG+ protein bound almost equally well to N-3 core and DC5 probes by itself; 0.05 ng of SOX2/HMG+ protein gave a barely detectable band of the protein-probe complex, and the band intensity of the complex increased with increased SOX2/HMG+ (Fig. 5D). When 2 ng of PAX6/PD protein was added, the band corresponding to the ternary complex including SOX2/HMG+, PAX6/PD and probe was formed using SOX2/HMG+ above 0.05 ng, although the efficiency of this ternary complex formation was higher with DC5 than with N-3 core probe. In either case, the efficiency of the ternary complex formation was significantly higher than would be predicted assuming independent binding of these two proteins to the probes, suggesting that SOX2/HMG+ and PAX6/PD bind cooperatively to the probes.
The cooperative binding of SOX2/HMG+ and PAX6/PD proteins to the probes was confirmed using a fixed amount of SOX2/HMG+ (0.25 ng) and varying amounts of the PAX6/PD protein (Fig. 5E). In confirmation of Fig. 5C, the PAX6/PD protein alone bound to the N-3 core probe more weakly than the DC5 sequence, and 2.5 ng of protein was required to demonstrate binding to the probe. However, when 0.25 ng of SOX2-HMG+, which bound equally to both probes, was added, 0.5 ng of PAX6/PD was sufficient to produce the PAX6/PD plus SOX2/HMG+-containing complexes. With increasing PAX6/PD in the presence of SOX2/HMG+, the band intensity of the ternary complex increased significantly using either probe, and the ternary complex was formed without a decrease of SOX2/HMG+ or PAX6/PD monomeric binding to the probes under this unsaturated binding condition. These observations clearly indicate that PAX6/PD and SOX2/HMG+ bind in a highly cooperative fashion to the N-3 core and DC5 sequences. The efficiency of the formation of the SOX2/HMG+/PAX6/PD/probe ternary complex is clearly higher for the DC5 sequence, reflecting the difference in the monomeric affinity of PAX6/PD to the probe.
Synergistic activation of the N-3 core and DC5 enhancers by SOX2 and PAX6
To confirm that the cooperative binding of PAX6 and SOX2 leads to the activation of the enhancer N-3 core, its tetramer was joined to the luciferase reporter with a minimal promoter, and transfected into fibroblasts and lens epithelial cells in primary cultures, with or without the exogenous SOX2/PAX6 effectors derived from the co-transfected respective expression vectors (Fig. 6A).
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Interestingly, the enhancer N-3 core was activated to some degree by the exogenous SOX2 alone in the fibroblasts. However, this is not because a singly bound SOX2 is sufficient for the activation of the N-3 core enhancer, since not only the mutation of SOX2 binding site (Mut-S2) but also that of PAX6 binding site (Mut-PAX) abolished the SOX2-dependent activation of the enhancer N-3 core (Fig. 6C, middle bars). This suggests that in fibroblasts, a factor binding to the PAX6 site partly substitutes for the function of PAX6.
In the lens cells expressing endogenous SOX2 and PAX6, the enhancer N-3 was activated by several-fold, and was activated further by their exogenous supply. Again, the response of the enhancer to exogenous SOX2 and PAX6 was stronger using DC5 enhancer (Fig. 6D).
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Discussion |
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The results of this study demonstrate that the activation of enhancer N-3 in all domains of the visual system primordia is dictated by the activity of the 36-bp-long enhancer core, consisting of SOX2- and non-canonical PAX6-binding sites. This suggests that the enhancer N-3-dependent Sox2 activation relies on prior Sox2 expression, and once the enhancer N-3 gains activity, the Sox2 expression is amplified through an auto-regulatory loop involving enhancer N-3 (Fig. 7).
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Major differences in the enhancer activity between the full-length N-3 and its 36-bp core are the level of activity, the core being very low and demonstrable only in multimeric forms, and much sharper tissue boundary of the activity using the full-length enhancer. Contribution of the non-core sequences in the activation of enhancer N-3 is, therefore, considered not only to amplify the enhancer core-based activity, but also to sharpen the cooperative effect elicited by SOX2 and PAX6.
This study demonstrated that the enhancer N-3 core is activated by cooperative interaction between SOX2 and PAX6 (Figs 5 and 6). This cooperative interaction is largely owing to the non-canonical PAX6 binding site of the N-3 core, which deviates considerably from the consensus sequence, which was determined by the in vitro binding of the PAX6 Paired domain (Epstein et al. 1994). This deviation of the PAX6-binding sequence from the consensus is essential for the cooperative interaction of SOX2 and PAX6 to occur, as demonstrated previously using the DC5 -crystallin minimal enhancer (Kamachi et al. 2001); the replacement of the non-canonical PAX6 binding site with the PAX6 consensus sequence not only disrupted the cooperative interaction between SOX2 and PAX6 but also abrogated the enhancer activity.
Comparison of the N-3 core sequence and the previously identified SOX2-PAX6 co-target DC5 sequence reveals interesting aspects of the cooperative action of SOX2 and PAX6 to such sequences. The nucleotide residues that deviate from the PAX6 binding consensus sequence are different in the two cases, although certain residues are common between the N-3 core and DC5 sequences. Importantly, the distance between the SOX2 and PAX6 binding sequences is perfectly conserved. In a previous study employing the DC5 sequence, we showed that strict spatial constraints exist in order for the cooperative action of these two factors to occur, and insertion of a few base pairs between the SOX2 and PAX6 binding sites inactivates the enhancer effect (Kamachi et al. 2001). It is likely that the N-3 core and DC5 elements observe the same principle for the cooperative action of these proteins. A Pax6 regulatory element has been reported that depends on SOX2 and PAX6 but is not subject to their cooperative interaction (Aota et al. 2003); however, in this case the relative orientations of the SOX2 and PAX6 sites are different from the one found for the N-3 core and DC5.
A significant difference exists between the N-3 core and DC5 elements in the threshold of the transcription factor levels required for the cooperation of SOX2 and PAX6 to occur, the threshold being several-fold higher with the N-3 core element (Fig. 5D,E), and this difference in the target DNA affinity was also reflected by the activation levels (Fig. 6B,D). This suggests a mechanism of differential gene regulation where the genes regulated by the cooperative interaction of SOX2 and PAX6 of different thresholds are activated differently in the spatial distribution and timing depending on the levels of these transcription factors. It is possible that a variety of such genes with different threshold levels for the action of SOX2 and PAX6 contribute to visual system development. This notion could account for the observation that eye development is very sensitive to the Pax6 gene dosage (not only haplo-insufficiency but also supernumery gene copies cause malformation of the eye (Schedl et al. 1996), and is corroborated by the recent observation of a Sox2 expression level-dependent abnormality of retinal development (Taranova et al. 2006).
There are ever-increasing reports of cases indicating that mutations of Sox2 also cause congenital disorder of eye morphogenesis analogous to Pax6 defects in heterozygous patients albeit with a low penetrance (Ragge et al. 2005; Hever et al. 2006). This could also be accounted for by the differential regulation of the SOX2/PAX6 cooperative target sites that are sensitive to subtle differences in the SOX2 and PAX6 levels. Identification of additional regulatory targets of the cooperative action of SOX2 and PAX6 will support this notion.
Two major findings have been made in this study, which are illustrated in Fig. 7. First, the expression of the Sox2 gene in the visual system development is regulated by the cooperative action of PAX6 and SOX2, indicating an autoregulatory loop involving enhancer N-3. This suggests that once PAX6/SOX2 levels exceed a threshold, their action is amplified in the same tissues, optic vesicle and surface ectoderm, underscoring the importance of non-hierarchical nature of developmental gene regulation. Another important finding made in this study is that the same SOX2- and PAX6-dependent regulation of the enhancer N-3 operates in the optic vesicle and lens placode-forming surface ectoderm, but the enhancer N-3 activity in the latter is elicited only after the contact by the optic vesicle. This suggests that a short-range extracellular signal (indicated as "X" in Fig. 7) is emitted from the optic vesicle, and activates the enhancer N-3-dependent Sox2 autoregulatory loop in the cephalic surface ectoderm. The hypothetical signal, a player in classical "lens induction," could be identified as one that activates enhancer N-3 in an isolated piece of pre-placodal head ectoderm.
In conclusion, the enhancer N-3 core element was identified as the second representative system after DC5 activated by the cooperative binding by SOX2 and PAX6, and this discovery revealed new important aspects of the embryonic visual system development dictated by SOX2 and PAX6.
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Experimental procedures |
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Chicken embryos at stage 4 were placed in New's culture condition, and electroporated in the epiblastic layer with plasmid DNAs as described previously (Uchikawa et al. 2003, 2004). The plasmids used were ptk-EGFP or ptk-mRFP1 (Campbell et al. 2002) with insertion of various enhancer sequences.
Electrophoretic mobility shift assay (EMSA)
Full-length SOX2 and PAX6 were synthesized using a transcription/translation-coupled reticulocyte lysate system (TNT; Promega, Madison, WI) using the cDNAs inserted in pCITE-3 (Novagen, San Diego, CA) as templates. Recombinant proteins SOX2/HMG+ (aa. 41–138 of mouse SOX2), and PAX6/PD (aa. 4–136 of chicken PAX6, identical to the mouse sequence), were tagged with glutathione-S-transferase at the N-terminus, synthesized in Escherichia coli, affinity-purified using a glutathione-Sepharose column, cleaved from the glutathione-S-transferase tag, and further purified to near homogeneity by gel filtration. EMSA using the purified recombinant proteins without competitors was carried out as described by Kamachi et al. (2001).
Cell culture and transfection
Primary cultures of fibroblasts and lens epithelial cells from 14-day-old chicken embryo were prepared in wells of 6-well plates as described in Kamachi & Kondoh (1993) and Kamachi et al. (1999). Transfection of these cells was done using a 1.5-µg mixture of the p51LucII firefly luciferase reporter vector (Kamachi & Kondoh 1993) with insertion of the tetrameric N-3 core sequence (1.3 µg), phRG-TK (0.1 µg) expressing Renilla luciferase (Promega), and expression vectors for SOX2 and PAX6 (0.1 µg), using a calcium–phosphate co-precipitation method (Kamachi et al. 1999). The expression vectors for SOX2 and PAX6 using pCMV/SV2 have been described by Kamachi et al. (2001). Luciferase activities were measured 2 days after transfection, and the firefly luciferase activity was normalized by the Renilla luciferase.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: kondohh{at}fbs.osaka-u.ac.jp
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References |
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Received: 5 April 2007
Accepted: 10 June 2007
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