Single Molecule Visualization and Characterization of Sox2–Pax6

Letter pubs.acs.org/NanoLett

Single Molecule Visualization and Characterization of Sox2−Pax6 Complex Formation on a Regulatory DNA Element Using a DNA Origami Frame Seigi Yamamoto,† Debojyoti De,‡ Kumi Hidaka,§ Kyeong Kyu Kim,*,‡ Masayuki Endo,*,§,∥ and Hiroshi Sugiyama*,†,§,∥ †

Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan ‡ Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea § Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan ∥ CREST, Japan Science and Technology Corporation (JST), Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan S Supporting Information *

ABSTRACT: We report the use of atomic force microscopy (AFM) to study Sox2−Pax6 complex formation on the regulatory DNA element at a single molecule level. Using an origami DNA scaffold containing two DNA strands with different levels of tensile force, we confirmed that DNA bending is necessary for Sox2 binding. We also demonstrated that two transcription factors bind cooperatively by observing the increased occupancy of Sox2−Pax6 on the DNA element compared to that of Sox2 alone.

KEYWORDS: Sox2, Pax6, AFM, single molecule, DNA origami, protein−DNA

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of all effects.12 The possibility of each molecule behaving differently from the average effect of the bulk molecules remains largely unexplored. At the molecular level, most of the processes are more discrete and random, and the bulk properties in most cases do not replicate the properties of a single molecule. In complex biological systems, this effect is magnified, and hence, a single molecule approach is critical to understand complex biochemical processes. In the past few years, an exponential surge in the development of single molecule studies and associated techniques has created new possibilities for more accurately and quantitatively analyzing various biological phenomena.13−23 The capability of directly visualizing molecules in motion,24 the ease of data procurement, and the flexibility to study samples prepared at physiologically relevant conditions provide an extra edge in rapid utilization of single molecule analysis.25,26 Force-based techniques like atomic force microscopy (AFM) have also gained prominence24 and allow direct visualization and precise localization of molecular changes in real time on a nanometer scale, which is difficult using traditional biophysical methods.

he complexity of organism development is enormous, with several key transcription factors forming multiprotein complexes that guide the precise spatiotemporal expression of a variety of genes controlling development and thus managing the enormous complexity of life.1−5 It has also been realized that transcriptional regulation is largely influenced by the combinatorial effect of these factors, rather than individual effects of each factor.1−5 One good example that fits these criteria is Sox2, which has a role that varies across developmental stages depending on which transcription factor forms a complex with Sox2. In embryonic stem cells, Sox2, a core transcription factor that maintains pluripotency,6,7 works along with Oct4 by tandem binding to DNA elements in the numerous stemnessrelated genes. However, it is also present in more committed lineages like neural stem cells to carry out specific functions depending on the context of the developmental stage.8 For example, in neuronal lineages, Sox2 forms a pair with other transcription factors such as Pax6,9 Otx2,10 Tlx,11 or Brn28 to activate the genes required for neuronal differentiation. Therefore, the intriguing questions are what the binding mode of Sox2 to different transcription factors is and how Sox2 is capable of swapping partners. The traditional approach to studying biochemical and biological processes does not take into consideration effects at the single molecule level but rather gives an ensemble or average © 2014 American Chemical Society

Received: December 4, 2013 Revised: March 11, 2014 Published: March 24, 2014 2286

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Figure 1. Overall experimental schema. (a) The model structure of the Sox2(HMG)/Pax6(DBD) complex on their putative DNA element, which was built by superimposing the Pax6 structure onto an Oct1 model adapted from the structural study of Sox2.49 This model shows the DNA binding mode of Sox2(HMG) and its interaction with Pax6(DBD) as well as the conspicuous bend mediated by Sox2. The bend axis is marked in red. (b)The DNA origami frame with the vacant area (40 nm × 40 nm) inside contained four connection sites, namely, “A, B, C, and D.” The two DNA strands of 64-mer duplex and 74-mer duplex differ with respect to the length of linkers represented in red and orange, respectively. Different elements of these duplexes are represented below the frame. (c) The overall schema of the origami frame with the two DNA strands inside the vacant area. The red and orange coils represent the double-stranded DNA corresponding to the tensed 64-mer and relaxed 74-mer dsDNA with the DC5 elements, respectively. Protein is represented as a blue oval. When added to the empty origami frame (left), proteins bind to the 64-mer or 74-mer DNA duplexes (right). The DNA frame lacks the right bottom corner (marked by an orange triangle) which is used as an orientation mark for the identification of 64-mer and 74-mer duplexes.

Table 1. Quantification of Protein Occupancy on Various DNA Frames Sox2(HMG) only 64-mer DNA 69-mer DNA 74-mer DNA

Pax6(DBD) only

Sox2(HMG)/Pax6(DBD)

occupied/total (frames)

percentage (%)

occupied/total (frames)

occupied/total (frames)

percentage (%)

24/981 21/564 46/981

2.5 3.7 4.7

0/∼300 0/∼300 0/∼300

8/225 15/326 22/225

3.6 4.6 9.8

Pax6 and Sox2 are coexpressed in neuronal and retinal tissues42 and work together in neuronal and retinal development.10,43−45 The expression of δ-crystallin, the most abundant lens protein, depends precisely on the cooperative complex formation and synergistic activity of Sox2 and Pax6 on the minimal enhancer element (DC5) of the δ1-crystallin gene.9 Transcription generally involves several factors occupying positions in a tandem fashion over a large stretch of DNA. The long DNA stretches occupied by these factors often fold back to form loops, thus bringing these factors into the range of interaction.46 To make such architecture, DNA must be bent by a protein. In stem cells and their various lineages, Sox2, a transcription factor containing a high mobility group (HMG) box, plays a role in DNA bending,47 providing other transcription factors with access to DNA and binding to Sox2.48 From the crystal structure of the HMG domain of Sox2 [Sox2(HMG)] in complex with DNA, it is suggested that Sox2 binds to the minor groove of DNA and bends DNA toward the major groove, as shown in Figure 1a.49 DNA must be long enough to accommodate the DNA bending

In particular, AFM is advantageous for studying protein−DNA interaction when combined with DNA origami, a scaffold formed by single stranded viral M13 DNA hybridized with several small predesigned “staple” oligomers.27 This method has been applied to construct various 2D and 3D structures with the provision of precise positioning of various functional molecules28−33 and harnessing their applications. Since its first development, DNA origami has been well-utilized in several single molecular studies like chemical,34 photochemical,35 and biochemical reactions36−38 and for the identification of SNPs39 and conformational changes in DNA.40,41 The addressability of these structures prompts their use to accurately position various functional molecules within the framework, and thus, these molecules can be easily located on the scaffold using atomic force microscopy. By using these approaches, it is possible to easily track molecular changes in real time. In this study, we attempted to observe the transcription process at the single molecule level by analyzing Sox2 binding to DNA and Sox2−Pax6 interaction using the AFM method. 2287

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Figure 2. Sox2(HMG) loading on the DC5 element. (a) AFM images and (b) individual images of the Sox2(HMG)-loaded DNA frames. Yellow arrows in the AFM images represent Sox2(HMG) bound to the dsDNA in the DNA frame. The orange triangle represents the orientation mark, which precisely identifies the presence of Sox2(HMG) on 64-mer and 74-mer dsDNA. (c) Representative AFM image of Sox2(HMG) bound onto a bent 74-mer dsDNA (top left) and the drawing of dsDNAs and Sox2(HMG) (green) directly traced from the AFM image (top right). Threedimensional image of the dsDNA-Sox2(HMG) complex (bottom). An arrow points to a kink made by Sox2(HMG) binding.

Figure 3. Pax6(DBD) loading on the DC5 element. (a) AFM images of Pax6(DBD) loaded on the DC5 elements in the origami frame (left). Pax6 alone failed to occupy any of the frames tested, confirming the low intrinsic binding capacity for their recognition element. Enlarged images of the representative DNA frames are also displayed (right). (b) AFM (left) and individual (right) images of Pax6(DBD) loaded on the DC5 element in the presence of Sox2(HMG). Green arrows in the AFM image indicate protein complexes bound to the duplex in the frame after addition of Sox2(HMG) and Pax6(DBD). The orange triangle represents the orientation mark. 2288

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necessary to provide access to Sox2. The structural study of Sox2(HMG) in complex with the DNA binding domain of Oct1 also reveals that the DNA-binding domain of Sox2 uses a separate interface to mediate its diverse interactions.49 It was also reported that Sox2 forms a complex with interacting partners in a cooperative fashion.9−11,48 Although the binding mode of Sox2 to DNA and its mechanism of action and cooperativity were proposed based on the crystal structure and bulk solution studies, it is necessary to quantitatively confirm the proposed mechanism of action at the single molecule level to precise understanding of their interaction and accompanying transcription process. Furthermore, because the high-resolution AFM technique has never been applied to analyze the dynamics of transcription factors, this study can contribute on extending the application repertoire of high-resolution AFM to transcriptome analysis. To monitor the Sox2(HMG) binding to DNA and to follow changes in the architecture at the single molecule level, we prepared DNA origami frames and scanned them with atomic force microscopy (AFM) in the presence of Sox2(HMG) (Figure 1). For this purpose, a DNA frame of rectangular shape (100 nm × 80 nm) was constructed with a vacant rectangular area (40 nm × 40 nm) that contained four connection sites for attaching two double-stranded DNA segments (dsDNA) (Figure 1b and S1). Each dsDNA contained the DC5 element, which is the binding site of Sox2 and Pax6 as the transcription element of δ-crystallin10 (Figure 1a). To examine the need for DNA bending for Sox2 binding, we prepared a tensed 64-mer and a relaxed 74-mer dsDNA by varying the length of linker connecting DC5 element to the origami frame (Figure 1b,c). The tensed 64-mer dsDNA, which restricts appropriate bending due to a high tensile force, showed less Sox2(HMG) occupancy, whereas the relaxed 74-mer DNA containing the DC5 element displayed higher occupancy of Sox2(HMG) (Figures 2 and S2, Table 1). Sox2(HMG) occupied 2.5% of the 64-mer dsDNA and 4.7% of the 74-mer dsDNA. Since the calculated length of 64mer dsDNA is similar to the distance between two connectors (“A” and “B” in Figure 1a), the 64mer dsDNA is expected to exhibit the highest tensile force. Therefore, it was assumed that Sox2(HMG) can still bind to the stiff DNA although the binding affinity or occupancy is significantly reduced. Previously, we also observed, using the DNA origami frames, that the tension of the substrate dsDNA is critical for controlling the activity of DNA methyl transferases and repair enzymes.36,37 Using these results together, we proved the advantage of using AFM and DNA origami frames for the study of protein−DNA interaction in the single molecule level, which is necessary to investigate their conformation and reactivity. In addition, we obtained several images of Sox2(HMG) bound to DNA with a kinked conformation and displayed one of representative images (Figure 2c). It has been reported that most of the partners of Sox2 have low intrinsic DNA-binding affinity, but they can form a stable ternary complex in the presence of Sox2.9−11,48 To confirm this phenomenon, we incubated DNA frames with the DNA binding domain of Pax6 [Pax6(DBD)] with and without Sox2(HMG). While Pax6(DBD) alone showed no binding to DNA (Figure 3a), incubation with Sox2 resulted in complex formation (Figure 3b and S3, Table 1). The repeated failure to locate Pax6(DBD) alone in the frame suggests that Pax6 alone is incapable of binding to DNA or has very weak DNA binding affinity. In contrast, high occupancy of the complex in the DNA

Figure 4. Quantification of the size and numbers of protein complexesloaded on the DC5 element. (a) Calculation of the protein volume. Each dimension is indicated in the representative AFM image of protein loaded on DNA. The equation is adopted from the report of Luda et al.50 (b) A histogram representing the volume distribution of complexes formed by Sox2(HMG) alone (top) and also in the presence of Pax6(DBD) (bottom). The complexes formed in the presence of Pax6(DBD) were greater in volume as compared to the complexes formed by Sox2(HMG) alone. Also, the portion of larger complexes found in the presence of both Sox2(HMG) and Pax6(DBD) was higher when compared to complexes formed by Sox2(HMG) alone.

frame incubated with Pax6(DBD) and Sox2(HMG) suggests that Pax6(DBD) can bind DNA that is associated with Sox2(HMG). Similar with Sox2(HMG) alone, Pax6(DBD)/ Sox2(HMG) complex also showed the binding selectivity to the 74-mer DNA. In a quantitative aspect, 3.6% of 64-mer dsDNA was occupied by the Sox2(HMG)/Pax6(DBD) complex, while 9.8% of 74-mer dsDNA harbored the complex (Table 1). The complex bound in the DNA frame in the presence of both Sox2(HMG) and Pax6(DBD) is distinguished from the complex incubated with Sox2(HMG) alone in terms of size and volume (Figures 2 and 3b, Table 1). The size of the complex observed when the frame was incubated with Sox(HMG), and Pax6(DBD) is larger than the complex seen when the frames were incubated with Sox2 alone (Figures 2 and 3b). To quantify this change, the volume of complex bound to DNA was calculated from lengths of complex in each of the three 2289

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Figure 5. Graphical representation of DNA only (a), Sox2(HMG) on DNA (b), and Sox2(HMG)/Pax6(DBD) complexes on DNA (c). Red and orange lines represent 64-mer and 74-mer DNA, respectively. Sox2(HMG) and Pax6(DBD) are shown as blue and green balls, respectively. The shape of the 64-mer and 72-mer dsDNA in each frame and position of proteins in each DNA were drawn based on the AFM images. Distributions of position occupied by proteins bound to 64-mer and 74-mer dsDNA are represented as histograms in respective cases.

and this binding further reduced the flexibility of the DNA (Figure 5c). The greater abundance of this complex on the 74mer dsDNA compared to the 64-mer DNA element further demonstrates that a stable Sox2(HMG)−DNA complex is a prerequisite for Pax6(DBD) recruitment to DNA as Pax6(DBD) alone could not bind to any DNA frames. Cooperativity is a characteristic feature exhibited by Sox2 and its cofactors when they form functional ternary complexes. Pax6 cooperates with Sox2 to associate with DNA, as shown through classical EMSA assays9 and as presently confirmed through AFM analysis. Cooperative binding of Pax6(DBD) is observed in the presence of Sox2(HMG) (Figures 4 and 5b,c). The binding frequency of Pax6(DBD) to the 74-mer dsDNA coincubated with Sox2(HMG) was 9.8% (Table 1), which is much higher than that of Sox2 alone (4.7%, Table 1). These results suggest that, in addition to Sox2, Pax6 enhances the binding affinity of the binding partner to DNA. The increased DNA binding frequency of Sox2(HMG)/Pax6(DBD) to DNA compared to that of Sox2(HMG) or Pax6(DBD) alone is evidence for cooperative binding. In contrast, the overall occupancy of the Sox2(HMG)/Pax6(DBD) complex on the 64-mer DNA element increased marginally to 3.6% (Figure 3b, Table 1) compared to 2.5% in the case of Sox2 alone (Figure 2, Table 1). As it is difficult for Sox2 to sufficiently bend the DNA in the already tensed 64-mer DNA element, which is necessary to create space for Pax6 binding, most protein bound to the 64-mer dsDNA in Figure 3b is not in the Sox2(HMG)/ Pax6(DBD) complex but rather is Sox2(HMG) alone. Thus, this finding implies that cooperativity also depends on prior binding and bending of DNA by Sox2, which subsequently makes DNA accessible for Pax6 and leads to further enhancement of Sox2/Pax6 complex loading on the DNA.

dimensions using the equation shown in Figure 4a. We systematically counted the number of frames occupied by entities of different volumes and presented the data in a histogram (Figure 4b). The number of frames harboring the complexes in the 74-mer DNA were 46 out of 981 frames in the presence of Sox2 (HMG) only which is about 4.7%, but it was increased to 9.8% (22 out of 225 frames) when both Sox2(HMG) and Pax6(DBD) were incubated with DNA (Table 1). We also noticed that Sox2(HMG)-occupied frames harbored complexes with a volume in the range of 40−120 nm3, whereas the volumes of complexes incubated with Sox2(HMG) and Pax6(DBD) were within the range of 80−160 nm3. The major size of the complexes in the frames incubated with Sox2 alone was 81 ± 28 nm3, while that of the Sox2/Pax6 complex was 130 ± 44 nm3, suggesting that many proteins in the DNA origami frames incubated with Pax6(DBD) and Sox2(HMG) are heterodimers, although a certain portion of them correspond to Sox2(HMG) alone (Figure 4b). Our results are summarized in Figure 5 with a graphical representation of Sox2 and Pax6 loading on both 64-mer and 74-mer DC5 elements. Figure 5a shows the frame containing DNA elements before protein loading. The 74-mer dsDNA element showed high flexibility, whereas the restrained 64-mer dsDNA element showed restricted flexibility when compared to the 74-mer element. When Sox2(HMG) was incubated with frames, it showed selective binding to the 74-mer dsDNA, which reduced the overall flexibility possibly due to the collaborative effect of DNA bending and protein loading (Figure 5b). The 74-mer dsDNA element provided room for proper bending, thus facilitating the formation of stable Sox2(HMG) complex with DNA. The Pax6(DBD)−Sox2(HMG) complex also preferentially bound the 74-mer dsDNA, 2290

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To further generalize our observation, we also examined the binding of these proteins to the 69-mer DNA in the DNA frame. The binding of Sox2(HMG) (3.7%) and Sox(HMG)/ Pax6(DBD) (4.6%) showed an intermediate level compared to those of 64-mer and 74-mer DNA (Table 1). Similar to the tensed 64-mer DNA, the ability of bending capability of the 69-mer DNA did not appear to be sufficient for the effective binding of these proteins. Our observations not only corroborated previously known facts but also verified some of them at the single molecule level. The most important revelation obtained through this single molecule level study is the importance of DNA bending for the formation of stable complexes. We showed that a compromise in bending ability seriously affected the stable complex formation by Sox2. This seems to also affect the loading of Pax6 onto the DNA element. While bulk studies through different biophysical techniques have predicted these possibilities, to our knowledge, our study is the first demonstration at the single-molecule level. By applying AFM along with the efficient use of DNA origami, we were able to address questions that would otherwise be difficult to answer through a simpler direct approach. This study is also important in the field of single molecule studies because the inclusion of DNA frames allows us to solve the many challenging problems that occur in most AFM studies. The origami frames actually restrict the region in which to look for a possible complex formation, providing high-level enhancement of the study clarity. Another advantage of using these DNA frames is the control of the tensile strengths of DNA, which play a role in various biological processes. A similar use of tensile strength to guide enzymatic reactions has been previously reported.36 Thus, this study can be considered another stepping stone toward the application of AFM to study and answer intriguing questions about various important and complex life processes, including transcriptional regulation.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental procedures, additional AFM images, and DNA sequences. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (K.K.K.). *E-mail: [email protected] (M.E.). *E-mail: [email protected] (H.S.). Author Contributions

S.Y. and D.D. contributed equally to this work. Author Contributions

S.Y. and K.H. carried out the AFM experiments. D.D. prepared the protein samples and wrote the paper. D.D., K.K.K., M.E., and H.S. analyzed the data and wrote the paper. All authors contributed to the interpretation and commented on the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Samsung Science & Technology Foundation (SSTF-BA1301-01) to K.K.K. and the CREST grant from Japan Science and Technology Corporation (JST) and JSPS KAKENHI (Grant Nos. 24310097, 24104002, 25253004) to M.E. and H.S. 2291

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