HMG-17 interacts with the PITX2 HD
HMG-17 was identified as a PITX2 interacting partner using the yeast two-hybrid assay. Over 500 000 clones were screened using an oral epithelial cell (LS-8 cells) library as prey and the PITX2A full-length, HD and C-terminal tail were used as the bait. A stringent screening process identified 40 clones that grew on histidine/tryptophan/leucine minus plates. After sequencing 17 were confirmed to be HMG-17. HMG-17 interacted with the PITX2A full-length protein, PITX2 HD and the PITX2 C-terminal tail. Transformants containing the HMG-17 clone were re-streaked on an SD agar plate without leucine, trptophan and histidine. LacZ expression of the cotransformants was determined by the filter lift assay (yeast two-hybrid data not shown).
To map the PITX2 interaction region, GST-pull-down experiments were performed using purified bacteria expressed proteins. A schematic of the immobilized GST-PITX2A wild type and truncated proteins are shown in A. The structure of the HMG-17 protein and Coomassie blue stained gel of the purified HMG-17 protein used in the assays are shown in B. Immobilized PITX2 HD (GST-PITX2 HD) bound the HMG-17 protein (C). GST-PITX2 ΔC173, which has the complete C-terminal tail deleted bound HMG-17 (C). HMG-17 bound to the PITX2°C-terminal tail (GST-PITX2 C173) and specifically to the last C-terminal 39 residues (GST-PITX2 C39) (C). As a control, GST beads or the PITX2 N terminus did not bind the HMG-17 protein (C, data not shown). We have designated these two binding sites in the PITX2 protein as HMG-17-binding domain (BD) #1 and #2 (A).
Figure 1. HMG-17 interacts with two regions of the PITX2 protein. (A) Schematic of the PITX2A protein and the N-terminal and C-terminal truncated proteins. The location of the two HMG-17-binding domains (BD) in the PITX2 protein are shown. (B) Schematic of the (more ...)
Because HMG-17 interacts with the PITX2 HD, which confers DNA-binding activity, we asked if HMG-17 affected the DNA-binding properties of PITX2. PITX2 specifically binds to the Dlx2
promoter and this element (TAATCC) was used as the probe in the EMSA. HMG-17 is a small peptide (89 residues) and does not cause a large shift in the probe band and only a small amount of HMG-17/DNA complex is observed immediately above the free probe (D). HMG-17–DNA binding is transient and may not be stable in these experiments (4
). However, HMG-17 inhibited PITX2A and PITX2C isoform (80 ng) DNA-binding activity (D). PITX2A and PITX2C are the major isoforms involved in embryogenesis and have identical HDs and C-terminal tails (19
). HMG-17 was titrated at 20, 40 and 60 ng to 80 ng of PITX2 isoforms and the higher HMG-17 concentration inhibited over 90% of PITX2A and PITX2C binding to the Dlx2
promoter probe (D).
β-catenin de-represses HMG-17 inhibition of PITX2 transcriptional activity
CHO cells were co-transfected with PITX2A, HMG-17 and the full-length Dlx2 3276 luc promoter to determine if HMG-17 regulated the transcriptional activity of PITX2A. CHO cells do not endogenously express Pitx2 and only low levels of β-catenin (22
). Therefore, CHO cells were used in the assay without interference from endogenous Pitx2 activity. The pluripotent C3H10T1/2 cells, CHO cells and LS-8 cells endogenously express HMG-17 demonstrating the ubiquitous expression pattern of this factor (B) (4
). PITX2A activated the Dlx2
promoter by ~30-fold (A). Co-expression of PITX2A and HMG-17 revealed a 3-fold decrease in PITX2A activation from 30-fold to 10-fold (A). We have previously shown that β-catenin interacts with PITX2 to synergistically activate gene expression (22
). Furthermore, β-catenin directly interacts with the PITX2 HD to regulate PITX2 transcriptional activation (33
). We asked if β-catenin S37A (constitutively active form) would regulate the HMG-17 repression of PITX2. Co-transfection of PITX2A and β-catenin S37A resulted in a 43-fold synergistic activation of the Dlx2
promoter (A). Co-transfection of PITX2A, HMG-17 and β-catenin S37A activated the Dlx2
promoter by 65-fold compared to 10-fold activation for PITX2 and HMG-17 co-transfection (A). The activity of the PITX2/HMG-17 complex is switched from a repressor to an activator in the presence of β-catenin (65-fold, A). The action of β-catenin was not cell specific as β-catenin de-repressed the HMG-17 inhibition of PITX2 in transfected C3H10T1/2 and LS-8 cells (data not shown). A western blot demonstrates equal expression of transfected PITX2, β-catenin and HMG-17 (C).
Figure 2. The repression of PITX2 activity by HMG-17 is modulated by β-catenin. (A) CHO cells were transfected with the Dlx2 3.2 kb luciferase reporter gene (5 μg) and co-transfected with CMV-PITX2, CMV-β-catenin S37A, CMV-HMG-17, or the (more ...)
Consistent with increased PITX2A transcriptional activation by transfected β-catenin, cells treated with LiCl increased PITX2C activation of the Dlx2
promoter (D). HMG-17 repression is not PITX2 isoform dependent as would be expected since both isoforms contain identical HDs and C-terminal tails (19
). LiCl is a potent GSK-3 inhibitor and inhibits β-catenin phosphorylation and stabilizes the pool of cellular β-catenin similar to Wnt signaling (34
). HMG-17 did not activate the Dlx2
promoter after LiCl treatment of CHO cells however, PITX2C activation of the Dlx2
promoter in the presence of HMG-17, increased from 10- to 37-fold after treatment with LiCl (D). The increase in PITX2C activation with LiCl treatment and HMG-17 was less than the activation by transfected β-catenin S37A with PITX2A and HMG-17 observed in A. This is presumably due to the increased pool of β-catenin S37A from the transfected plasmid compared to the endogenous β-catenin pool stabilized by LiCl treatment. Furthermore, LiCl treatment of PITX2C and HMG-17 transfected cells did not activate the Dlx2
promoter at the levels of PITX2C expression alone with LiCl due to the limited endogenous β-catenin pool and exogenous HMG-17 (D). However, both methods of increasing β-catenin activity reveal a role for β-catenin in de-repressing HMG-17 inhibition of PITX2. Similar results were observed in LS-8 transfected cells (E).
Knockdown of endogenous HMG-17 increased PITX2 transcriptional activity
We next asked if endogenous HMG-17 was repressing PITX2 activation. Inhibition of endogenous HMG-17 expression by HMG-17 siRNA (siHMG-17) increased PITX2C activation of the Dlx2 promoter from 33- to 45-fold (A). A negative control (siNEG supplied by Ambion), and siGADPH did not affect the activity of PITX2C in CHO cells (A).
Figure 3. Reduced endogenous HMG-17 protein increased PITX2 transcriptional activation. (A) CHO cells were transfected as in with the Dlx2 3.2 kb luciferase reporter (5 μg). The cells were co-transfected with expression vectors, shRNA expression (more ...)
Western blot analyses were performed to demonstrate reduced endogenous HMG-17 protein by HMG-17 siRNA. Endogenous HMG-17 protein is detected in mock and PITX2 transfected lysates (B, lanes 2 and 3). Transfected HMG-17 migrates slightly slower than endogenous HMG-17 in the gel due to a myc/his tag on the transfected protein (B, lanes 4 and 5). Interestingly, PITX2 stabilized both endogenous and transfected HMG-17 (B, compare lanes 4 and 5). Endogenous HMG-17 was completely inhibited by siHMG-17 expression (B, lane 6). The siNEG control had no effect on endogenous HMG-17 expression (B, lane 7). As a loading control the western blot was stripped and re-probed with the GAPDH antibody. Similar levels of GAPDH were observed in all lanes demonstrating that equal protein amounts were assayed (C). As another control a western blot demonstrates that PITX2 and HMG-17 expression did not affect GAPDH expression. More importantly siNEG and siHMG-17 did not affect GAPDH expression demonstrating the specificity of these siRNAs (D). siGADPH inhibited endogenous GAPDH expression ~50% after only 24 h (D). It should be noted that all siRNA experiments were assayed after 24 h, and the complete loss of HMG-17 protein after 24 h suggests that it is a labile protein. A western blot of transfected PITX2C demonstrates PITX2C expression (E).
PITX2 and HMG-17 co-localize in the cell nucleus
HMG-17 protein was visualized using FITC in the CHO cell nucleus associated with chromatin structures and in foci (A), (35
). There are two adjacent nuclei on the left side of the panels. PITX2 was visualized with Texas red in distinct regions of the nucleus and associated with chromatin structures (B). Merging the two fluorescent proteins reveals a strong overlap in their nuclear localization (C). DAPI staining reveals the nuclear/chromatin structure (D), and merging of the DAPI stain with HMG-17 and PITX2 expression correlates with these factors recruitment to nuclear chromatin structures (E).
Figure 4. PITX2 and HMG-17 co-localize in the nucleus. (A) HMG-17 expression was detected using an HMG-17 antibody and visualized with the Alexa Fluor 555 goat anti-rabbit IgG. There are two adjacent nuclei on the left side of the panels. (B) PITX2 expression was (more ...)
Endogenous Pitx2 and HMG-17 expression was observed in LS-8 nuclei (H and I). Endogenous Pitx2 expression appears diffusely throughout the nucleus (H), whereas HMG-17 expression is more localized to small foci (I). Merging the two images reveals that Pitx2 is co-localized to nuclear regions where HMG-17 resides (J). While all HMG-17 appears to co-localize with Pitx2 not all Pitx2 staining co-localizes with HMG-17.
Furthermore, HMG-17 expression occurs in the dental and oral epithelial cells of an E14.5 molar tooth bud (data not shown). The tooth epithelial specific expression of HMG-17 directly overlaps that of PITX2 (37
Specificity of DNA binding and the PITX2/HMG-17 interaction
The specificity of PITX2 and HMG-17 for DNA were determined by binding to 5′- fluoresceinated oligonucleotides corresponding to the PITX2-binding site (bicoid element). Fluorescence polarization experiments determined the binding curves for PITX2 and HMG-17 (A and B). The calculated equilibrium dissociation constant, Kd is 129 ± 17 nM for PITX2 binding to the bicoid element (A). The calculated equilibrium dissociation constant, Kd is 1.3 ± 0.40 μM for HMG-17 binding to the bicoid element (B). Thus, HMG-17 has a low affinity for DNA and correlates with HMG-17 binding to DNA non-specifically.
Figure 5. Binding isotherms and surface plasmon resonance (SPR) analysis of HMG-17/PITX2 interaction. Millipolarization (mP) is plotted against the concentration of PITX2 and HMG-17 or both binding to fluoresceinated oligodeoxynucleotides (F-bicoid DNA) in HEPES-binding (more ...)
HMG-17 binding to PITX2 was determined after PITX2 DNA binding reached equilibrium. A specific PITX2/HMG-17 complex was identified by titrating HMG-17 protein to DNA bound PITX2. Surprisingly, HMG-17 addition to the PITX2/DNA complex resulted in decreased polarization, demonstrating that HMG-17 was removing PITX2 from the DNA (C). These data correlate with the EMSA data demonstrating that the PITX2/HMG-17 complex cannot bind DNA. The inhibitory constant (binding constant) of HMG-17 for PITX2 is, Ki 105 ± 5 nM (C). Thus, HMG-17 has 10-fold higher affinity for PITX2 than for DNA and HMG-17 has a higher binding affinity for PITX2 than PITX2 has for DNA. HMG-17 physically interacts with the PITX2 HD and this interaction releases PITX2 from the DNA.
The interaction between HMG-17 and PITX2 HD (HD only) was examined by real-time SPR-binding assay. The sensorgrams fit well to a 1:1 binding model (Chi2 = 0.61) (D). The dissociation constant (KD = 3.04 pM) obtained from the analysis indicated the HMG-17–PITX2 HD interaction to be of high affinity. These data indicate a stronger affinity of HMG-17 for PITX2 than the polarization experiments due to binding of the PITX2 HD peptide compared to the full-length protein. Moreover, the kinetic parameters suggested that this strong interaction was due to a fast complex formation with association rate kon of (4.39 ± 0.03) × 105 M−1s−1, and a very stable complex with dissociation rate koff of (1.33 ± 0.09) × 10−6 s−1. However, the stability of the HMG-17/PITX2 HD complex could be easily destroyed by slight increase of salt concentration. This is based on the observation that using the regeneration buffer, which contained 200 mM or 500 mM of NaCl, removed bound HMG-17 from the HMG-17/PITX2 HD complex (E). This indicated that HMG-17 and PITX2 HD bind to each other through ionic interactions and the interaction can be regulated by salt concentration. Therefore, strong interacting proteins can modify PITX2 and HMG-17 ionic interactions. The HMG-17/PITX2 interaction is measured differently in the two assays, however both measurements demonstrate a high affinity association between the two proteins.
HMG-17 antibody (Ab) immunoprecipitates PITX2 only in the presence of β-catenin. The previous experiments demonstrated specific PITX2 and HMG-17 protein interactions. Interestingly, the HMG-17 Ab weakly co-immunoprecipitated a PITX2/HMG-17 complex (A). A band was detected on longer exposure times indicating that an interaction occurred between PITX2 and HMG-17. This weak interaction could correspond to the HMG-17 interaction with the PITX2 C-terminal tail as revealed in the GST-pull-down experiments in or due to low levels of endogenous β-catenin in the cells. These results are often observed when the Ab cannot recognize an epitope buried in a protein complex. However, when β-catenin was co-expressed with PITX2 and HMG-17 the HMG-17 Ab immunoprecipitated PITX2 suggesting that the complex formed between these three proteins caused a conformational change allowing the Ab to recognize the HMG-17 protein (A). As a control the HMG-17 Ab does not recognize β-catenin in a western blot (data not shown). Because β-catenin is a high-molecular weight protein (~89 KDa), its interaction with PITX2 may displace or reposition HMG-17 and allow the HMG-17 Ab to recognize it in the complex.
Figure 6. PITX2, HMG-17 and β-catenin form a complex. (A) Co-immunoprecipitation (IP) experiments demonstrate a PITX2/HMG-17/β-catenin complex in CHO cells. PITX2A, HMG-17 and/or β-catenin (2.5 μg) were transfected into CHO cells. (more ...)
GST-pull-down assays provide an alternative method to identify a PITX2/HMG-17/β-catenin complex. Immobilized GST–PITX2A, GST–PITX2 HD and GST–PITX2 C173 were incubated with both HMG-17 (75 ng) and β-catenin (400 ng) in one reaction (protein concentrations were adjusted to equal similar amounts of protein molecules) and after incubation and extensive washing the reaction was divided into two aliquots, each aliquot resolved separately on a polyacrylamide gel. One western blot was probed using the HMG-17 antibody (B). As controls, GST–PITX2A alone and GST–PITX2A incubated with only β-catenin did not produce an HMG-17 protein band (B). However, HMG-17 bound to PITX2A in the presence of β-catenin (B). HMG-17 bound to PITX2 HD and PITX2A C173 demonstrating that HMG-17 binds to both regions of PITX2 in the presence of β-catenin. The second western blot revealed β-catenin binding to PITX2A and the PITX2A HD in the presence of HMG-17 (C). However, β-catenin did not bind to the PITX2 C-terminal tail and is specific only for the HD. Furthermore, HMG-17 and β-catenin do not interact as β-catenin would be pulled down by HMG-17 binding to the PITX2 C-terminal tail. GST–pull downs and IP assays did not detect a β-catenin/HMG-17 complex (data not shown). While it is possible HMG-17 and β-catenin bind to PITX2 separately in these assays, excess amounts of HMG-17 and β-catenin were added to each reaction with a limited amount of immobilized PITX2. If the proteins were binding separately we would not expect the high levels of binding by each protein to PITX2 when both are present.
GST–β-catenin was immobilized on beads and did not interact with purified HMG-17 protein (D). However, when PITX2 protein was added to the binding reaction, HMG-17 bound to PITX2 ΔC173 (does not contain the C-terminal tail), which bound to β-catenin. As expected PITX2 ΔC173 bound to β-catenin and interestingly addition of HMG-17 to the binding reaction increased PITX2 ΔC173 binding to β-catenin (D). Thus, HMG-17 appears to facilitate the interaction between PITX2 and β-catenin. These data correlate with the IP data to demonstrate a PITX2/HMG-17/β-catenin complex forms by binding to the PITX2 HD.
A triple sequential ChIP assay demonstrates that the Pitx2/HMG-17/β-catenin ternary complex resides on the Dlx2 promoter chromatin. LS-8 cells were used in the ChIP assay as these cells endogenously express all three factors. The first ChIP assay used the β-catenin antibody, followed by the HMG-17 antibody and then the PITX2 antibody. After the last IP with the PITX2 antibody, the Dlx2 promoter chromatin was amplified by PCR using primers specific for the Dlx2 promoter flanking a PITX2-binding site. The primers amplified a 390 bp product from the triple antibody IP (E, lane 2). As a control the Dlx2 primers only did not produce a PCR product, however the primers did produce the correct size band from the input chromatin (E, lane 4). Control primers to an unrelated gene did not produce a product from the triple IP chromatin (E, lane 5). The control primers did work with the input chromatin (E, lane 6). Thus, the endogenous complex of Pitx2/HMG-17/β-catenin binds to the Dlx2 promoter in vivo. These data correlate with the IP data to demonstrate a PITX2/HMG-17/β-catenin complex forms by binding to the PITX2 HD.