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1.
Figure 7.

Figure 7. From: Chromatin-associated HMG-17 is a major regulator of homeodomain transcription factor activity modulated by Wnt/?-catenin signaling.

Model for the novel regulation of homeodomain transcriptional activity by HMG-17 modulated by β-catenin. Without Wnt signaling, β-catenin is degraded in the cytoplasm, HMG-17 binds to PITX2 with a high affinity and prevents PITX2 from binding DNA. PITX2 target genes are repressed due to the inability of PITX2 to bind and activate the target gene promoters. In the presence of Wnt signaling, β-catenin is translocated to the nucleus and interacts with the HMG-17/PITX2 complex changing it from a repressor complex to an activator complex. Subsequently when Wnt signaling is turned off and nuclear β-catenin is limited or turned over then HMG-17 forms an inactive complex with PITX2. This mechanism would allow for the tight coordinated control of homeodomain transcriptional activity during development.

Melanie Amen, et al. Nucleic Acids Res. 2008 February;36(2):462-476.
2.
Figure 4.

Figure 4. From: Chromatin-associated HMG-17 is a major regulator of homeodomain transcription factor activity modulated by Wnt/?-catenin signaling.

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 detected with the PITX2 antibody (25) and the Alexa Fluor 488 goat anti-rabbit IgG(H+L) (Molecular Probes, Invitrogen detection technologies). (C) The corresponding stained nuclei from panel A and B were merged to yield the image in panel C, demonstrating co-localization of HMG-17 and PITX2. (D) DAPI staining of the nuclei in panels A–C, demonstrating intact nuclei. (E) Merging of panels A, B and C showing the DAPI stain overlaps with HMG-17 and PITX2 staining. (F) Negative control or secondary only antibody for Alexa Fluor 555 goat anti-rabbit IgG and (G) is the negative control for secondary only antibody using the Alexa Fluor 488 goat anti-rabbit IgG. (H) Confocal image (×40) of endogenous Pitx2 expression in LS-8 nuclei and (I) endogenous HMG-17 expression. (J) Merged images of Pitx2 and HMG-17 expression.

Melanie Amen, et al. Nucleic Acids Res. 2008 February;36(2):462-476.
3.
Figure 3.

Figure 3. From: Chromatin-associated HMG-17 is a major regulator of homeodomain transcription factor activity modulated by Wnt/?-catenin signaling.

Reduced endogenous HMG-17 protein increased PITX2 transcriptional activation. (A) CHO cells were transfected as in Figure 2 with the Dlx2 3.2 kb luciferase reporter (5 μg). The cells were co-transfected with expression vectors, shRNA expression vectors or shRNA vectors alone (–) (2.5 μg). The activities are shown as mean fold activation compared to the Dlx2 promoter plasmids without PITX2, siRNA or HMG-17 expression and normalized to β-galactosidase activity (±SEM from five independent experiments). (B) Western blots of endogenous and transfected HMG-17 in CHO cells. Lane 1 is purified HMG-17 protein, lane 2 is endogenous HMG-17, lane 3 is endogenous HMG-17 after PITX2C transfection, lane 4 is both endogenous and transfected HMG-17. The transfected HMG-17 migrates slightly slower in the gel due to a myc/his tag on the C-terminal tail of the protein. Lane 5 is endogenous and transfected HMG-17 with transfected PITX2C, lane 6 demonstrates a complete inhibition of endogenous HMG-17 expression by transfected HMG-17 siRNA. Lane 7 is endogenous HMG-17 with the transfected siNegative control and PITX2C. (C) The western blot in panel B was stripped and re-probed with GAPDH antibody to demonstrate equal loading of the cell lysates. (D) Western blot of cell lysates probed with the GAPDH antibody. (E) Western blot of transfected PITX2C with and without HMG-17 siRNA. The transfected PITX2C protein migrates slightly slower in the gel due to a myc/his tag on the C-terminal tail of the protein.

Melanie Amen, et al. Nucleic Acids Res. 2008 February;36(2):462-476.
4.

Figure 2. From: Chromatin-associated HMG-17 is a major regulator of homeodomain transcription factor activity modulated by Wnt/?-catenin signaling.

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 CMV plasmid without PITX2, HMG-17 or β-catenin (–) (2.5 μg). To control for transfection efficiency, all transfections included the SV-40 β-galactosidase reporter (0.5 μg). Cells were incubated for 24 h, and then assayed for luciferase and β-galactosidase activities. The activities are shown as mean fold activation compared to the Dlx2 promoter plasmids without PITX2, β-catenin or HMG-17 expression and normalized to β-galactosidase activity (±SEM from five independent experiments). (B) HMG-17 is endogenously expressed in LS-8, C3H10T1/2 and CHO cells. (C) Western blot of transfected cell lysates (20 μg); PITX2A was detected using the PITX2 Ab (25) as previously described; β-catenin was detected using the antibody from Santa Cruz Biotech; and HMG-17 was detected using an antibody from Chemicon International. The proteins were visualized using ECL reagents from Amersham. Molecular weight markers are shown on the left of each blot. (D) Endogenous β-catenin de-repressed the PITX2/HMG-17 complex. CHO cells were transfected as described above and transfected cells were treated without (–) and with (+) LiCl at a final concentration of 10 mM. The activities are shown as mean fold activation compared to the Dlx2 promoter plasmid without PITX2 or HMG-17 expression and normalized to β-galactosidase activity (±SEM from four independent experiments). (E) LS-8 oral epithelial cells were transfected as in panel D to determine if the activation was cell dependent. The activities are shown as mean fold activation compared to the Dlx2 promoter plasmids without PITX2 or HMG-17 expression and normalized to β-galactosidase activity (±SEM from eight independent experiments).

Melanie Amen, et al. Nucleic Acids Res. 2008 February;36(2):462-476.
5.
Figure 5.

Figure 5. From: Chromatin-associated HMG-17 is a major regulator of homeodomain transcription factor activity modulated by Wnt/?-catenin signaling.

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 buffer. (A) PITX2 binding to F-bicoid DNA. (B) HMG-17 binding to F-bicoid DNA. (C) HMG-17 binding to DNA bound PITX2. (D) SPR-binding assay. HMG-17 at indicated concentrations was injected over immobilized PITX2 HD (HD, homeodomain) (GST-tagged) at a flow rate of 30 μl/min. After 8 min of injection (indicated by the arrow), the HMG17/PITX2 HD complex was washed with running buffer for 4 min. Shown here are overlay of baseline-adjusted sensorgrams (red jagged lines) and fitted curves (black smooth lines) using 1:1 Langmuir-binding model. Kinetic rate constants kon = (4.39 ± 0.03) × 105 M−1s−1 and koff = (1.33 ± 0.09) × 10−6 s−1 were derived from the fitting, and the dissociation constant KD of 3.04 pM (KD = kon/koff) was obtained. (E) Dissociation of HMG17–PITX2 HD complex by salt. HMG17 (128 nM) was injected over the PITX2 HD surface (i) for 8 min (ii), and followed by 4 min wash with running buffer (iii). After regeneration with two consecutive 30 s pulses of buffers containing 200 mM (iv) and 500 mM of NaCl (v), bound HMG17 was removed from the HMG17/PITX2 HD complex. RU, resonance units. Data are one representative of two experiments.

Melanie Amen, et al. Nucleic Acids Res. 2008 February;36(2):462-476.
6.
Figure 1.

Figure 1. From: Chromatin-associated HMG-17 is a major regulator of homeodomain transcription factor activity modulated by Wnt/?-catenin signaling.

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 HMG-17 protein; NLS, nuclear localization signal; NBD, nucleosomal-binding domain; CHUD, chromatin-unfolding domain (4). The purified HMG-17 protein used in the various assays was visualized by Coomassie blue staining. (C) GST–PITX2A protein pull-down assay with bacterial expressed and purified HMG-17 protein (100 ng). To demonstrate HMG-17 binding to PITX2A, PITX2A protein was incubated with purified HMG-17 protein. HMG-17 binds to the PITX2 homeodomain (HD) and this region is termed HMG-17 BD #1. HMG-17 binds to a region containing the C-terminal OAR domain (conserved 14 residue motif), which is termed HMG-17 BD #2. As a control GST-beads were incubated with purified HMG-17 to demonstrate the specificity of HMG-17 binding to the GST–PITX2A fusion proteins. (D) HMG-17 protein (20, 40 and 60 ng) was incubated with the Dlx2 promoter sequence containing a PITX2-binding element (TAATCC) as the radioactive probe in an electrophoretic mobility shift assay (EMSA). HMG-17 did not bind in this assay to DNA however, PITX2A and PITX2C proteins (80 ng) bound to the DNA. HMG-17 titration (20, 40 and 60 ng) with 80 ng of either PITX2A or PITX2C revealed that HMG-17 inhibited PITX2 binding in a dose-responsive manner. The EMSA experiments were analyzed in 8% native polyacrylamide gels. The free and bound forms of DNA were quantitated using the Molecular Dynamics STORM PhosphoImager. The free probe and bound DNA are indicated.

Melanie Amen, et al. Nucleic Acids Res. 2008 February;36(2):462-476.
7.
Figure 6.

Figure 6. From: Chromatin-associated HMG-17 is a major regulator of homeodomain transcription factor activity modulated by Wnt/?-catenin signaling.

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. Cell lysates were incubated with HMG-17 antibody and the IP complex was isolated and resolved on a 10% SDS–polyacrylamide gel and probed for PITX2 using the PITX2 Ab. The HMG-17 antibody was unable to immunoprecipitate the PITX2/HMG-17 complex. Co-expression of β-catenin with PITX2 and HMG-17 allowed the HMG-17 antibody to recognize the PITX2/HMG-17/β-catenin complex and precipitated the complex. HMG-17 Ab does not bind to β-catenin. The proteins were visualized using ECL reagents from Amersham. (B) PITX2A, HMG-17 and β-catenin complex formation by purified proteins. GST–PITX2A and truncated PITX2A protein pull-down assay with bacterial expressed and purified HMG-17 (75 ng) and β-catenin proteins (400 ng). Binding reactions contain both HMG-17 and β-catenin and after incubation the reactions were divided into two equal aliquots and resolved on separate 10% SDS–polyacrylamide gels and transferred to PVDF membranes. One aliquot was probed for HMG-17 interactions using the HMG-17 antibody. HMG-17 interacted with the PITX2 HD and C-terminal tail in the presence of β-catenin. (C) The duplicate aliquot was probed for β-catenin interactions using the β-catenin antibody. β-catenin interacted with the PITX2 HD but not the C-terminal tail. These results demonstrate that both HMG-17 and β-catenin bind to the PITX2 HD as a complex. As a control GST-beads were incubated with purified HMG-17 and β-catenin to demonstrate the specificity of binding to the GST–PITX2A fusion proteins. (D) GST-β-catenin was immobilized on beads and incubated with HMG-17 to demonstrate that these two proteins do not physically interact. Addition of PITX2 ΔC173 protein to GST-β-catenin and HMG-17 demonstrated that HMG-17 interacts with PITX2 bound to β-catenin. Furthermore, PITX2 ΔC173 interacts with β-catenin and addition of HMG-17 increases the PITX2 ΔC173 interaction with β-catenin. The proteins were visualized using ECL reagents from Amersham. Molecular weight markers are noted. (E) Triple sequential ChIP using β-catenin Ab first, followed by HMG-17 Ab and PITX2 Ab immunoprecipitations. The Dlx2 primers produced the correct PCR product from the triple antibody IP (lane 2), Dlx2 primer only control (lane 3), Dlx2 primers with chromatin input (lane 4), unrelated control primers with the triple IP chromatin (lane 5) and control primers with input control (lane 6). All bands were sequenced to confirm their identity.

Melanie Amen, et al. Nucleic Acids Res. 2008 February;36(2):462-476.

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