January 5, 2018;
HMG-box factor SoxD/Sox15 and homeodomain-containing factor Xanf1/Hesx1 directly interact and regulate the expression of Xanf1/Hesx1 during early forebrain development in Xenopus laevis.
The homeodomain-containing transcription factor Anf (also known as Rpx/Hesx1 in mammals) plays an important role during the forebrain development in vertebrates. Here we demonstrate the ability of the Xenopus laevis Anf, Xanf1/Hesx1, to directly bind SRY-related HMG-box-containing transcription factor SoxD/Sox15 and to cooperate with the latter during regulating of the expression of Xanf1/Hesx1 own gene. As we have shown by GST pull-down, EMSA and the luciferase reporter assays, Xanf1/Hesx1 and SoxD/Sox15 bind the Xanf1/Hesx1 promoter region counteracting the inhibitory effect of Xanf1/Hesx1 alone. This finding explains how Xanf1/Hesx1 could escape the repressive activity of its own protein during early patterning of the forebrain rudiment.
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Fig. 1. SoxD/Sox15 binds to Xanf1/Hesx1.
(A) SoxD/Sox15 binds to Xanf1/Hesx1 in the yeast two-hybrid assay. Schematic of the Y2H experiment.
(B) co-immunoprecipitation of Flag-SoxD/Sox15 and Myc-Xanf1/Hesx1 proteins: Protein extracts from embryos injected with 100 pg of either Flag-SoxD/Sox15 mRNA (lanes 1 and 4) or both Flag-SoxD/Sox15 and Myc-Xanf1/Hesx1 mRNAs (lanes 2 and 3) were lysed at early neurula stage and immunoprecipitated with anti-Myc antibodies, followed by western blotting using anti-Flag antibodies. Translation levels of Flag-SoxD/Sox15 and mixture of Flag-SoxD/Sox15 and Myc-Xanf1/Hesx1 mRNAs were measured in lines 1 and 2, respectively. Line 3 indicates the presence of SoxD/Sox15-Xanf1/Hesx1 complex (arrowhead); line 4 serves as a negative control.
(C, Right) Principal scheme of SoxD/Sox15 – GST N-fusion fragments used for In vitro pull-down assay (proteins were produced in E. coli):
1. Full-size SoxD/Sox15 factor.
2. HMG (high mobility group) domain (1–96 a.a.) of SoxD/Sox15 factor.
3. The N-terminal part of the HMG domain (1–55 a.a.) of SoxD/Sox15.
4. The C-terminal domain of SoxD/Sox15 (93–196 aa).
5. Truncated C-terminal part of the SoxD/Sox15 C-domain (139–196 aa).
(С, left) Principal scheme of N-terminal Myc-Xanf1/Hesx1 fragments, obtained from lysates of Xenopus embryos injected with the corresponding mRNAs:
1. The full-size Myc-Xanf1/Hesx1.
2. The N-terminal part Myc-Xanf1/Hesx1 (1–85 аа).
3. The C-terminal part Myc-Xanf1/Hesx1 (72–187 аа).
(D) In vitro complex formation between Myc-Xanf1/Hesx1 and GST-hybrid fragments of the SoxD/Sox15 factor.
(E) Complex formation between Myc-N-terminal and Myc-C-terminal domains of Xanf1/Hesx1 and Gst-hybrid fragments of SoxD/Sox15.
Fig. 2. Confirmation of association factors Myc-Xanf1/Hesx1, Flag-SoxD/Sox15 and their complex with regulatory elements in the Xanf1/Hesx1 promoter using EMSA method.
(a) Electrophoretic Mobility Shift Assays demonstrating binding of SoxD/Sox15 but not Sox2 to Box189 element from the Xanf1/Hesx1 promoter. An extract from embryos microinjected with mRNA of the proteins being tested was incubated with Box189 and Box 203 end-labeled elements from the Xanf1/Hesx1 promoter and electrophoresed on a nondenaturing polyacrylamide gel. The reaction mixtures were the following: controls - extract from EGFP injected embryos with Box 203 (lane 1) and Box189 (lane 4) elements; extract from SoxD/Sox15 injected embryos with Box 203 (lane 2) and Box189 (lane 5) elements or Sox2 injected embryos with Box 203 (lane 3) and Box189 (lane 6).
(b) Confirmation of association of Myc-Xanf1/Hesx1-Flag-SoxD/Sox15 complex with Box189 and Box 203 regulatory elements in the Xanf1/Hesx1 promoter. Lines 1–4: Box 203 regulatory element with (1) EGFP (as control); (2) Flag-SoxD/Sox15; (3) Myc-Xanf1/Hesx1; (4) both Myc-Xanf1/Hesx1 and Flag-SoxD/Sox15 containing embryo lysates.
Lines 5–8: Box189 regulatory element with (5) EGFP (as control); (6) Flag-SoxD/Sox15; (7) Myc-Xanf1/Hesx1; (8) both Myc-Xanf1/Hesx1 and Flag-SoxD/Sox15 containing embryo lysates.
The appearance of slowly migrating bands are marked with arrows.
(c) Confirmation the formation of supershifts by addition of specific antibodies.
Lines 1–3: Box 203 regulatory element with (1) Myc-Xanf1/Hesx1 + Flag-SoxD/Sox15 containing embryo lysates; (2) reaction mixture with anti-Flag monoclonal antibodies and (3) reaction mixture with anti-Myc monoclonal antibodies.
Lines 4–6: Box 189 regulatory element with (4) Myc-Xanf1/Hesx1 + Flag-SoxD/Sox15 containing embryo lysates; (5) reaction mixture with anti-Flag monoclonal antibodies and (6) with anti-Myc monoclonal antibodies. Shift and supershift bands are indicated by arrows. S, X, and S + X specify SoxD/Sox15-DNA, Xanf1/Hesx1-DNA and SoxD/Sox15-Xanf1/Hesx1-DNA complexes, respectively.
Fig. 3. The effect of Xanf1/Hesx1 + SoxD/Sox15 complex formation on expression of the reporter gene (luciferase) under control of the regulatory elements of the Xanf1/Hesx1 promoter.