Gbx2 Directly Restricts Otx2 Expression to Forebrain and Midbrain, Competing with Class III POU Factors

Abstract

Otx2 plays essential roles in rostral brain development, and its counteraction with Gbx2 has been suggested to determine the midbrain-hindbrain boundary (MHB) in vertebrates. We previously identified the FM enhancer that is conserved among vertebrates and drives Otx2 transcription in forebrain/midbrain from the early somite stage. In this study, we found that the POU homeodomain of class III POU factors (Brn1, Brn2, Brn4, and Oct6) associates with noncanonical target sequence TAATTA in the FM enhancer. MicroRNA-mediated knockdown of Brn2 and Oct6 diminished the FM enhancer activity in anterior neural progenitor cells (NPCs) differentiated from P19 cells. The class III POU factors associate with the FM enhancer in forebrain and midbrain but not in hindbrain. We also demonstrated that the Gbx2 homeodomain recognizes the same target TAATTA in the FM enhancer, and Gbx2 associates with the FM enhancer in hindbrain. Gbx2 misexpression in the anterior NPCs repressed the FM enhancer activity and inhibited Brn2 association with the enhancer, whereas Gbx2 knockdown caused ectopic Brn2 association in the posterior NPCs. These results suggest that class III POU factors and Gbx2 share the same target site, TAATTA, in the FM enhancer and that their region-specific binding restricts Otx2 expression at the MHB.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00083-12.

ACKNOWLEDGMENTS

We are grateful to K. Yamasu and N. Takasaki for critical discussions and thank M. A. Frohman for providing us with pGbx2 plasmid. We thank K. Shinmyozu (Mass Spectrometry Analysis Unit, RIKEN CDB) for LC-MS/MS analysis and are also grateful to the Laboratory for Animal Resource and Genetic Engineering for transgenic mouse production and the housing of mice.

This work was supported by a Grant-in-Aid for Creative Scientific Research from the Japan Society for the Promotion of Science.