Embryonic development is tightly controlled. The clustered genes of the Hox family of homeobox proteins play an important part in regulating this development and also proliferation. They specify embryonic structures along the body axis, and are associated with normal and malignant cell growth. The cell cycle regulator, Geminin, controls replication by binding to the licensing factor Cdt1, and is involved in neural differentiation. Recently, the interaction between Geminin and Hox proteins was found to prevent Hox proteins from binding to DNA, inhibit Hox-dependent transcriptional activation of reporter and endogenous downstream target genes, and displace Cdt1 from its complex with Geminin. By establishing competitive regulation, Geminin functions as a coordinator of developmental and p...[ Read more ]

Embryonic development is tightly controlled. The clustered genes of the Hox family of homeobox proteins play an important part in regulating this development and also proliferation. They specify embryonic structures along the body axis, and are associated with normal and malignant cell growth. The cell cycle regulator, Geminin, controls replication by binding to the licensing factor Cdt1, and is involved in neural differentiation. Recently, the interaction between Geminin and Hox proteins was found to prevent Hox proteins from binding to DNA, inhibit Hox-dependent transcriptional activation of reporter and endogenous downstream target genes, and displace Cdt1 from its complex with Geminin. By establishing competitive regulation, Geminin functions as a coordinator of developmental and proliferative control.

To understand molecular basis of interaction between Geminin and Hox proteins, structural information of this complex is essential. NMR titration experiment was used to demonstrate the interaction between the coiled-coil domain of Geminin and homeodomain of Hox protein and to map the interaction regions on Hox proteins. Based on the backbone assignment of Hox, it indicates that the N-terminal and C-terminal section of homeodomain is involved in strong binding. Almost all the assignment of backbone and sidechain atoms of both domains was completed. The structures of bound forms of both domains were calculated by Cyana with NOE distance constraints and backbone dihedral angle constraints obtained from TALOS. Intermolecular NOE constraints were used to dock these two bound forms together. In addition, the luciferase assay system was used to examine whether Geminin and the coiled-coil domain inhibit the transcriptional activation promoted by Hox in vivo.

In the second part, the Hox-Smad complex was studied. Hox proteins have been identified as downstream transcription factors of TGFβ/BMP signaling pathways. Smad family proteins mediate signaling initiated by BMPs and upon TGFβ/BMP stimulation the common Smad, Smad4, can interact directly with Hox proteins and suppresses their DNA-binding activity. Although the interaction between the Smad4 MH1 domain and Hox homeodomain was found to regulate the transcription activity of Hox proteins, the mechanism is not well characterized and direct contact residues remain to be elucidated.

In the present study, the interaction between the recombinant Hoxc9 homeodomain and Smad4 MH1 domain was investigated using GST pull-down assay, surface plasmon resonance (SPR) analysis as well as multidimensional nuclear magnetic resonance (NMR) techniques. Hoxc9 was precipitated with the GST-Smad4 MH1 fusion protein but not with GST alone, demonstrating a direct interaction between the two domains in vitro. Titration experiments showed that a strong and specific binding occurred between the two domains. NMR triple-resonance experiments and backbone assignments revealed that the N terminal arm of Hoxc9 was involved in the interaction with Smad4 MH1. In addition, SPR measurement verified a strong interaction, bearing the dissociation constant of 400 nM, between the two domains. These results are in good agreement and yield the first detailed insight into the interaction between the homeodomain of Hox proteins and the conserved MH1 domain of Smad family proteins.