Caudal-related homeodomain proteins CDX1/2 bind to DNA replication-related element binding factor.

In the intestinal epithelium, the CDX1 and CDX2 homeodomain genes play proliferative and tumor suppressor roles, respectively. The transcription factor DNA replication-related element binding factor (DREF), is an 80kDa polypeptide homodimer that plays an important role in regulating cell proliferation-related genes. Homeodomain genes encode DNA-binding proteins that play crucial roles during development by defining the body plan and determining cell fate. However, until now, the regulation of DREF function by caudal-related homeodomain proteins is poorly understood. In this study, recombinant CDX1/2 homeodomains (CDX1, amino acids [aa] 152-216 and CDX2, aa 184-248) and the DNA-binding domain of Drosophila DREF (dDREF; aa 1-125) were isolated in order to investigate the regulatory mechanism of their interaction. The expression and purification of the truncated CDX1/2 and DREF proteins were successfully performed in Escherichia coli. Models of the CDX1/2 homeodomain and dDREF were constructed using SWISS-MODEL software, a program for relative protein structure modeling. The binding of CDX1/2 and DREF proteins was detected by fluorescence measurement, size-exclusion column (SEC) chromatography, His-tagged pull-down assay, and surface plasmon resonance spectroscopy (BIAcore). In addition, we identified that four different mutants of CDX1 (S185A, N190A, T194A, and V212A) were bound to dDREF with different degrees of interaction. Our results indicate that CDX1/2 homeodomains interact with the DNA-binding domain of dDREF, thereby regulating its transcription activity.