Two distinct DNA sequences recognized by transcription factors represent enthalpy and entropy optima.

Most transcription factors (TFs) can bind to a population of sequences closely related to a single optimal site. However, some TFs can bind to two distinct sequences that represent two local optima in the Gibbs free energy of binding (ΔG). To determine the molecular mechanism behind this effect, we solved the structures of human HOXB13 and CDX2 bound to their two optimal DNA sequences, CAATAAA and TCGTAAA. Thermodynamic analyses by isothermal titration calorimetry revealed that both sites were bound with similar ΔG. However, the interaction with the CAA sequence was driven by change in enthalpy (ΔH), whereas the TCG site was bound with similar affinity due to smaller loss of entropy (ΔS). This thermodynamic mechanism that leads to at least two local optima likely affects many macromolecular interactions, as ΔG depends on two partially independent variables ΔH and ΔS according to the central equation of thermodynamics, ΔG = ΔH - TΔS.

Ubiquitin Ligase, Fbw7, Targets CDX2 for Degradation via Two Phosphodegron Motifs in a GSK3ß-Dependent Manner.

Drosophila caudal-related homeobox transcription factor 2 (CDX2) drives differentiation of the intestinal epithelium. Loss of CDX2 expression has been reported in several colorectal cancers and cancer cell lines with a potential inverse correlation between CDX2 levels and tumor stage. Ubiquitination of CDX2 leading to its downregulation has been implicated in several studies; however, the E3 ubiquitin ligases involved in CDX2 ubiquitination have largely remained unknown. Here, it is mechanistically determined that the E3 ubiquitin ligase Fbw7 promotes CDX2 ubiquitination and degradation through two phosphodegron motifs present within CDX2 in a GSK3ß-dependent manner leading to its reduced expression and function in colon cancer cells. Fbw7, through its WD domain, interacted with CDX2 both in a heterologous HEK293T cell system and in colon cancer cells. GSK3ß was also present in the same complex as determined by coimmunoprecipitation. Furthermore, overexpression of both Fbw7 or GSK3ß down regulated endogenous CDX2 expression and function; however, both failed to inhibit endogenous CDX2 when either of them were depleted in colon cancer cells. Fbw7-mediated inhibition of CDX2 expression also led to reduced CDX2 transactivation and growth arrest of colon cancer cells. Both GSK3ß and Fbw7 degraded mutant-CDX2 having either of the Cdc4-phosphodegron (CPD) motifs disrupted (CDX2-S60A or CDX-S281A), but were unable to degrade mutant-CDX2 having both CPDs disrupted (CDX2-S60,64,281A). IMPLICATIONS: Taken together, these findings demonstrate that Fbw7 negatively regulates CDX2 expression in a GSK3ß-dependent manner through two CPDs present in CDX2. Mol Cancer Res; 14(11); 1097-109. ©2016 AACR.