Redundant and synergistic effect of Cdx-2 and Brn-4 on regulating proglucagon gene expression

Cdx-2 and Brn-4 are recognized as transcriptional activators for the proglucagon gene. These two homeodomain (HD) proteins are able to interact with the correspondent motifs on the G1 enhancer element of proglucagon promoter, separated by only 8 bp. We have examined Brn-4 expression in proglucagon-producing cells, isolated hamster Brn-4 cDNA, and localized its activation domain. Ectopic expression of either Cdx-2 or Brn-4 in the pancreatic B cell line In111 provoked it to express proglucagon mRNA, whereas ectopically expressing both of them further stimulated proglucagon mRNA expression in this cell line. Furthermore, Brn-4 was found to synergize with Cdx-2 in activating proglucagon promoter, and the Brn-4 activation domain was not required for this synergistic activation. When the binding site for either Cdx-2 or Brn-4 was mutated, the synergistic activation by these two HD proteins was significantly attenuated, but not abolished. We propose that both cooperative DNA binding and mutual recruitment between Cdx-2 and Brn-4 are involved in this synergistic activation and have detected physical interaction between Cdx-2 and Brn-4 by glutathione-S-transferase-fusion protein pull-down assay. Our observations suggest that Cdx-2 and Brn-4, two HD proteins that belong to two different families, exert a synergistic and redundant effect on proglucagon gene expression.