Heterodimeric Pbx-Prep1 homeodomain protein binding to the glucagon gene restricting transcription in a cell type-dependent manner

Homeodomain proteins specify developmental pathways and cell-specific gene transcription whereby proteins of the PBC subclass can direct target gene s pecificity of Hox proteins. Proteins encoded by nonclustered homeobox genes have been shown to be essential for cell lineage differentiation and gene expression in pancreatic islets. Using specific antiserum in an electrophor etic mobility shift assay and in vitro transcribed/ translated proteins, th e nuclear proteins binding domain B of the G3 enhancer-like element of the glucagon gene were identified in the present study as heterodimers consisti ng of the ubiquitously expressed homeodomain protein Prep1 and the also wid ely expressed PBC homeoprotein Pbx (isoform 1a, 1b, or 2), These heterodime ric complexes were found to bind also to the glucagon cAMP response element and to a newly identified element termed G5 (from -169 to -140). Whereas t he expression of Prep1 or Pbx forms alone had no effect, coexpression of Pb x1a/1b-Prep1 inhibited the glucagon promoter when activated by cotransfecte d Pax6 or another transcription factor in non-glucagon-producing cells. In contrast, in glucagon-producing pancreatic islet cells, Pbx-Prep1 had no ef fect on GAL4-Pax6-induced mutant glucagon promoter activity or on Pax6-depe ndent wild-type glucagon promoter activity. Furthermore, 5'-deletion of G5 enhanced glucagon promoter activity in a non-glucagon producing cell line b ut not in glucagon-producing islet cells. This study thus identifies a nove l target and Hox-independent function of Pbx-Prep1 heterodimers that, throu gh repression of glucagon gene transcription in non-glucagon-producing cell s, may help to establish islet cell-specific expression of the glucagon gen e.