Regulation of alphaA-crystallin via Pax6, c-Maf, CREB and a broad domain of lens-specific chromatin

Pax6 and c-Maf regulate multiple stages of mammalian lens development. Here, we identified novel distal control regions (DCRs) of the alphaA-crystallin gene, a marker of lens fiber cell differentiation induced by FGF-signaling. DCR1 stimulated reporter gene expression in primary lens explants treated with FGF2 linking FGF-signaling with alphaA-crystallin synthesis. A DCR1/alphaA-crystallin promoter (including DCR2) coupled with EGFP virtually recapitulated the expression pattern of alphaA-crystallin in lens epithelium and fibers. In contrast, the DCR3/alphaA/EGFP reporter was expressed only in 'late' lens fibers. Chromatin immunoprecipitations showed binding of Pax6 to DCR1 and the alphaA-crystallin promoter in lens chromatin and demonstrated that high levels of alphaA-crystallin expression correlate with increased binding of c-Maf and CREB to the promoter and of CREB to DCR3, a broad domain of histone H3K9-hyperacetylation extending from DCR1 to DCR3, and increased abundance of chromatin remodeling enzymes Brg1 and Snf2h at the alphaA-crystallin locus. Our data demonstrate a novel mechanism of Pax6, c-Maf and CREB function, through regulation of chromatin-remodeling enzymes, and suggest a multistage model for the activation of alphaA-crystallin during lens differentiation.     

TCF-4 mediates cell type-specific regulation of proglucagon gene expression by beta-catenin and glycogen synthase kinase-3beta

The proglucagon gene (glu) encodes glucagon, expressed in pancreatic islets, and the insulinotropic hormone GLP-1, expressed in the intestines. These two hormones exert critical and opposite effects on blood glucose homeostasis. An intriguing question that remains to be answered is whether and how glu gene expression is regulated in a cell type-specific manner. We reported previously that the glu gene promoter in gut endocrine cell lines was stimulated by beta-catenin, the major effector of the Wnt signaling pathway, whereas glu mRNA expression and GLP-1 synthesis were activated via inhibition of glycogen synthase kinase-3beta, the major negative modulator of the Wnt pathway (Ni, Z., Anini, Y., Fang, X., Mills, G. B., Brubaker, P. L., & Jin, T. (2003) J. Biol. Chem. 278, 1380-1387). We now show that beta-catenin and the glycogen synthase kinase-3beta inhibitor lithium do not activate glu mRNA or glu promoter expression in pancreatic cell lines. In the intestinal GLUTag cell line, but not in the pancreatic InR1-G9 cell line, the glu promoter G2 enhancer-element was activated by lithium treatment via a TCF-binding motif. TCF-4 is abundantly expressed in the gut but not in pancreatic islets. Furthermore, both TCF-4 and beta-catenin bind to the glu gene promoter, as detected by chromatin immunoprecipitation. Finally, stable introduction of dominant-negative TCF-4 into the GLUTag cell line repressed basal glu mRNA expression and abolished the effect of lithium on glu mRNA expression and GLP-1 synthesis. We have therefore identified a unique mechanism that regulates glu expression in gut endocrine cells only. Tissue-specific expression of TCF factors thus may play a role in the diversity of the Wnt pathway.     

Brn-4 transcription factor expression targeted to the early developing mouse pancreas induces ectopic glucagon gene expression in insulin-producing beta cells

The endocrine pancreas is comprised of beta and alpha cells producing the glucostatic hormones insulin and glucagon, respectively, and arises during development by the differentiation of stem/progenitor cells in the foregut programmed by the beta cell lineage-specific homeodomain protein Idx-1. Brain-4 (Brn-4) is expressed in the pancreatic anlaga of the mouse foregut at e10 in the alpha cells and transactivates glucagon gene expression. We expressed Brn-4 in pancreatic precursors or beta cell lineage in transgenic mice by placing it under either Idx-1 or insulin promoter (rat insulin II promoter) control, respectively. Idx-1 expression occurs at developmental day e8.5, and insulin expression occurs at e9.5, respectively. Misexpression of Brn-4 by the Idx-1 promoter results in ectopic expression of the proglucagon gene in insulin-expressing pancreatic beta cells, whereas misexpression by rat insulin II promoter did not. The early developmental expression of Brn-4 appears to be a dominant regulator of the glucagon expressing alpha cell lineage, even in the context of the beta cell lineage.