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MafA is a key transcriptional activator of islet beta cells, and its exclusive expression within beta cells of the developing and adult pancreas is distinct among pancreatic regulators. Region 3 (base pairs -8118 to -7750 relative to the transcription start site), one of six conserved 5' cis domains of the MafA promoter, is capable of directing beta-cell-line-selective expression. Transgenic reporters of region 3 alone (R3), sequences spanning regions 1 to 6 (R1-6; base pairs -10428 to +230), and R1-6 lacking R3 (R1-6(DeltaR3)) were generated. Only the R1-6 transgene was active in MafA(+) insulin(+) cells during development and in adult cells. R1-6 also mediated glucose-induced MafA expression. Conversely, pancreatic expression was not observed with the R3 or R1-6(DeltaR3) line, although much of the nonpancreatic expression pattern was shared between the R1-6 and R1-6(DeltaR3) lines. Further support for the importance of R3 was also shown, as the islet regulators Nkx6.1 and Pax6, but not NeuroD1, activated MafA in gel shift, chromatin immunoprecipitation (ChIP), and transfection assays and in vivo mouse knockout models. Lastly, ChIP demonstrated that Pax6 and Pdx-1 also bound to R1 and R6, potentially functioning in pancreatic and nonpancreatic expression. These data highlight the nature of the cis- and trans-acting factors controlling the beta-cell-specific expression of MafA.

Proteins Glis1 to -3 constitute a subfamily of Kruppel-like zinc finger transcriptional regulators that share a highly conserved five-C2H2-type zinc finger domain with members of the Gli and Zic subfamilies (6, 25, 27, 30-32, 35, 38, 39, 43, 56). Glis1 to -3 regulate gene transcription by binding specific DNA sequences referred to as Glis-binding sites (Glis-BS) in promoter regulatory regions of target genes (10, 30, 31). Although their precise physiological functions are still poorly understood, genetic studies have implicated Glis1 to -3 in several pathologies (7, 8, 24, 29, 33, 39, 45). Glis3 is abundantly expressed in the adult kidney, pituitary, pancreas, uterus, and thyroid gland (31, 45). During mouse embryonic development, Glis3 is expressed in a spatiotemporal pattern suggesting that Glis3 regulates gene expression at specific stages during development (31). Genetic alterations in the human GLIS3 gene have been linked to a rare syndrome characterized by neonatal diabetes and congenital hypothyroidism (NDH) (45, 50). Depending on the nature of the GLIS3 mutation, NDH patients can also display facial abnormalities, glaucoma, liver fibrosis, and polycystic kidney disease. Recently, a genome-wide association study identified the GLIS3 gene as a susceptibility locus for type 1 diabetes (8). These studies, together with evidence that Glis3 is expressed in pancreatic β cells, suggest that Glis3 has an important regulatory role in the pancreas. Although major advances have been made in understanding pancreatic development, many of the molecular mechanisms that regulate progenitor cell dynamics and cell differentiation are still not precisely understood (1, 18, 19, 21, 26, 28, 37). At approximately embryonic day 9 (E9) of mouse embryogenesis, the pancreas first appears from distinct ventral and dorsal anlagen as evaginations of the distal foregut endoderm (21, 36). The buds grow and initiate branching morphogenesis at about E11.5. Early multipotent pancreatic progenitors, marked by Pdx1, Ptf1a, Nkx2.2, and Cpa1 expression (12, 14, 41, 57), are the source of all differentiated cells of the exocrine, ductal, and endocrine cell lineages. Lineage determination is a complex process that involves many transcription factors and signaling pathways. Induction of Ngn3 marks the differentiation of pancreatic progenitors into proendocrine progenitors (15, 17, 22, 36, 51). Differentiation into the different endocrine cell lineages involves the induction of a combination of additional transcription factors, including Myt1, NeuroD, Isl1, Pax4, Pax6, and Arx (2, 13, 22, 34, 36, 37, 48, 49). Defects in the expression or activity of these transcription factors in mice and humans often result in abnormal pancreatic development and function that can lead to diabetes. To obtain greater insights into the physiological and molecular functions of Glis3, we recently generated Glis3zf/zf mutant mice that are deficient in Glis3 transactivating activity (24). In this study, we characterize the pancreatic phenotype of these mice and analyze the role of Glis3 in pancreatic development. We demonstrate that, in addition to cyst formation in the pancreatic ducts, Glis3zf/zf mutant mice develop neonatal diabetes that is associated with an almost total loss of β cells. We provide evidence which indicates that Glis3 plays a key role in cell lineage specification, particularly in the development of mature pancreatic β cells. We further identify Glis3 as a regulator of insulin 2 gene expression. Our study shows that Glis3 has multiple functions in the pancreas and suggests that Glis3 might provide a new therapeutic target to intervene in diabetes.