Islet β cell-specific transcription of the insulin gene is mediated through the binding of the islet-enriched PDX-1, BETA2, and MafA transcription factors to conserved 5′-flanking region regulatory elements. However, additional non-conserved sequences within this region are also significant in regulating expression. Thus, PDX-1 binds to and activates the GG2 element located between nucleotides −145 and −140 of the human gene, while the corresponding, but non-identical, site in the rodent insulin genes are negatively regulated by the Nkx2.2 transcription factor. Here, we show that despite binding PDX-1 approximately 20-fold less effectively than the conserved insulin A3 and A1 sites in gel mobility shift assays, human GG2 appears to be more important for the activation of transfected human insulin enhancer-driven reporter constructs in β cell lines. Furthermore, functional interaction analysis in non-islet cell lines demonstrated that PDX-1 binding to GG2, A1, and A3 contributes to synergistic activation of insulin gene expression with MafA. Our analysis also illustrated the requirement of poorly conserved human sequences between −293 and −251 in mediating activity through the more upstream A3 binding site. Collectively these experiments have revealed distinct features in control of the human and rodent insulin genes by PDX-1, processes that may be involved in regulating insulin expression under both normal and diabetic conditions in humans.
John Le Lay and Roland Stein
Isabella Artner, Yan Hang, Min Guo, Guoqiang Gu and Roland Stein
As successful generation of insulin-producing cells could be used for diabetes treatment, a concerted effort is being made to understand the molecular programs underlying islet β-cell formation and function. The closely related MafA and MafB transcription factors are both key mammalian β-cell regulators. MafA and MafB are co-expressed in insulin+β-cells during embryogenesis, while in the adult pancreas only MafA is produced in β-cells and MafB in glucagon+α-cells. MafB−/− animals are also deficient in insulin+ and glucagon+ cell production during embryogenesis. However, only MafA over-expression selectively induced endogenous Insulin mRNA production in cell line-based assays, while MafB specifically promoted Glucagon expression. Here, we analyzed whether these factors were sufficient to induce insulin+ and/or glucagon+ cell formation within embryonic endoderm using the chick in ovo electroporation assay. Ectopic expression of MafA, but not MafB, promoted Insulin production; however, neither MafA nor MafB were capable of inducing Glucagon. Co-electroporation of MafA with the Ngn3 transcription factor resulted in the development of more organized cell clusters containing both insulin- and glucagon-producing cells. Analysis of chimeric proteins of MafA and MafB demonstrated that chick Insulin activation depended on sequences within the MafA C-terminal DNA-binding domain. MafA was also bound to Insulin and Glucagon transcriptional control sequences in mouse embryonic pancreas and β-cell lines. Collectively, these results demonstrate a unique ability for MafA to independently activate Insulin transcription.