PAX6 is a transcription factor that plays an important role during
pancreatic morphogenesis. The aim of the present study is to
identify the upstream activator(s) of the PAX6 gene possibly
involved in the early stages of pancreatic differentiation. Recently,
individual elements regulating PAX6 gene activity in the pancreas
have been identified in a 1100 bp Spe/HincII fragment 4.6 kb
upstream of exon 0. Preliminary sequence analysis of this region
revealed some potential DNA-binding sites (E boxes) specific for
the binding of basic helix–loop–helix transcription factors. By
using electrophoretic mobility shift assays, we demonstrated that
both nuclear protein extracts from insulin-secreting RINm5F cells
and in vitro-translated NeuroD/BETA2 can bind specifically to
these E boxes. Furthermore, by transient transfection experiments
we demonstrated that the expression of basic helix–loop–helix
transcription factor NeuroD/BETA2 can induce activation of the
PAX6 promoter in the NIH-3T3 cell line. Thus we show that
NeuroD/BETA2 is involved in the activation of the expression
of PAX6 through E boxes in the PAX6 promoter localized in a
1.1 kb sequencewithin the 4.6 kb untranslated region upstream of
exon 0.
