Glis3 is a member of Krüppel-like ZF proteins and has the potential to function as a transcription activator or repressor (3
). It is highly expressed in human pancreatic β cells (8
) and mouse islets and rat insulinoma 832/13 cells. The recent discovery that mutations in the GLIS3 gene are responsible for neonatal diabetes and congenital hypothyroidism (NDH) in humans (8
) motivated us to focus on the function of this factor in β cells.
In this study, we showed that Glis3 overexpression in rat 832/13 insulinoma cells markedly upregulated Ins2 gene expression. However, both Ins1 and Ins2 were downregulated when endogenous Glis3 was knocked down by siRNA, suggesting that it is involved directly or indirectly in the regulation of both genes. Our data demonstrated that Glis3 is able to directly bind the Glis3RE sequence of the rat Ins2 promoter using ChIP and EMSA, so it likely has a direct effect. The lack of response of Ins1 to Glis3 overexpression may indicate that its expression levels are already near or at maximum in 832/13 cells and an increase in Glis3 levels cannot further enhance Ins1 expression. A similar phenomenon was noted previously for the lack of activation of mouse Ins1, Ins2 and Pdx1 expression by MafB overexpression in βTC3 cells even though it binds to the promoters (26
). We cannot identify a Glis3RE sequence in the rat Ins1 promoter region. However, Glis3 can increase MafA mRNA expression and as it is known that MafA is capable of activating Ins1 promoter, we speculate that, although the Ins1 promoter is not directly bound by Glis3, Glis3 has effects on other β cell transcription factors which may in turn influence Ins1 expression.
We also showed that Glis3 expression stimulated the rat insulin 2 promoter (RIP-Gluc) in both NIH3T3 cells and 832/13 cells and mapped the regulatory region required for Glis3 activation to a 15-bp sequence (−255 to −241) of the promoter. The site GTCCCCTGCTGTGAA shares 100% homology with a similar region of the mouse Ins2 promoter, and is 80% conserved with a sequence from the human insulin promoter. We showed that Glis3 binds to this sequence and can activate the transcription of both human and rodent (Glis3RE)5-TATA box constructs, confirming its place as a bona fide Glis3 binding and transcription activation site. By ChIP analysis, we confirmed that Glis3 was able to bind to the region of interest in the endogenous Ins2 promoter in 832/13 cells as well.
The sequence we identified differs from the 11 bp consensus identified by Beak et al.
) (G/C)TGGGGGGGT(A/C) using PCR amplification of random oligonucleotide sequences. Their consensus binds to Glis3 with high affinity and can mediate transcription activation in vitro
). However, it is an artificial binding sequence that has not been identified as of yet in any 5′ regulatory regions. A computer search also failed to find any matching homologous sequences in the rat Ins2 promoter (−696/+8). The FGF18 promoter contains a Glis3-binding site with the sequence AACCCCCAAA (6
). We found a similar sequence AACCCCCAG at −7 to +1 of RIP. However, deletion analysis showed that the proximal promoter region (−75/+1) of RIP is minimally activated by co-transfected Glis3 and this sequence overlaps the transcription initiation site +1, making it unlikely that this region is involved in Glis3-activated gene transcription. The Glis3RE (−269 to −255) identified in this paper partially overlaps with a previously characterized element (GAGACATTTGCCCCCAGCTGT) known as a n
lement (NRE, −279 to −258) (28
) that lies within the glucose sensing Z element (−292 to −243) of the human insulin promoter (30
The human NDH1 mutation of GLIS3 contains a single base insertion at nucleotide 2067. It encodes a mutant protein that is truncated at its C-terminus at aa 844 due to a translation frameshift (8
). The mechanism by which this mutation causes NDH has not been elucidated. Our analysis shows that the analogous mutation of the mouse Glis3 abrogates the transcription activity of the factor, consistent with localization of the activation domain at the C-terminus (7
). Furthermore, Glis3-NDH1 acts as a dominant negative factor when expressed together with wild-type Glis3 in insulinoma cells and NIH3T3 cells. The putative mechanism is that Glis3-NDH1 binds to DNA and competes with the wild-type Glis3, but cannot activate transcription. We speculate that the NDH1 mutant protein might profoundly affect wild-type Glis3 function during early pancreas development.
In addition to direct effects on insulin promoter activity, we showed that Glis3 is capable of interacting with other key β-cell-specific transcription factors. MafA is a crucial factor for the assembly and function of the insulin gene transcription complex. Previous studies have shown that Pdx1, MafA and NeuroD1 synergistically activate the insulin promoter (12–14
). Our present results show that Glis3 physically interacts with Pdx1, MafA and NeuroD1 in co-precipitation experiments. In addition, Glis3 shows cooperative interactions with Pdx1, MafA and NeuroD1 for insulin promoter activation. These results lead us to conclude that Glis3 modulates insulin gene expression, both directly through binding to the insulin promoter and indirectly by modulating the activity of other β-cell-enriched transcription factors.
Manipulation of Glis3 levels through stable overexpression or siRNA knock-down also affected the levels of several other Glis3 target genes in β cells, including MafA, Nkx6-1 and Pax6. Therefore, Glis3 also may modulate insulin gene transcription indirectly through changes in the levels of these factors. The mRNA expression of MafA was upregulated in Glis3-overexpressing cells, and downregulated in Glis3-knockdown cells, suggesting that MafA could be a Glis3 target gene. Nkx6-1 is initially expressed broadly in the developing pancreatic bud, but eventually is restricted exclusively to β cells (32
). It has been shown that Nkx6-1 can repress insulin promoter activity in β cells (33
). Nkx6-1 mRNA expression was significantly downregulated by Glis3 overexpression, and upregulated in cells treated with Glis3 siRNA. Thus, Nkx6-1 could be another mediator of Glis3 action in β cells. Pax6 works through the C2 element of the rat Ins1 promoter (16
). Similar to Nkx6-1, Pax6 was decreased in Glis3-overexpressing β cells and increased in Glis3-knockdown cells. We only find that Glis3-NDH1 inhibits wild-type Glis3 when co-expressed during transient transfection. We do not see Glis3-NDH1 inhibition of MafA expression or stimulation of expression of Nkx6-1 and Pax-6 when overexpressed in 832/13 cells. Whether authentic Glis3RE sequences are present in the 5′ regulatory regions of the genes for the affected factors and will require further investigation.
In conclusion, we have demonstrated for the first time that Glis3 regulates insulin gene transcription in a cell line derived from β cells. Furthermore, we have identified a 15-bp sequence of the Ins2 promoter required for Glis3 binding, and this sequence is highly conserved among mouse, rat and human, underscoring its role in insulin gene regulation. Our data indicate that Glis3 can act in concert with other known insulin gene transactivators, such as Pdx1, MafA and NeuroD1. Finally, Glis3 expression also may modulate insulin gene transcription by effects on the levels of other β-cell-enriched transcription factors such as MafA, Nkx6-1 and Pax6.