Disruption of a novel Kruppel-like transcription factor p300-regulated pathway for insulin biosynthesis revealed by studies of the c.-331 INS mutation found in neonatal diabetes mellitus

Abstract

Krüppel-like transcription factors (KLFs) have elicited significant attention because of their regulation of essential biochemical pathways and, more recently, because of their fundamental role in the mechanisms of human diseases. Neonatal diabetes mellitus is a monogenic disorder with primary alterations in insulin secretion. We here describe a key biochemical mechanism that underlies neonatal diabetes mellitus insulin biosynthesis impairment, namely a homozygous mutation within the insulin gene (INS) promoter, c.-331C>G, which affects a novel KLF-binding site. The combination of careful expression profiling, electromobility shift assays, reporter experiments, and chromatin immunoprecipitation demonstrates that, among 16 different KLF proteins tested, KLF11 is the most reliable activator of this site. Congruently, the c.-331C>G INS mutation fails to bind KLF11, thus inhibiting activation by this transcription factor. Klf11(-/-) mice recapitulate the disruption in insulin production and blood levels observed in patients. Thus, these data demonstrate an important role for KLF11 in the regulation of INS transcription via the novel c.-331 KLF site. Lastly, our screening data raised the possibility that other members of the KLF family may also regulate this promoter under distinct, yet unidentified, cellular contexts. Collectively, this study underscores a key role for KLF proteins in biochemical mechanisms of human diseases, in particular, early infancy onset diabetes mellitus.

FIGURE 1.

Mutation and functional analysis of the INS promoter in NDM. a, diagram shows the human INS promoter sequence surrounding the c.-331 site (c.-229 to c.-351). The previously described cis-regulatory elements, E1 and A1, are in capital letters and underlined. The TATA box is also in capital letters, and the transcription start site (TSS) is indicated with an arrow (c.-238). The CACCC box, which is recognized by the KLF protein family is in capital letters and highlighted in a gray box with the c.-331C>G mutation of this element shown above the sequence. The numbering is described relative to the translational start site (c.1) according to Human Genome Variation Society guidelines. b, pedigree of Family 1. Proband-1-CE is indicated by a black arrow. c, loss of INS promoter activity with the c.-331C>G mutation in both INS-1 and β-TC-3 cells. Luciferase-based reporter assays were performed using the WT and c.-331C>G mutant INS promoters. The mean, standard error, and p values are shown for three replicates performed independently at least three times.

FIGURE 2.

Expression profiling of KLF genes reveals enrichment of distinct KLF proteins in human islets and β cells. Illumina-based KLF gene family-wide array profiling was used to determine the expression profile in whole human islets and FACS-assisted sorted human β cells. a, heat map indicating the relative value of expression of each KLF gene in a particular sample (see supplemental Table S3 for numerical data). Color scale of values is shown below. b, expression of KLF proteins found to be expressed by Illumina array was confirmed by RT-PCR in two cDNA samples of each, sorted human β cells and human islets. Positive control (+) is total human cDNA. MW, molecular weight marker. c, histogram showing the relative enrichment of KLF genes specifically in human β cells within the islet as defined by the ratio between the median level of expression in sorted β cells compared with whole islets (MLEβ/MLEi). KLF3, KLF5, KLF11, and KLF13 had values close to 1 (>0.85), indicating that most of the expression signal detected in islets for these genes was recovered in the sorted β cells. Note, however, that the abundance of KLF3 transcript was not significant in either whole islets or β cells, as shown in Fig. 2A.

FIGURE 3.

KLF11 activates the human INS promoter but not the c.-331C>G mutant promoter. a, rat INS-1 β cells were co-transfected with either the WT or c.-331C>G mutant INS promoter reporter construct and each of the KLF family members (KLF1–KLF16) or empty vector (EV) control. Luciferase activity was measured, and the means and standard errors (as indicated by the error bars) were determined for each experimental condition from triplicates of three independent experiments. The dashed line represents the 2-fold cut-off value for significant activation above empty vector control. b, KLF11 was the only KLF family member to increase WT INS promoter activity more than 2-fold in β-TC-3 cells, but this activity was abolished with the c.-331C>G mutant promoter. His- and FLAG-tagged KLF protein expression was confirmed by Western blot (lower panel). c, KLF11 requires functional p300 to activate the INS promoter. INS-1 cells were co-transfected with the WT INS promoter and either empty vector control or KLF11 along with an empty vector control or a DN-p300. Note that plasmid DNA amounts were adjusted to accommodate either control vector or DN-p300; thus KLF11 activation of the WT INS promoter was adjusted to 1.6-fold above empty vector control. The mean promoter activity ± standard error is shown relative to WT INS promoter transfected with control empty vector.

FIGURE 4.

KLF11 binds to the c.-331 novel KLF binding site, which is disrupted by the c.-331C>G NDM mutation. a, EMSA was performed using either the wild-type human INS CACCC promoter site (WT hINS; lanes 1, 3, 4, and 7–9) or the c.-331C>G mutation (mut hINS; lanes 2, 5, and 6) with GST protein (lanes 3 and 5), recombinant KLF11 (KLF11; lanes 4 and 6–9), or probe alone (lanes 1 and 2). The specific complexes that form between KLF11 and the probe are indicated on the left. Although the anti-GST antibody shifted the KLF11-WT hINS complex (lane 7), the same amount of an anti-mIgG antibody did not (lane 8). Notably, KLF11 did not shift the c.-331C>G mutant hINS probe (lane 6). Furthermore, the addition of an excess of unlabeled WT hINS probe competed for the binding (lane 9). Finally, the corresponding region of the rat Ins2 promoter was utilized as a probe for gel shift assays (WT rIns2, lanes a–c) with control GST protein alone (lane b), KLF11 (KLF11; lane c), or probe alone (lane a), which demonstrated that KLF11 indeed forms a complex with this region of the rat Ins2 promoter in vitro (lane c). b, KLF11 occupies the CACCC box of the rat Ins2 promoter in vivo. The region of the Ins2 promoter fragment containing the conserved CACCC box was amplified by PCR after ChIP from KLF11-infected cells but not the empty vector-infected sample immunoprecipitated (IP) with the same anti-His antibody (negative control), demonstrating that this region of the rIns2 promoter is a target of KLF11 in INS-1 β cells. Positive amplification of PCR products is shown in the input DNA lanes, demonstrating that this region of the rIns2 promoter is present in all samples before immunoprecipitation. Anti-mouse IgG was used as an additional negative control (data not shown). MW, molecular weight marker.

FIGURE 5.

Klf11^−/− mice display defects in insulin biosynthesis and blood levels. a, nonfasting insulin levels in Klf11^−/− animals were significantly lower when compared with Klf11^+/+ littermates. b, insulin levels were also lower in Klf11^−/− animals than in Klf11^+/+ littermates after overnight fasting. c, immunohistochemistry shows decreased insulin immunoreactivity within Klf11^−/− islets, on the right as compared with Klf11^+/+ littermates, on the left. Representative images are shown, and semiquantitative evaluation is depicted as a bar graph on the right. Scale bar, 200 μm. d, Ins2 mRNA levels were significantly lower in pancreata from Klf11^−/− mice compared with Klf11^+/+ littermate values by microarray (left panel) and RT-PCR (right panel). e, OGTT in Klf11^−/− mice show peak glucose levels at 20 min in both groups, although values in Klf11^−/− mice were lower than in Klf11^+/+ littermates (left panel). Lower values were also observed at 60 and 120 min. Serial plasma insulin levels corresponding to each time point of OGTT (right panel) are also shown.