Disruption of a Novel Krüppel-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.

Mechanistic Role for a Novel Glucocorticoid-KLF11 (TIEG2) Protein Pathway in Stress-induced Monoamine Oxidase A Expression

“…DNA was recovered from the beads by elution buffer (1% SDS and 0.1 m NaHCO 3 ) and analyzed by real-time PCR as described previously (22). Klf11 Ϫ/Ϫ Mice-The Klf11 homozygous knockout model was generated at the University of Washington, Seattle following standard homologous recombination techniques to inactivate the endogenous Klf11 gene in embryonic stem cells, generating chimeras, and isolating colony founders carrying the knockout gene (28,36). This animal was originally generated in a mixed background and subsequently transferred to the Mayo Animal Facilities where it was crossed back into a pure C57BL/6 background for more than 20 generations to produce the inbred strain used in this study.…”

“…These MAO A promoter-luciferase reporter gene constructs were cotransfected with the KLF11 vector (or the pcDNA3.1 vector) (22) and the p300-expression vector (28) in SH-SY5Y cells using Superfect transfection reagent (Qiagen) following the protocol of the manufacturer (21,22).…”

Human Krüppel-like factor 11 differentially regulates human insulin promoter activity in β-cells and non-β-cells via p300 and PDX1 through the regulatory sites A3 and CACCC box

“…Variability of these results may be at least in part explainable by the close structural and functional relationship of KLF11 to KLF10 (Subramaniam et al, 2007). As an example for shared function among KLFs, coexpressed KLF10 as well as KLF13, KLF14 and KLF16 have recently been demonstrated to activate the hInsP (Bonnefond et al, 2011).…”

“…However, PDX1 function strictly depends on the transcriptional cofactor p300, and KLF11 has been demonstrated to interact with p300 (Fernandez-Zapico et al, 2009). The importance of p300 is underscored by a reduction of KLF11-mediated hInsP activation if a mutant dominant negative p300 is coexpressed (Bonnefond et al, 2011).…”

“…The initial report by Neve et al (2005) and the recent study of Bonnefond et al (2011) characterise KLF11 as a stimulator of cotransfected hInsP reporter in b-cell lines. Our own research, however, could not confirm these findings.…”