Although mutations in HNF4A were identified as the cause of Maturity Onset Diabetes of the Young 1 (MODY1) two decades ago, the mechanisms by which this nuclear receptor regulates glucose homeostasis remain unclear. Here we report that loss of Drosophila HNF4 recapitulates hallmark symptoms of MODY1, including adult-onset hyperglycemia, glucose intolerance and impaired glucose-stimulated insulin secretion (GSIS). These defects are linked to a role for dHNF4 in promoting mitochondrial function as well as the expression of Hex-C, a homolog of the MODY2 gene Glucokinase. dHNF4 is required in the fat body and insulin-producing cells to maintain glucose homeostasis by supporting a developmental switch toward oxidative phosphorylation and GSIS at the transition to adulthood. These findings establish an animal model for MODY1 and define a developmental reprogramming of metabolism to support the energetic needs of the mature animal.
dHNF4_RNAseq_DiffExpr_1.5fold
Total RNA was extracted from dHNF4 mutants and genetically-matched control adult males (8 days old) using TriPure isolation reagent (Roche) followed by further purification/cleanup by RNeasy kit (QIAGEN). RNA from 4 biological replicates (n=40 flies per sample) per genotype was polyA-selected and assembled into barcoded libraries and pooled into a single-flow cell lane for Illumina HighSeq2000 50-cycle single-read sequencing, which produced ≥ 21.9 million reads per sample. Standard replicate RNAseq analysis was performed using USeq and DESeq analysis packages with alignment to the Drosophila melanogaster dm3 genome assembly. Transcripts meeting a cutoff of ≥1.5 fold difference in mRNA abundance (~Log2 ≥ +/- 0.6) and FDR ≤ 1% were considered as differentially expressed genes. RNA quality control, library preparation, sequencing, and data analysis were performed at the University of Utah High Throughput Genomics and Bioinformatics Core Facilities.
dHNF4_ChIPseq_NearestNeighbor
dHNF4-DNA bound complexes were immunoprecipitated using polyclonal antibody against endogenous dHNF4 protein. Barcoded libraries for dHNF4-IP and input control samples were generated by the University of Utah High Throughput Genomics core facility and sequenced in a single lane Illumina HiSeq 5-cycle single-read sequencing. Data analysis was performed by the Bioinformatics Core at the University of Utah School of Medicine using USeq and Model-based Analysis for ChIP-seq (MACS2) run with a peak shift of 100 bp. Enrichment peaks were identified with an FDR cutoff of 1% and nearest neighboring genes were compiled using USeq FindNeighboringGenes and UCSC dm3 EnsGenes gene tables. Nearest neighbor genes were compared to our RNA-seq dataset to identify neighbors that are also misexpressed in dHNF4 mutants, highlighting these as direct targets of dHNF4.
