Diabetes results from the failure of insulin producing β cells to compensate for increased metabolic demand and stress. The homeodomain protein PDX1 is important for the function and survival of β cells. Juliana et al. identify previously undiscovered stress responsive transcriptional complexes containing PDX1, activating transcription factor 4 (ATF4), and ATF5 that regulate expression of stress and apoptosis genes to influence β cell fate decisions during stress.
A PDX1-ATF transcriptional complex governs β cell survival during stress
Objective: Loss of insulin secretion due to failure or death of the insulin secreting β cells is the central cause of diabetes. The cellular response to stress (endoplasmic reticulum (ER), oxidative, inflammatory) is essential to sustain normal β cell function and survival. Pancreatic and duodenal homeobox 1 (PDX1), Activating transcription factor 4 (ATF4), and Activating transcription factor 5 (ATF5) are transcription factors implicated in β cell survival and susceptibility to stress. Our goal was to determine if a PDX1-ATF transcriptional complex or complexes regulate β cell survival in response to stress and to identify direct transcriptional targets.
Methods: Pdx1, Atf4 and Atf5 were silenced by viral delivery of gRNAs or shRNAs to Min6 insulinoma cells or primary murine islets. Gene expression was assessed by qPCR, RNAseq analysis, and Western blot analysis. Chromatin enrichment was measured in the Min6 β cell line and primary isolated mouse islets by ChIPseq and ChIP PCR. Immunoprecipitation was used to assess interactions among transcription factors in Min6 cells and isolated mouse islets. Activation of caspase 3 by immunoblotting or by irreversible binding to a fluorescent inhibitor was taken as an indication of commitment to an apoptotic fate.
Results: RNASeq identified a set of PDX1, ATF4 and ATF5 co-regulated genes enriched in stress and apoptosis functions. We further identified stress induced interactions among PDX1, ATF4, and ATF5. PDX1 chromatin occupancy peaks were identified over composite C/EBP-ATF (CARE) motifs of 26 genes; assessment of a subset of these genes revealed co-enrichment for ATF4 and ATF5. PDX1 occupancy over CARE motifs was conserved in the human orthologs of 9 of these genes. Of these, Glutamate Pyruvate Transaminase 2 (Gpt2), Cation transport regulator 1 (Chac1), and Solute Carrier Family 7 Member 1 (Slc7a1) induction by stress was conserved in human islets and abrogated by deficiency of Pdx1, Atf4, and Atf5 in Min6 cells. Deficiency of Gpt2 reduced β cell susceptibility to stress induced apoptosis in both Min6 cells and primary islets.
Conclusions: Our results identify a novel PDX1 stress inducible complex (es) that regulates expression of stress and apoptosis genes to govern β cell survival.