- Browse by Author
Browsing by Author "Stein, Roland"
Now showing 1 - 3 of 3
Results Per Page
Sort Options
Item Identification of direct transcriptional targets of NFATC2 that promote β cell proliferation(The American Society for Clinical Investigation, 2021) Simonett, Shane P.; Shin, Sunyoung; Herring, Jacob A.; Bacher, Rhonda; Smith, Linsin A.; Dong, Chenyang; Rabaglia, Mary E.; Stapleton, Donnie S.; Schueler, Kathryn L.; Choi, Jeea; Bernstein, Matthew N.; Turkewitz, Daniel R.; Perez-Cervantes, Carlos; Spaeth, Jason; Stein, Roland; Tessem, Jeffery S.; Kendziorski, Christina; Keleş, Sündüz; Moskowitz, Ivan P.; Keller, Mark P.; Attie, Alan D.; Pediatrics, School of MedicineThe transcription factor NFATC2 induces β cell proliferation in mouse and human islets. However, the genomic targets that mediate these effects have not been identified. We expressed active forms of Nfatc2 and Nfatc1 in human islets. By integrating changes in gene expression with genomic binding sites for NFATC2, we identified approximately 2200 transcriptional targets of NFATC2. Genes induced by NFATC2 were enriched for transcripts that regulate the cell cycle and for DNA motifs associated with the transcription factor FOXP. Islets from an endocrine-specific Foxp1, Foxp2, and Foxp4 triple-knockout mouse were less responsive to NFATC2-induced β cell proliferation, suggesting the FOXP family works to regulate β cell proliferation in concert with NFATC2. NFATC2 induced β cell proliferation in both mouse and human islets, whereas NFATC1 did so only in human islets. Exploiting this species difference, we identified approximately 250 direct transcriptional targets of NFAT in human islets. This gene set enriches for cell cycle–associated transcripts and includes Nr4a1. Deletion of Nr4a1 reduced the capacity of NFATC2 to induce β cell proliferation, suggesting that much of the effect of NFATC2 occurs through its induction of Nr4a1. Integration of noncoding RNA expression, chromatin accessibility, and NFATC2 binding sites enabled us to identify NFATC2-dependent enhancer loci that mediate β cell proliferation.Item The MafA transcription factor becomes essential to islet β-cells soon after birth(American Diabetes Association, 2014-06) Hang, Yan; Yamamoto, Tsunehiko; Benninger, Richard K. P.; Brissova, Marcela; Guo, Min; Bush, Will; Piston, David W.; Powers, Alvin C.; Magnuson, Mark; Thurmond, Debbie C.; Stein, Roland; Department of Pediatrics, IU School of MedicineThe large Maf transcription factors, MafA and MafB, are expressed with distinct spatial-temporal patterns in rodent islet cells. Analysis of Mafa(-/-) and pancreas-specific Mafa(∆panc) deletion mutant mice demonstrated a primary role for MafA in adult β-cell activity, different from the embryonic importance of MafB. Our interests here were to precisely define when MafA became functionally significant to β-cells, to determine how this was affected by the brief period of postnatal MafB production, and to identify genes regulated by MafA during this period. We found that islet cell organization, β-cell mass, and β-cell function were influenced by 3 weeks of age in Mafa(Δpanc) mice and compromised earlier in Mafa(Δpanc);Mafb(+/-) mice. A combination of genome-wide microarray profiling, electron microscopy, and metabolic assays were used to reveal mechanisms of MafA control. For example, β-cell replication was produced by actions on cyclin D2 regulation, while effects on granule docking affected first-phase insulin secretion. Moreover, notable differences in the genes regulated by embryonic MafB and postnatal MafA gene expression were found. These results not only clearly define why MafA is an essential transcriptional regulator of islet β-cells, but also why cell maturation involves coordinated actions with MafB.Item Transcriptional Activity of the Islet β Cell Factor Pdx1 is Augmented by Lysine Methylation Catalyzed by the Methyltransferase Set7/9(2015-04) Maganti, Aarthi V.; Maier, Bernhard; Tersey, Sarah A.; Sampley, Megan L.; Mosley, Amber L.; Özcan, Sabire; Pachaiyappan, Boobalan; Woster, Patrick M.; Hunter, Chad S.; Stein, Roland; Mirmira, Raghavendra G.; Department of Cellular & Integrative Physiology, IU School of MedicineThe transcription factor Pdx1 is crucial to islet β cell function and regulates target genes in part through interaction with coregulatory factors. Set7/9 is a Lys methyltransferase that interacts with Pdx1. Here we tested the hypothesis that Lys methylation of Pdx1 by Set7/9 augments Pdx1 transcriptional activity. Using mass spectrometry and mutational analysis of purified proteins, we found that Set7/9 methylates the N-terminal residues Lys-123 and Lys-131 of Pdx1. Methylation of these residues occurred only in the context of intact, full-length Pdx1, suggesting a specific requirement of secondary and/or tertiary structural elements for catalysis by Set7/9. Immunoprecipitation assays and mass spectrometric analysis using β cells verified Lys methylation of endogenous Pdx1. Cell-based luciferase reporter assays using wild-type and mutant transgenes revealed a requirement of Pdx1 residue Lys-131, but not Lys-123, for transcriptional augmentation by Set7/9. Lys-131 was not required for high-affinity interactions with DNA in vitro, suggesting that its methylation likely enhances post-DNA binding events. To define the role of Set7/9 in β cell function, we generated mutant mice in which the gene encoding Set7/9 was conditionally deleted in β cells (SetΔβ). SetΔβ mice exhibited glucose intolerance similar to Pdx1-deficient mice, and their isolated islets showed impaired glucose-stimulated insulin secretion with reductions in expression of Pdx1 target genes. Our results suggest a previously unappreciated role for Set7/9-mediated methylation in the maintenance of Pdx1 activity and β cell function.