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Proper regulation of cytosolic Ca2+ is critical for pancreatic acinar cell function. Disruptions in normal Ca2+ concentrations affect numerous cellular functions and are associated with pancreatitis. Membrane pumps and channels regulate cytosolic Ca2+ homeostasis by promoting rapid Ca2+ movement. Determining how expression of Ca2+ modulators is regulated and the cellular alterations that occur upon changes in expression can provide insight into initiating events of pancreatitis. The goal of this study was to delineate the gene structure and regulation of a novel pancreas-specific isoform for Secretory Pathway Ca2+ ATPase 2 (termed SPCA2C), which is encoded from the Atp2c2 gene. Using Next Generation Sequencing of RNA (RNA-seq), chromatin immunoprecipitation for epigenetic modifications and promoter-reporter assays, a novel transcriptional start site was identified that promotes expression of a transcript containing the last four exons of the Atp2c2 gene (Atp2c2c). This region was enriched for epigenetic marks and pancreatic transcription factors that promote gene activation. Promoter activity for regions upstream of the ATG codon in Atp2c2’s 24th exon was observed in vitro but not in in vivo. Translation from this ATG encodes a protein aligned with the carboxy terminal of SPCA2. Functional analysis in HEK 293A cells indicates a unique role for SPCA2C in increasing cytosolic Ca2+. RNA analysis indicates that the decreased Atp2c2c expression observed early in experimental pancreatitis reflects a global molecular response of acinar cells to reduce cytosolic Ca2+ levels. Combined, these results suggest SPCA2C affects Ca2+ homeostasis in pancreatic acinar cells in a unique fashion relative to other Ca2+ ATPases. J. Cell. Physiol. 231: 2768–2778, 2016. © 2016 Wiley Periodicals, Inc.

Strict regulation of cytosolic Ca2+ is essential to regulated exocytosis and proper pancreatic acinar cell function, controlled in part by pumps that shuttle Ca2+ out of the cytosol. Our laboratory identified a novel isoform of Secretory Ca2+ ATPase 2 (SPCA2) containing only the carboxy terminus. Pancreatic SPCA2, is an approximately 17-20 kDa, protein encoded by the Atp2c2 gene and is completely absent in Mist1-/- acini.. The focus of this thesis was to understand transcriptional regulation of Atp2c2 in the pancreas. Pancreatic Atp2c2 appears to be transcribed from an alternative transcriptional start site (TSS) and is regulated by MIST1. Bioinformatic analyses identified a truncated Atp2c2 isoform initiated within the 23rd intron of the Atp2c2 gene. Comparative analysis suggested this isoform differs between humans and mice. ChIP-seq analysis identified enrichment for trimethylated (Me3) histone 3(H3) at lysine 4 (K4) in intron 23 in pancreatic tissue, which was confirmed by ChIP-qPCR. ChIP-qPCR also identified enrichment of Acetylated Histone 3 (H3Ac), H3K36Me3, MIST1 and RNA PolII within this region. Luciferase assays and immunofluorescence confirmed this region possessed promoter activity and produced a protein of the correct size. Our results identify a unique TSS for Atp2c2 within the pancreas that is regulated by MIST1.