The X-axis from the graph indicates fold-activation over normalized luciferase activities. Forskolin produced even greater changes in luciferase activity than glucose. is a critical, but not the unique signaling pathway involved in IA-2 gene expression. Keywords: Dense-core vesicle, Insulin, IA-2, Promoter, PKA pathway, CREB binding site == Introduction == IA-2 and IA-2 (also known respectively as ICA512 and Phogrin) are dense-core vesicle (DCV) transmembrane proteins and major autoantigens in type 1 diabetes [1, 2]. Human IA-2 and IA-2 are 979 and 1015 amino acids, respectively, in length (GenBank acc. no ., NM_002846. 3andNM_002847. 3). Both proteins are encoded by 23 exons and consist of an intracellular, transmembrane and luminal domain [3]. The genomic DNA sequence of the human IA-2 gene is 20kb, whereas that of IA-2 spans approximately 1, 000kb. Both genes are widely distributed in neuroendocrine cells throughout the body, and are abundantly expressed in the pancreatic islets, pituitary and hippocampus. Recently we showed that deletion from the IA-2 and/or IA-2 genes in mice results in a decrease in the number of DCV, impairs insulin secretion [4], leads to female infertility, alters behavior and results circadian dysregulation [5]. In contrast to the deletion of these genes, overexpression of IA-2 in insulin-secreting MIN6 cells results in an increase in the number of DCV and the secretion of insulin [6]. These findings show that IA-2 and IA-2 are important molecules involved in the secretion of insulin and other neurohormones and neuropeptides. Despite the extensive studies on the properties and function of IA-2, little is known about the promoter region or transcriptional factors that regulate the expression of this gene. In the present experiments, using deletion fragments from the 5-upstream region of the IA-2 transcription start site, we identified a short sequence with strong promoter activity and transcriptional factors that hole to this region. == Materials and Methods == == IA-2 promoter-luciferase assay == Eight consecutive human IA-2 promoter fragments (1300 to +115) were cloned in the pGL3 vector to drive the expression of the Rabbit polyclonal to MMP9 firefly luciferase reporter gene (Promega, Madison WI, USA) [3]. All constructs were confirmed by Sanger sequencing. A dual-luciferase reporter system was used intended for promoter activity measurement according to the manufacturer’s instruction (Promega). Renilla luciferase was used as an internal control intended for transfection efficiency. Normalized luciferase data (firefly/renilla) was compared with the empty pGL3-basic vector. The construct pIA2-8 or pGL3 vector served as a control intended for GW843682X basal levels. The cell line HEK293 also was included as a control because endogenous IA-2 expression was not detected. In a separate experiment, inserts of pIA2-1, pIA2-2, pIA2-6, pIA2-7 and pIA2-8 also were inserted into the pGL4 luciferase reporter vector (Promega) intended for comparison of luciferase activities measured from the corresponding pGL3-based constructs. Unless otherwise indicated the pGL3 vector was used in all experiments. == Cell culture and transfection == Four cell lines (HEK293, U87MG, AtT20, and MIN6) were cultured in RPMI 1640 or Dulbeccos modified Bald eagle medium supplemented with 5. 6 mM glucose and 10% fetal bovine serum as explained previously [6, 7]. AtT20 cells were commercially obtained (ATCC, Manassas, VA). MIN6 cells were obtained from Dr . Jun-ichi Miyazaki [8] and used between passages 37~42, which responded to high-glucose by a 5~6 fold increase of insulin secretion and also responded to forskolin and IBMX treatment [9]. Cells were transfected with a few g of appropriate luciferase reporter plasmid by lipofectamin2000 (Invitrogen Corp., Carlsbad, CA, USA), which resulted in approximately 50% transfection efficiencies at 48 h. Plasmid coding for Renilla luciferase (0. 03 g) was co-transfected with each of the reporter gene constructs. After transfection, cells were cultured for 2448 hours and lysed in Passive Lysis Buffer (Promega) for measurement of luciferase activity using a Microplate GW843682X Luminometer (Berthold Technologies, Germany). == Site-directed mutagenesis of the IA-2 promoter == The GeneEditor site-directed mutagenesis system (Promega) was used to generate mutations in the sequence of Cre-L and Cre. 2 binding sites. The GW843682X sequences of the primers used for deletion-mutation are shown inSupplementary Table 1 . Constructs were confirmed by Sanger sequencing at the Sequencing Core Facility of NIDCR/NIH. == ChIP assay and Western blots == Approximately 2 106MIN6 cells were used per immunoprecipitation assay [7] with small modifications. Briefly, nuclei were collected from formaldehyde-fixed cells by homogenization. DNAs were sonicated into 2001000 bp. Precleared chromatin was immunoprecipitated with 10 g of anti-CREB monoclonal antibody.
