Diabetologia "2002) 45: 309±326 Ó Springer-Verlag 2002 Regulation of insulin gene transcription D.Melloul, S.Marshak, E.Cerasi Department of Endocrinology and Metabolism, Hadassah University Hospital, Jerusalem, Israel Abstract binding activity and their transactivating potency can be modified in response to nutrients "glucose, The mammalian insulin gene is exclusively expressed NEFA) or hormonal stimuli "insulin, leptin, glucagon in the beta cells of the endocrine pancreas. Two de- like peptide-1, growth hormone, prolactin) through cades of intensive physiological and biochemical kinase-dependent signalling pathways "PI3-K, studies have led to the identification of regulatory se- p38MAPK, PKA, CaMK) modulating their affinities quence motifs along the insulin promoter and to the for DNA and/or for each other. From the overview isolation of transcription factors which interact to ac- of the research presented, it is clear that much more tivate gene transcription. The majority of the islet-re- study is required to fully comprehend the mecha- stricted "BETA2, PDX-1, RIP3b1-Act/C1) and ubi- nisms involved in the regulated-expression of the in- quitous "E2A, HEB) insulin-binding proteins have sulin gene in the beta cell to prevent its impairment been characterized. Transcriptional regulation results in diabetes. [Diabetologia "2002) 45: 309±326] not only from specific combinations of these activa- tors through DNA-protein and protein-protein inter- Keywords Insulin gene, beta cell, transcription factor, actions, but also from their relative nuclear concen- promoter, PDX-1, glucose, diabetes, NEFA, leptin, trations, generating a cooperativity and transcription- GLP-1. al synergism unique to the insulin gene. Their DNA Received: 23 May 2001 and in revised form: 15 November 2001 Insulin, synthesized by the beta cells of pancreatic is- Corresponding author: D. Melloul, Department of Endocrinol- lets, is of major physiological importance in metabolic ogy and Metabolism, Hadassah University Hospital, P.O. Box homeostasis. While mature insulin consists of two 12000, 91120 Jerusalem, Israel, e-mail: [email protected] Abbreviations: PDX-1, Pancreatic duodenal homeobox-1; polypeptide chains joined by disulphide bridges, the RIPE3, rat insulin promoter element 3; NRE, negative regula- gene encodes for a highly conserved single chain pre- tory element; IEF-1, insulin enhancer factor-1; IPF-1, insulin cursor, preproinsulin [1]. In most species preproinsu- promoter factor-1; STF-1, somatostatin transcription factor-1; lin exists as a single gene, whereas in the mouse and IDX-1, islet duodenum homeobox-1; GSF, glucose sensitive the rat two non-allelic insulin genes are present. The factor; IUF-1, insulin upstream factor-1; Isl-1, islet-1; Cdx-3, human insulin gene is located on the short arm of chro- caudal homeobox-3; Imx-1, LIM homeobox-1; HNF-1a, hepa- tocyte nuclear factor-1a; HMGI"Y), high mobility group pro- mosome 11 "p15.5) [2], the rat insulin I and II genes are tein I "Y); bHLH, basic helix-loop-helix; USF, upstream stimu- colocalized on chromosome 1 [3] and the mouse genes latory factor; GLUT 2, glucose transporter 2; IAPP, islet amy- are positioned on two different chromosomes, insulin loid polypeptide; GK, Goto-Kakisaki; ZDF, Zucker diabetic I on chromosome 19 [4] and insulin II on chromosome fatty; GLP-1, glucagon like peptide-1; GH, growth hormone; 7 [5]. In adult islets, the nonallelic genes appear to be PRL, prolactin; ILPR, insulin linked polymorphic region; coordinately expressed and regulated [6, 7]. The ro- VNTR, variable number of tandem repeats; HVR, hypervari- dent insulin II and the human genes contain three ex- able region; PI3-K, phosphatidylinositol 3-kinase; p38MAPK, p38 mitogen activated protein kinase; PKA, protein kinase A; ons and two introns, whilst insulin I lacks the second CaMK, Ca/calmodulin-dependent protein kinase; PDE3B, intron. The organisation and structure of the insulin phosphodiesterase 3B gene has been reviewed in detail [8]. Insulin is regulat- 310 Melloul et al.: Regulation of insulin gene transcription Fig.1. Organization of human, rat I and rat II insulin enhancer hundred basepairs "bp) upstream of the transcrip- and promoter regions. Cis-acting regulatory elements along tional start site are sufficient to confer beta cell spe- the promoter regions of these genes are boxed. Proteins bind- cific expression to an exogenous gene in transfected ing to these sequences are indicated above each box. A1-A5, E1-E2, C1-C2, and G1 elements are termed according to the cells and transgenic mice. Thus, 660 bp of the rat insu- nomenclature in [20]. E2* is an E2-like element; GR, glucocor- lin II promoter region "RIP) was shown to direct the ticoid receptor; GLE, gamma-interferon-activated sequence expression of simian virus 40 T-antigen "Tag) to beta "GAS)-like element; ISP"Un), islet-specific protein "unidenti- cells [12]. The expressed viral oncogene induced tu- fied); NRE, negative regulatory element; NCS, negative con- mour formation, permitting the establishment of trol sequences; Z, Z minienhancer valuable beta cell lines "b-TCs). Moreover, correct initiation of insulin transcripts in mice together with cell-specific expression and physiological regulation of either the integrated human insulin gene [13, 14] ed at several levels, from gene transcription to insulin or transgenes driven by the 5' flanking regions of in- secretion. This review focuses on the transcriptional sulin genes [12, 15, 16] implied functional conserva- regulation of the insulin gene. tion of regulatory sequences between different spe- cies. Deletion and mutational analyses of various in- sulin promoter regions, conducted in transfected cell Beta cell-specific expression of the insulin gene lines and in transgenic mice, led to the identification of cis-acting regulatory sequences necessary for spe- In the mouse embryo, insulin mRNA is first detected cific-gene expression. Regulated insulin gene tran- at stage E9 "20 somites) [9] in the foregut area from scription relies on the interaction of sequence motifs which the pancreas develops. In humans, insulin-pos- in the promoter with a number of ubiquitous and islet itive cells are already present by the 8th week of em- specific transcription factors "Fig.1). These interac- bryonic development [10]. tions determine the positive and negative regulation In post-natal life, insulin gene expression is re- of insulin gene expression and its inducibility by phys- stricted to the beta cell. Most studies on the regulated iological stimuli. The binding of proteins to regulato- expression of insulin used human and rat genes. Tis- ry elements has led to the identification and cloning sue specificity has been shown to be controlled by 5' of putative insulin transcription factors. The charac- flanking regulatory regions of the gene [11]: a few terization of such factors "and their genes) contribut- Melloul et al.: Regulation of insulin gene transcription 311 ed to our current understanding not only of insulin family. While the E47/BETA2 complexcontrols gene expression, but also of endocrine pancreas de- both insulin and glucagon gene transcription, it was velopment and of beta cell differentiation. shown to bind to the glucagon promoter with much less affinity [27]. Of interest, overexpression of E47 inhibited only E box-mediated glucagon gene expres- Positive regulation sion whereas it activated insulin gene transcription, suggesting that the E47/BETA2 ratio is important Regulatory cis-acting elements and trans-acting factors for regulated gene expression in islet cells. Thus, un- involved in insulin gene transcription. Insulin genes ique cooperative interactions allow the insulin E box share a number of conserved DNA motifs in their 5' enhancer to discriminate between different bHLH flanking region [11], implying that they might be reg- factors for achieving tissue-specific activation of the ulated by similar trans-acting factors. Amongst these, gene. E, A and C1/RIPE3b elements seem to be major de- In contrast to the rat I gene, rat II and human insu- terminants of beta cell-specific expression of the insu- lin 5' flanking regions do not contain the conserved lin gene. E2 box, but a related sequence in the human gene binds the HLH protein USF "upstream stimulatory E boxes "E1 and E2). Systematic mutagenesis of the factor) [28]. rat insulin I promoter and transfection studies led to the identification of two closely related repeats, A boxes "A1-A5). Additional prominent regulatory found to play an important role in beta cell-specific elements, containing AT rich sequences, are the A expression [17]. These elements, located between boxes "A1-A5) [11]. Except for A2 "GGAAAT), ±104 and ±112 "IEB1/E1) and between ±233 and they all contain the core TAAT sequence which binds ±241 "IEB2/E2), contain the core sequence belonging factors belonging to the homeodomain-containing to the class of regulatory motifs designated E boxes, protein family. Whereas the A1 and A3 boxes are the sharing the consensus sequence CANNTG. Similarly, most conserved, mutated A3 had a more dramatic ef- mutagenesis of the rat insulin II 5' flanking region fect on transcription [29±31]. These promoter ele- also showed that the conserved E1 motif "-100 to ments bind the pancreatic duodenal homeobox±1 ±91) is essential for selective expression of the gene "PDX-1) transcription factor, the mammalian homo- [18, 19]. As to the human insulin gene, a promoter logue of the Xenopus laevis XlHbox8 [32], previously fragment containing