A single element of the elastase I enhancer is sufficient to direct transcription selectively to the pancreas and gut.

Abstract

The elastase I (El) gene is expressed at high levels in the exocrine pancreas and at lower levels in other regions of the gut. The transcriptional enhancer of the EI gene, from nucleotides -205 to -72, recapitulates the expression of the endogenous gene in transgenic mice; it directs not only pancreatic acinar cell expression of a human growth hormone (hGH) transgene but also expression to the stomach, duodenum, and colon. This pattern of selective expression limited to the gastroenteropancreatic organ system is specified by the A element, one of three functional elements in the El enhancer. When multimerized, the A element directed expression of a hGH reporter gene selectively to the pancreas, stomach, and intestine in transgenic mice. Immunofluorescent localization of hGH indicated that the A element multimer transgenes were expressed in the acinar cells of the pancreas as well as in Brunner's gland cells of the duodenum. The A element binds a pancreatic acinar cell-specific factor, PIF1. Our results show that while the A element is responsible for directing tissue and cell type specificity, other elements of the enhancer must be involved in the regulation of the level of gene expression.Cell-specific transcriptional enhancers are complex assemblies of multiple DNA elements and their bound transcription factors. To understand their mechanism of action, it is necessary to understand the role of the individual functional elements and their bound factors in controlling the cell type and strength of activation of the basal transcriptional machinery at the promoter. The tissue-specific transcriptional enhancer of the pancreatic elastase I (El) gene confers high-level expression in transgenic mice independent of its position relative to the promoter and with a heterologous promoter (15). The enhancer forms a tissue-specific chromatin DNase I-hypersensitive site indicative of a nucleoprotein transcription complex which follows the enhancer when it is moved in transgene constructs either near the promoter, far upstream, or within the reporter gene.The minimal region that retains these properties spans 134 bp (from -72 to +205) and is composed of three functional elements (A, B, and C). All three elements are necessary for expression in cultured pancreatic acinar cells (20) and important in pancreatic expression in transgenic mice (38). The A element comprises the pancreatic consensus element (PCE), a 17-bp sequence found in the 5' flanking region of other pancreatic acinar cell digestive enzyme genes as well (2,8,37 648-8856. amylase gene was used to partially purify a binding activity from pancreatic nuclear extracts (6). This binding activity, designated PTF1, is specific for DNA sequences containing a PCE (6, 16). PTF1 contains two DNA-binding proteins, a 48-kDa subunit which binds to the A box and a 64-kDa subunit which binds to the B box (34). The appearance of PTF1 during the development of the mouse pancreas coincides with the onset of expression of the digestive enzyme genes (32). These observations sugg...

Pancreas organogenesis: From bud to plexus to gland

“…The trimeric Pancreas Transcription Factor 1 complex (PTF1) was first identified as an acinar enzyme gene activator (Rose et al, 1994;Krapp et al, 1996) and comprises a tissuespecific bHLH protein, Ptf1a; an ubiquitous bHLH protein, E2A or HEB (formerly known as p75, which provides a constitutive nuclear import function for the PTF1 protein complex) (Rose et al, 1994, Krapp et al, 1996, Sommer et al, 1991; and the distinct mammalian Suppressor of Hairless (RBP-J) protein, or the RBP-JL paralog (Obata et al, 2001;Beres et al, 2006). Ptf1a is expressed as early as E9.5 in most cells of the nascent pancreatic buds, and is essential for pancreas organogenesis in mouse and human (Kawaguchi et al, 2002;Krapp et al, 1998;Sellick et al, 2004).…”

Maturation of Stem Cell-Derived Beta-cells Guided by the Expression of Urocortin 3

“…While it was discovered as an activator of acinar cell-specific genes [39], at this stage it helps define exocrine, endocrine, and ductal cell types within the pancreas by binding to and activating the PDX1 promoter [40]. Shortly after the specification of the pancreatic buds, further differentiation into pancreatic endoderm (PE) and subsequent endocrine cells is initiated by the expression of NK2 homeobox 2 (NKX2.2) [41] and NK6 homeobox 1 (NKX6.1) [42].…”

Integration of Tetracycline Regulation into a Cell-specific Transcriptional Enhancer

“…In animals, the specificity of the enhancer is the sum of that of the A and B elements, whereas its overall strength includes synergy between all three elements. In transfected pancreatic acinar tumor cells in culture, the elements play similar roles except that each element is essential, whereas in acinar tissue in situ the B and C elements are largely redundant (15,16).…”

“…In animals, the specificity of the enhancer is the sum of that of the A and B elements, whereas its overall strength includes synergy between all three elements. In transfected pancreatic acinar tumor cells in culture, the elements play similar roles except that each element is essential, whereas in acinar tissue in situ the B and C elements are largely redundant (15,16).Another strategy to probe the functional organization of complex enhancers is to substitute endogenous elements with an ectopic one to examine whether the enhancer complex is sufficiently flexible to integrate the function of the new element. In this report we describe the use of this strategy to test whether the tetracycline regulatory element, tetO, and its binding transactivator (tTA) (18) can integrate into the nucleoprotein complex of the EI enhancer to complement the transcriptional activities of the endogenous elements.…”

“…We have used this approach to dissect the functional elements of the transcriptional enhancer of the pancreatic elastase I (EI) 1 gene. The EI enhancer (14) comprises only three mutation-sensitive elements, termed A, B, and C (15,16) (Fig. 1).…”