Cooperation between Elements of an Organ-Specific Transcriptional Enhancer in Animals

Abstract

The elastase I gene enhancer that specifies high levels of pancreatic transcription comprises three functional elements (A, B, and C). When assayed individually in transgenic mice, homomultimers of A are acinar cell specific, those of B are islet specific, and those of C are inactive. To determine how the elements interact in the elastase I enhancer and to investigate further the role of the C element, we have examined the activity of the three possible combinations of synthetic double elements in transgenic animals. Combining the A and B elements reconstitutes the exocrine plus endocrine specificity of the intact enhancer with an increased activity in acinar cells compared with that in the A homomultimer. The B element therefore plays a dual role: in islet cells it is capable of activating transcription, whereas in acinar cells it is inactive alone but greatly augments the activity specified by the A element. The C element augments the activity of either the A or B element without affecting their pancreatic cell type specificity. The roles of each element were verified by examining the effects of mutational inactivation of each element within the context of the elastase I enhancer. These results demonstrated that when tested in animals, the individual enhancer elements can perform discrete, separable functions that combine additively for cell type specificity and cooperatively for the overall strength of a multielement stage-and site-specific transcriptional enhancer.The elastase I (EI) gene is one of the pancreatic digestive enzyme genes which are activated in a stage-and site-specific manner during development of the mammalian gut. EI mRNA is present at very high levels in pancreatic acinar cells, equivalent to approximately 1% of the mRNA population, and at 100-to 1,000-fold-lower levels in other parts of the gastrointestinal tract including the stomach, duodenum, and colon (16,26). EI mRNA first becomes detectable in the pancreas at about embryonic day 14 in rats (9), about 4 days after the appearance of the pancreatic bud on the developing gut. The transcriptional regulatory regions that confer this organ-and stage-specific expression lie immediately upstream of the EI structural gene (14,22). This region includes a 134-bp enhancer (Ϫ72 to Ϫ205) that is both necessary and sufficient for the spectrum of gastrointestinal transcription (8, 26) and a TATA box-containing promoter (ϩ8 to Ϫ71) that does not contribute to organ specificity (21, 26). Although organ specific, the isolated enhancer directs expression inappropriately to islet cells as well as acinar cells (14). The acinar cell specificity notable for the endogenous gene is enforced by a negative regulatory region immediately upstream of the enhancer (Ϫ206 to Ϫ501) that selectively suppresses islet cell expression.The EI transcriptional enhancer contains three functional elements (A, B, and C) of 12 to 25 bp each (26, 35). When multimerized, the A and B elements can direct the activation of a naive reporter gene in the pancreas of transgenic mice (14, ...

Exocrine specific expression of Connexin32 is dependent on the basic helix-loop-helix transcription factor Mist1

“…Mist1 Het mice were maintained on a C57BL/6 background. The elastasep-Mist1 mut basic mice were generated through standard pronuclear injection protocols using a -500 -+8 fragment of the rat elastase 1 promoter (Heller et al, 2001;Kruse et al, 1995). Mist1 mut basic mice express the Mist1 mut basic protein exclusively in pancreatic acinar cells.…”

Integration of Tetracycline Regulation into a Cell-specific Transcriptional Enhancer

“…15 and see below) or in transgenic animals (14). However, this promoter region can be activated by the addition of the EI enhancer (14), other enhancers (30), and artificial constructs of homomultimeric repeats of factor binding sites in both transfected cells (17) and transgenic animals (10,16,17). For the acinar 266-6 cells, the tetO-promoter construct was inactive even in the presence of cotransfected tTA.…”

“…Other enhancers appear to have much more flexible organizational requirements; individual elements play only incremental roles (5,6) and spacing requirements are much less stringent (7,8). When examined, the separate activities of these enhancers are evident in the individual elements (7)(8)(9)(10).…”

“…The 12-bp B element has a dual function; it directs expression itself selectively to pancreatic islet cells, whereas in acinar cells it augments the acinar specific activity of the A element approximately 20-fold. The 30-bp C element has no intrinsic ability to act on its own, but in combination with either A or B it augments their activity an order of magnitude without affecting cell specificity (10,17). Consequently, the specificity and strength of this enhancer is based on the assembly of individual elements of unique function.…”

“…1). By analyzing the elements individually as homomultimers or pairwise combinations of heteromultimers in transgenic mice, we have elucidated the role of each element in controlling the pancreas specificity exhibited by the complete enhancer (10,16,17). The 21-bp A element directs expression selectively to acinar cells.…”