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

The elastase I (EI) 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 EI 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, PTF1. 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.

Cooperation between elements of an organ-specific transcriptional enhancer in animals.

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 rat elastase I regulatory element is an enhancer that directs correct cell specificity and developmental onset of expression in transgenic mice.

A total of 134 base pairs of the 5' flanking sequence of the elastase I gene is sufficient and necessary to direct expression of the passive human growth hormone gene (hGH) to the exocrine pancreas. We demonstrate that this elastase I regulatory region contains a transcriptional enhancer which directs acinar cell-specific expression in transgenic animals. The elastase I enhancer specifies correct expression of the linked hGH gene in an orientation- and position-independent manner and can activate a heterologous promoter. The enhancer also directs the appropriate temporal activation of the hGH gene in the developing pancreas. Transcription is initiated correctly for the elastase I or hGH promoter, and the transcripts are correctly processed regardless of the enhancer position within or outside the fusion gene. The elastase I enhancer generates coincident DNase I-hypersensitive sites in pancreatic chromatin when moved 3 kilobases upstream or within the first intron of the hGH gene and when associated with the hGH promoter.

An endocrine-exocrine switch in the activity of the pancreatic homeodomain protein PDX1 through formation of a trimeric complex with PBX1b and MRG1 (MEIS2).

HOX proteins and some orphan homeodomain proteins form complexes with either PBX or MEIS subclasses of homeodomain proteins. This interaction can increase the binding specificity and transcriptional effectiveness of the HOX partner. Here we show that specific members of both PBX and MEIS subclasses form a multimeric complex with the pancreatic homeodomain protein PDX1 and switch the nature of its transcriptional activity. The two activities of PDX1 are exhibited through the 10-bp B element of the transcriptional enhancer of the pancreatic elastase I gene (ELA1). In pancreatic acinar cells the activity of the B element requires other elements of the ELA1 enhancer; in beta-cells the B element can activate a promoter in the absence of other enhancer elements. In acinar cell lines the activity is mediated by a complex comprising PDX1, PBX1b, and MRG1 (MEIS2). In contrast, beta-cell lines are devoid of PBX1b and MRG1, so that a trimeric complex does not form, and the beta-cell-type activity is mediated by PDX1 without PBX1b and MRG1. The presence of specific nuclear isoforms of PBX and MEIS is precisely regulated in a cell-type-specific manner. The beta-cell-type activity can be detected in acinar cells if the B element is altered to retain binding of PDX1 but prevent binding of the PDX1-PBX1b-MRG1 complex. These observations suggest that association with PBX and MEIS partners controls the nature of the transcriptional activity of the organ-specific PDX1 transcription factor in exocrine versus endocrine cells.

Analysis of transcriptional regulatory regions in vivo.

Understanding the transcriptional regulation of development and tissue-specific gene expression is a central goal of modern biology. Although the analysis of gene transcription in transfected cultured cells has been essential in establishing many key aspects of this gene control, only analysis in animals can determine developmental timing and cell-specificity of expression within a complex organ and in all the tissues of an animal. The advent of transgenesis made in vivo studies possible. A summary of the in vivo regulatory properties of the pancreas-specific transcriptional enhancer of the rat elastase 1 gene (ELA1) and the role individual elements in this enhancer play in directing high level, cell-specific transcription illustrates the nature, revelations and limitations of transgenic analysis.

Assessment of RNA quality by semi-quantitative RT-PCR of multiple regions of a long ubiquitous mRNA.

A simple method to assess the degree of degradation present in a total RNA preparation from cells or tissues is based on the increasing probability of RNA cleavage with increasing length of an RNA molecule. Under ideal conditions, reverse transcription of a particular mRNA species with oligo-dT as the primer generates a population of cDNAs, terminating at the 5' end of the mRNA if all template RNA molecules are intact, or at the first cleavage site 5' to the polyA if some template RNAs are partially degraded. Consequently, for cellular RNA preparations with some degradation, the 5' end of an mRNA is represented in the cDNA population to a lesser extent than the 3' end of the mRNA. We describe a sensitive assay of mRNA quality that compares the relative PCR amplification of 5' and 3' regions of a long and ubiquitous mRNA following oligo-dT-primed reverse transcription.

Rat pancreatic ribonuclease messenger RNA. The nucleotide sequence of the entire mRNA and the derived amino acid sequence of the pre-enzyme.

We have cloned via recombinant DNA technology the mRNA sequence of rat pancreatic ribonuclease, and have determined the entire nucleotide sequence of the mature message. Clones bearing RNase sequences within a double-stranded complementary DNA library of rat pancreatic mRNA were initially detected by hybridization with size-fractionated rat pancreatic polyadenylated RNA that included mRNA 0.85 to 1.0 kilobase in length. Recombinant plasmids bearing RNase mRNA sequences were conclusively identified by comparison of the amino acid sequence of the encoded protein with the known amino acid sequence of rat RNase. RNase mRNA is 783 nucleotides in length, plus a poly(a) tail with an average length of 140 nucleotides, and contains long 5' and 3' noncoding regions relative to other pancreatic mRNAs. It encodes a secretory preRNase of 152 amino acid residues including a signal peptide of 25 amino acids.

An element of the elastase I enhancer is an overlapping bipartite binding site activated by a heteromeric factor.

The B element of the elastase I transcriptional enhancer is active in both exocrine and endocrine cells of the pancreas. Cell transfection experiments revealed that in an acinar cell line the active sequence of the element is more extensive than in an endocrine cell line. Electrophoretic mobility shift assays identified three major complexes (designated C, M, and L) from acinar cell nuclear extracts bound to the element. The C complex appears to be responsible for the activity of the element in acinar cells because its binding site, determined by methylation interference and mobility shift competition experiments, matches the critical sequence identified by cell transfection analysis of mutated B elements. The DNA sequence requirements for formation of the C complex is the sum of those for the M and L complexes. Methylation interference experiments indicated that the sensitivity of the C complex to guanine methylation also was the sum of that of the M and L complexes. Diagonal electrophoretic mobility shift assays confirmed that L is a component of complex C. However, the M complex, which we identified as GATA-4, is not part of the C complex, because the C complex was neither competed by GATA-binding sites nor super-shifted by anti-GATA-4 antiserum. Both the C and L complexes are specific to the pancreatic acinar cell line.

DNA binding and transcriptional activation by a PDX1.PBX1b.MEIS2b trimer and cooperation with a pancreas-specific basic helix-loop-helix complex.

In pancreatic acinar cells, the HOX-like factor PDX1 acts as part of a trimeric complex with two TALE class homeodomain factors, PBX1b and MEIS2b. The complex binds to overlapping half-sites for PDX1 and PBX. The trimeric complex activates transcription in cells to a level about an order of magnitude greater than PDX1 alone. The N-terminal PDX1 activation domain is required for detectable transcriptional activity of the complex, even though PDX1 truncations bearing only the PDX1 C-terminal homeodomain and pentapeptide motifs can still participate in forming the trimeric complex. The conserved N-terminal PBC-B domain of PBX, as well as its homeodomain, is required for both complex formation and transcriptional activity. Only the N-terminal region of MEIS2, including the conserved MEIS domains, is required for formation of a trimer on DNA and transcriptional activity: the MEIS homeodomain is dispensable. The activity of the pancreas-specific ELA1 enhancer requires the cooperation of the trimer-binding element and a nearby element that binds the pancreatic transcription factor PTF1. We show that the PDX1. PBX1b.MEIS2b complex cooperates with the PTF1 basic helix-loop-helix complex to activate an ELA1 minienhancer in HeLa cells and that this cooperation requires all three homeoprotein subunits, including the PDX1 activation domain.

Two similar but nonallelic rat pancreatic trypsinogens. Nucleotide sequences of the cloned cDNAs.

We have cloned and identified mRNA sequences for two rat pancreatic trypsinogens. Nucleotide sequence analysis of the cloned sequences revealed two mRNAs that encode similar, though noallelic, pretrypsinogens. Trypsinogen I mRNA is 804 nucleotides in length, plus an estimated poly(A) tract of 100 nucleotides, and contains a short (13 nucleotide) 5' noncoding region and a 3' noncoding region of 54 nucleotides. It encodes a preproenzyme of 246 amino acids comprising a hydrophobic prepeptide (signal peptide) of 15 amino acids, an activation peptide characteristic of trypsinogens, and an active form of trypsin, 223 amino acids in length, that has 78% amino acid sequence identity with porcine trypsin. Trypsinogen II mRNA has a nucleotide sequence 88% homologous with that of trypsinogen I mRNA and encodes a protein with 89% amino acid sequence identity with trypsinogen I. The enzymes encoded by trypsinogen I and II mRNAs retain the key amino acid residues that determine the characteristic substrate cleavage preference of trypsins and, therefore, represent the rat counterparts of this digestive enzyme. Trypsinogen I mRNA is a major pancreatic mRNA comprising an estimated 2-5% of the total, whereas trypsinogen II mRNA is present at much lower levels.

Tissue-specific expression of the rat pancreatic elastase I gene in transgenic mice.

The gene for rat pancreatic elastase I is selectively expressed to high levels in the rat exocrine pancreas. When the cloned rat elastase I gene with 7 kb upstream and 5 kb downstream flanking sequences was introduced into mice by microinjection into fertilized eggs, the gene was expressed in a pancreas-specific manner. In four of five transgenic mice, the level of rat elastase I mRNA in the pancreas was equal to or greater than the normal rat level (10,000 mRNAs per cell) and correlated with the number of integrated gene copies. In nonpancreatic tissues the levels were at least 10(3)-fold lower, except for expression in the liver of one mouse. Thus transfer of a 23 kb genomic DNA segment containing the rat elastase I gene to a foreign chromosomal location in the mouse can give rise to qualitatively and quantitatively normal expression.

Differential requirements for cell-specific elastase I enhancer domains in transfected cells and transgenic mice.

The 134-bp enhancer region of the pancreatic elastase I gene is sufficient to direct pancreatic acinar cell-specific transcription in transgenic mice and in transfected cells in culture. Ten-base-pair scanner mutations in three separate enhancer domains that inactivate enhancer function in transfected pancreatic cells in culture have no significant effect in transgenic mice. Because any pair of the three domains is sufficient to direct pancreas-specific expression in mice, no one domain is required for pancreas-specific transcription. Disruption of any two domains does inactivate the enhancer function in transgenic mice. Therefore, the elastase I enhancer domains essential for function in transfected cells in culture are not essential in animals but have a redundant function not apparent in transfected cells. This redundant function is not because of the particular acinar cell line used for transfections, the nature of the reporter gene, or the state of integration of the foreign test gene. We conclude that a trans-acting transcription factor or a modification of a factor(s) present in pancreatic cells of an animal is absent in pancreatic acinar cell lines.

A binary mechanism for the selective action of a pancreatic beta -cell transcriptional silencer.

The pancreatic elastase I gene (ELA1) is selectively transcribed to high levels in pancreatic acinar cells. Pancreatic specificity is imparted by a 100-base pair enhancer that activates transcription in beta-cells of the islets of Langerhans as well as in acinar cells. Adjacent to the enhancer is a silencer that renders transcription specific to acinar cells by selectively suppressing the inherent beta-cell activity of the enhancer. We show that the selective repression of beta-cell transcription is due neither to a beta-cell specific activity of the silencer nor to selective interference with beta-cell-specific transcriptional activators acting on the enhancer. Rather, the silencer is effective in both pancreatic endocrine and acinar cell types against all low and moderate strength enhancers and promoters tested. The silencer appears to act in a binary manner by reducing the probability that a promoter will be active without affecting the rate of transcription from active promoters. We propose that the ELA1 silencer is a weak off switch capable of inactivating enhancer/promoter combinations whose strength is below a threshold level but ineffective against stronger enhancer/promoters. The apparent cell-specific effects on the ELA1 enhancer appear due to the ability of the silencer to inactivate the weak beta-cell activity of the enhancer but not the stronger acinar cell activity.

An endocrine-specific element is an integral component of an exocrine-specific pancreatic enhancer.

We have analyzed the function of individual elements of the elastase I transcriptional enhancer in transgenic animals. This pancreas-specific enhancer comprises three functional elements, one of which (the B element) plays a dual role. Within the context of the enhancer, the B element contributes to appropriate acinar cell expression. However, when separated from the other enhancer components, the B element selectively directs transcription in islet cells of transgenic animals. This islet-specific activity is normally suppressed by an upstream repressor domain. The B element binds a novel islet-specific factor, and similar B-like elements are present in other pancreatic genes, both exocrine and endocrine specific. We suggest that a principal role of this transcriptional element and its associated factors is to activate many pancreatic genes as part of the program of pancreatic determination prior to the divergence of the acinar and islet cell lineages.

Primary structure of two distinct rat pancreatic preproelastases determined by sequence analysis of the complete cloned messenger ribonucleic acid sequences.

The mRNA sequences for two rat pancreatic elastolytic enzymes have been cloned by recombinant DNA technology and their nucleotide sequences determined. Rat elastase I mRNA is 1113 nucleotides in length, plus a poly(A) tail, and encodes a preproelastase of 266 amino acids. The amino acid sequence of the predicted active form of rat elastase I is 84% homologous to porcine elastase 1. Key amino acid residues involved in determining substrate specificity of porcine elastase 1 are retained in the rat enzyme. The activation peptide of the zymogen does not appear related to that of other mammalian pancreatic serine proteases. The mRNA for elastase I is localized in the rough endoplasmic reticulum of acinar cells, as expected for the site of synthesis of an exocrine secretory enzyme. Rat elastase II mRNA is 910 nucleotides in length, plus a poly(A) tail, and encodes a preproenzyme of 271 amino acids. The amino acid sequence is more closely related to porcine elastase 1 (58% sequence identity) than to the other pancreatic serine proteases (33-39% sequence identity). Predictions of substrate preference based upon key amino acid residues that define the substrate binding cleft are consistent with the broad specificity observed for mammalian pancreatic elastase 2. The activation peptide is similar to that of the chymotrypsinogens and retains an N-terminal cysteine available to form a disulfide link to an internal conserved cysteine residue.

Specific expression of an elastase-human growth hormone fusion gene in pancreatic acinar cells of transgenic mice.

Transfection of genes into tissue culture cell lines has demonstrated that relatively short DNA sequences can allow expression of immunoglobulin, insulin and chymotrypsin genes in their appropriate cell types. A definitive test of cell-specific gene expression, however, requires testing genes in every possible cell type, an experiment performed easily by introducing the gene in question into the germ line of an animal. Transfer of intact genes into mice has demonstrated that a mouse immunoglobulin kappa gene is expressed specifically in B lymphocytes, a rat elastase I gene is expressed specifically in pancreas and a chicken transferrin gene is expressed preferentially in liver. Mouse metallothionein-growth hormone fusion genes introduced into mice are preferentially expressed in the liver, consistent with the expression of endogenous metallothionein genes, but initial experiments with beta-globin genes have not revealed proper regulation. To identify the DNA elements required for pancreas-specific expression of the rat elastase I gene, we joined the 5'-flanking region of this gene to the human growth hormone (hGH) structural gene and introduced the fusion gene into mice. Here we demonstrate that a fusion gene containing only 213 base pairs (bp) of elastase I gene sequence directs expression of hGH in pancreatic acinar cells.

Rat pancreatic kallikrein mRNA: nucleotide sequence and amino acid sequence of the encoded preproenzyme.

We have cloned via recombinant DNA technology the mRNA sequence for rat pancreatic preprokallikrein. Four cloned overlapping double-stranded cDNAs gave a continuous mRNA sequence of 867 nucleotides beginning within the 5'-noncoding region and extending to the poly(A) tail. The mRNA sequence reveals that pancreatic kallikrein is synthesized as a prezymogen of 265 amino acids, including a proposed secretory prepeptide of 17 amino acids and a proposed activation peptide of 11 amino acids. The activation peptide, although similar in length, is distinct from those of the other classes of pancreatic serine proteases. The amino acid sequence of the predicted active form of the enzyme is closely related to the partial sequences obtained for other kallikrein-like serine proteases including rat submaxillary gland kallikrein, pig pancreatic and submaxillary gland kallikreins, the gamma subunit of mouse nerve growth factor, and rat tonin. Key amino acid residues thought to be involved in the substrate-cleavage specificity of kallikreins are retained. Hybridization analysis showed relatively high levels of kallikrein mRNA in the rat pancreas, submaxillary and parotid glands, spleen, and kidney, indicating the active synthesis of kallikrein in these tissues.