The bHLH protein PTF1-p48 is essential for the formation of the exocrine and the correct spatial organization of the endocrine pancreas.

We have generated a mouse bearing a null allele of the gene encoding basic helix-loop-helix (bHLH) protein p48, the cell-specific DNA-binding subunit of hetero-oligomeric transcription factor PTF1 that directs the expression of genes in the exocrine pancreas. The null mutation, which establishes a lethal condition shortly after birth, leads to a complete absence of exocrine pancreatic tissue and its specific products, indicating that p48 is required for differentiation and/or proliferation of the exocrine cell lineage. p48 is so far the only developmental regulator known to be required exclusively for committing cells to an exocrine fate. The hormone secreting cells of all four endocrine lineages are present in the mesentery that normally harbors the pancreatic organ until day 16 of gestation. Toward the end of embryonic life, cells expressing endocrine functions are no longer detected at their original location but are now found to colonize the spleen, where they persist in a functional state until postnatal death of the organism occurs. These findings suggest that the presence of the exocrine pancreas is required for the correct spatial assembly of the endocrine pancreas and that, in its absence, endocrine cells are directed by default to the spleen, a site that, in some reptiles, harbors part of this particular cellular compartment.

Constitutive expression of the gene for the cell-specific p48 DNA-binding subunit of pancreas transcription factor 1 in cultured cells is under control of binding sites for transcription factors Sp1 and alphaCbf.

We have cloned and characterized the rat gene that encodes the p48 DNA-binding subunit of pancreas transcription factor 1 (Ptf1), a cell-specific basic region helix-loop-helix (bHLH) protein. The ptf1-p48 gene measures 1.8 kilobases in size and occurs as a single copy in the haploid genome. Run-on transcription assays suggest that this gene is subject to transcriptional control since no activity of its promoter is detected in nonproducing cells. The gene specifies two mRNAs that encode the same protein and originate from transcription initiation at alternative sites. Expression analysis of hybrid genes bearing deletions of the gene's 5'-flanking region fused to a reporter gene defines a promoter region within the gene-proximal 260 base pairs of DNA. The cis-acting elements that control promoter activity include binding sites for transcription factors Sp1 and alphaCbf, a 60-kDa CCAAT box-binding protein. The gene promoter, however, functions not only in exocrine pancreatic cells but also in cells of other origin. No cell-specific transcriptional control element was detected in as much as 10 kilobases of 5'-flanking region. We discuss models of how the cell-specific expression of the endogenous ptf1-p48 gene might be established during development of the animal.

The p48 DNA-binding subunit of transcription factor PTF1 is a new exocrine pancreas-specific basic helix-loop-helix protein.

We report the isolation of cDNA for the p48 DNA-binding subunit of the heterooligomeric transcription factor PTF1. A sequence analysis of the cDNA demonstrates that p48 is a new member of the family of basic helix-loop-helix (bHLH) transcription factors. The p48 bHLH domain shows striking amino acid sequence similarity with the bHLH domain of proteins that act as developmental regulators, including the twist gene product, myogenic factors and proteins involved in hematopoietic differentiation. We show that reduced p48 synthesis correlates with a diminished expression of genes encoding exocrine pancreas-specific functions. The synthesis of p48 mRNAs, and therefore also the protein, is restricted to cells of the exocrine pancreas in the adult and to the pancreatic primordium in the embryo. Thus the pancreas-specific DNA-binding activity of PTF1 originates from the synthesis of at least one cell-specific component rather than from a cell-specific assembly of more widely distributed proteins.

Binding sites for hepatocyte nuclear factor 3 beta or 3 gamma and pancreas transcription factor 1 are required for efficient expression of the gene encoding pancreatic alpha-amylase.

Efficient expression of genes under the control of alpha-amylase 2 5'-flanking sequences in exocrine pancreatic cells requires, in addition to the pancreas transcription factor 1 binding site (M. Cockell, B.J. Stevenson, M. Strubin, O. Hagenbüchle, and P. K. Wellauer, Mol. Cell. Biol. 9:2464-2476, 1989), another cis-acting element at positions -60 to -86. This DNA element, which contains an AT-rich core, site for nuclear proteins present not only in the pancreas but also in other tissues and cell lines derived from the endoderm. Purification of binding activities from pancreatic cells by DNA affinity chromatography reveals several distinct proteins ranging in size from 45 to 54 kDa (p45, p47/48, and p54). All of these proteins interact with the specific DNA sequence upon renaturation in vitro. Protein sequencing, electrophoretic mobility shift assay, and immunoblot analyses identify p54 and p47/48 as members of the hepatocyte nuclear factor 3 (HNF3 [forkhead]) family of transcription factors. p54 belongs to the subfamily of HNF3 beta proteins, while p47/48 binding activity includes HNF3 gamma. The cDNAs for two HNF3 beta proteins differing only in N-terminal amino acid sequences were isolated from a pancreatic cDNA library. The mRNAs encoding the two protein species accumulate to different steady-state levels in poly(A)+ RNA of pancreatic cells. Our results support a model by which the pancreas-specific expression of the alpha-amylase gene is mediated by a combination of cell-specific and cell lineage-specific transcription factors.

A rapid method for the isolation of DNA-binding proteins from purified nuclei of tissues and cells in culture.

We describe a rapid and general method for isolating DNA-binding proteins in high yield from purified nuclei of animal cells. The method has been tested for the isolation of a series of different DNA-binding activities including those of transcription factors PTF1 and SP1. The rationale consists of first preparing purified nuclei from tissue or cells in culture by centrifugation over sucrose cushions. A synthetic, biotinylated oligonucleotide bearing the binding site for the protein of interest is then added directly to nuclei resuspended in binding buffer. At the end of the binding reaction, nuclei are removed by centrifugation; and protein-DNA complexes present in the postnuclear supernatant are attached to streptavidin-agarose. Two rounds of DNA-affinity chromatography are carried out to yield highly purified preparations of DNA-binding proteins.

Nuclear targeting of the transcription factor PTF1 is mediated by a protein subunit that does not bind to the PTF1 cognate sequence.

The pancreas-specific transcription factor PTF1 is a heterooligomer that exists as two variants, alpha and beta, both of which bind DNA. The nucleus contains exclusively alpha while the cytoplasm contains both forms. Alpha and beta differ in protein composition. Reconstitution of alpha in vitro requires, in addition to the DNA-binding subunits common to both forms, a 75 kd glycosylated protein that apparently does not bind DNA. Here we show that this protein is essential for targeting PTF1 to the nucleus. Upon injection into frog oocytes, alpha is translocated quantitatively to the nucleus while beta remains in the cytoplasm. However, if beta is coinjected with purified 75 kd protein or a particular size fraction of pancreatic mRNA, it can be converted to alpha and imported into the nucleus.

The DNA-binding activity of transcription factor PTF1 parallels the synthesis of pancreas-specific mRNAs during mouse development.

We have studied the expression of the alpha-amylase, trypsin, and elastase II genes in the acinar pancreas during mouse development. Transcriptional control is the major mechanism by which the differential accumulation of alpha-amylase, trypsin, and elastase II mRNAs is determined during late embryogenesis. The synthesis of pancreatic mRNAs is detected around day 15 of gestation and involves most if not all acinar cells. The DNA-binding activity of the pancreas-specific transcription factor PTF1, which binds to enhancers of genes expressed in this tissue, is detected for the first time at day 15 of gestation. The appearance of the factor at this early stage of development suggests that it plays an important role during pancreas differentiation.

The cell-specific transcription factor PTF1 contains two different subunits that interact with the DNA.

The cognate sequence of transcription factor PTF1, which plays a key role in pancreas-specific gene expression, has a bipartite organization. Two separate DNA domains, the A and the B boxes, are required for efficient binding of the factor. The structure of PTF1 was elucidated by cross-linking purified PTF1 to DNA templates that had been differentially substituted with azido-deoxyuridine (N3.dU). This site-directed UV cross-linking shows that PTF1 contains two DNA-binding proteins, distinct in size and sensitivity to Staphylococcus aureus V8 protease. A 64-kD protein is cross-linked with DNA containing N3.dU substitutions in the A box, and a 48-kD protein is cross-linked with DNA containing N3.dU substitutions in the B box. Both proteins bind simultaneously to the same DNA molecule. The data indicate that PTF1 is a heteromeric oligomer and that its cell-specific DNA-binding potential is the result of a concerted activity of two DNA-binding subunits.

Identification of a cell-specific DNA-binding activity that interacts with a transcriptional activator of genes expressed in the acinar pancreas.

Footprint analysis of the 5'-flanking regions of the alpha-amylase 2, elastase 2, and trypsina genes, which are expressed in the acinar pancreas, showed multiple sites of protein-DNA interaction for each gene. Competition experiments demonstrated that a region from each 5'-flanking region interacted with the same cell-specific DNA-binding activity. We show by in vitro binding assays that this DNA-binding activity also recognizes a sequence within the 5'-flanking regions of elastase 1, chymotrypsinogen B, carboxypeptidase A, and trypsind genes. Methylation interference and protection studies showed that the DNA-binding activity recognized a bipartite motif, the subelements of which were separated by integral helical turns of DNA. The alpha-amylase 2 cognate sequence was found to enhance in vivo transcription of its own promoter in a cell-specific manner, which identified the DNA-binding activity as a transcription factor (PTF 1). The observation that PTF 1 bound to DNA sequences that have been defined as transcriptional enhancers by others suggests that this factor is involved in the coordinate expression of genes transcribed in the acinar pancreas.

Sequence organisation and transcriptional regulation of the mouse elastase II and trypsin genes.

Elastase II and trypsin mRNAs were cloned in form of their cDNAs from pancreas of strain A/J mice, and their complete nucleotide sequences were determined. The elastase II mRNA is 912 nucleotides long and encodes a protein of 271 amino acids. The cloned trypsin mRNA species is 814 nucleotides long and encodes a protein of 246 amino acids. The elastase II gene, which exists as a single copy in the haploid mouse genome, measures 11.2 kb from cap to poly(A) site and is interrupted by at least seven introns. Between 5 and 10 trypsin genes exist in the mouse genome. Five different trypsin genes, two of which are closely linked in a tail-to-tail manner, were studied in detail. They vary in size between 3.4 and 4.0kb, and all are interrupted by four introns. DNA sequence comparison of the elastase II, trypsin and Amy-2a alpha-amylase genes reveals a conserved 13 nucleotide motif in their 5'-flanking regions. The differential accumulation of the elastase II and trypsin mRNAs in the cytoplasm of the acinar pancreatic cell is regulated predominantly at the transcriptional level.

Expression of mouse Amy-2a alpha-amylase genes is regulated by strong pancreas-specific promoters.

Three types of Amy-2-related DNA sequences, Amy-2a I, Amy-2a II and Amy-X, exist in the genome of mice of the inbred strain A/J. Amy-2a I and Amy-X are single copy sequences. Amy-2a II occurs as three copies per haploid genome. DNA sequence analysis reveals that both classes of Amy-2a genes specify the same unique pancreatic alpha-amylase mRNA species, since they share common exon sequences. Four independently cloned Amy-2a II isolates were found to be identical in all regions sequenced. This suggests that most, if not all, chromosomal Amy-2a II copies are identical. Amy-X is presumably a pseudogene, since its exon sequences, which are distinct from those of Amy-2a, are not detected in pancreatic alpha-amylase mRNA. We have determined the transcriptional activities of the Amy-2a genes by mapping in vitro elongated nascent transcripts to Amy-2a restriction fragments. Transcription initiation occurs at or close to the cap site. The expression of Amy-2a in vivo is under control of strong promoters, which are active exclusively in the pancreas. The accumulation of alpha-amylase mRNA in cells of the exocrine pancreas is regulated mainly at the transcriptional level. We have searched for pancreatic transcripts of Amy-1a, which specifies both parotid gland and liver-type alpha-amylase mRNAs. Surprisingly, the weak Amy-1a promoter, which directs the synthesis of the mRNA containing the liver-type leader sequence, also is active in the pancreas and, hence, in all alpha-amylase-producing tissues.

Members of the Amy-2 alpha-amylase gene family of mouse strain CE/J contain duplicated 5' termini.

Several members of the Amy-2 alpha-amylase multigene family from the CE/J strain of mouse have been cloned in cosmid vectors. Structural analysis of these recombinants reveals that the cloned Amy-2 copies contain tandem 5' termini. The duplicated 5'-terminal elements, which lie upstream from the Amy-2 cap site, are separated from their Amy-2 homologues by about 8000 bases. The orientation of these 5' orphons is the same as that of Amy-2. Gene titration and cloning experiments suggest that at least four of the approximately 15 Amy-2 copies present in the CE/J genome contain 5' orphon elements. The extent of sequence homology between 5' orphons and their gene homologues has been determined by DNA sequence analysis. All the orphons are identical and contain the entire 185 base-pairs of the first exon, 49 base-pairs of the first intron and more than 400 base-pairs of the Amy-2 5' flanking region. Intron and flanking-region sequences of the orphons differ by about 20% from their Amy-2 counterparts, and the exon by about 8%. The TATA box and the cap site are conserved, while the ATG translation initiation signal is mutated to ATA in the orphon. No transcription initiation has been detected at the orphon cap site using run-off transcription in isolated pancreatic nuclei in vitro.

Multiple non-allelic genes encoding pancreatic alpha-amylase of mouse are expressed in a strain-specific fashion.

The number of active Amy-2 genes has been estimated in strain CE/J mice which produce four distinct electrophoretic forms of alpha-amylase in their pancreas. cDNA cloning and DNA sequence analysis discloses five distinct mRNA sequences which differ by approximately 1% of their nucleotides. Two of these mRNAs specify the same protein. Changes in the nucleotide sequences result in amino acid replacements that alter the net charges of the deduced proteins. This has allowed a tentative assignment of individual mRNAs to isozymes detected by electrophoresis. Quantitative Southern blot hybridization using a DNA probe specific for the first exon of Amy-2 reveals the presence of greater than 10 Amy-2 related sequences per haploid CE/J genome. Models which could account for the mouse strain-specific differences with respect to the number of pancreatic alpha-amylase isozymes and their variable but genetically determined quantitative ratios are discussed.

Termination of transcription in the mouse alpha-amylase gene Amy-2a occurs at multiple sites downstream of the polyadenylation site.

We have delimited the region of transcription termination in the alpha-amylase gene Amy-2a. Mapping of in vitro elongated nascent transcripts to Amy-2a restriction fragments indicates that transcription terminates in a region between 2.5 and 4 kb downstream of the polyadenylation site. These runoff transcription experiments, combined with S1 nuclease mapping of nuclear transcripts at steady state, suggest that transcription termination occurs at multiple sites.

Two promoters of different strengths control the transcription of the mouse alpha-amylase gene Amy-1a in the parotid gland and the liver.

We show that two promoters of different strengths are involved in the tissue-specific expression of the alpha-amylase gene Amy-1a in the parotid gland and the liver of mouse. The weaker of the two promoters directs the synthesis of mRNA with a liver-type leader sequence. This promoter is active in both tissues. A promoter that is about 30-fold stronger is exclusively active in the parotid, where it directs the synthesis of an mRNA with a parotid-specific leader sequence. Neither the parotid nor the liver promoter is used in tissues that do not contain cytoplasmic alpha-amylase mRNAs, such as brain, kidney, and spleen. Nuclear transcripts that are initiated several kilobases upstream of the parotid cap site are detected in several tissues. They are most abundant in brain, and are apparently not processed into alpha-amylase mRNA.

Tissue specific expression of mouse alpha-amylase genes.

Two distinct alpha-amylase genes, Amy-1a and Amy-2a, are expressed in the mouse strain A/J. Amy-1a and Amy-2a are interrupted by 10 and 9 introns, respectively. With the exception of the first Amy-1a intron, which has no counterpart in Amy-2a, introns are located at analogous positions within the two genes. Comparable exons of Amy-1a and Amy-2a are more highly conserved in sequence than analogous introns. Amy-2a specifies pancreatic alpha-amylase mRNA. Two apparently identical copies of this gene exist in the haploid mouse genome. The single copy of Amy-1a is expressed in a tissue specific fashion in the salivary gland and the liver. It specifies alpha-amylase mRNA with identical translated and 3' nontranslated sequences but different 5' nontranslated sequences in the two tissues. These different mRNAs are generated by tissue specific splicing events. S1 nuclease mapping of nuclear transcripts from salivary gland and liver suggests the presence of at least two promotors of different strength in Amy-1a. A strong promoter appears to be active in the salivary gland exclusively, while a weak promoter is apparently used in both the salivary gland and the liver. The data suggest that regulation of Amy-1a expression occurs primarily at the transcriptional level.

Mouse liver and salivary gland alpha-amylase mRNAs differ only in 5' non-translated sequences.

The sequence of 1,773-nucleotide major and 1,806-nucleotide minor mouse liver alpha-amylase mRNAs differ only with respect to approximately 30 additional residues at the extreme 5' terminus of the minor species. Comparison of the liver alpha-amylase mRNAs with their salivary gland counterpart reveals that these mRNAs share identical coding and 3'-noncoding sequences, but contain distinct 5'-terminal residues. These data suggest that all three mRNAs might be transcribed from the same gene.