Galvin H. Swift

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.

PTF1 Is an Organ-Specific and Notch-Independent Basic Helix-Loop-Helix Complex Containing the Mammalian Suppressor of Hairless (RBP-J) or Its Paralogue, RBP-L

ABSTRACT PTF1 is a trimeric transcription factor essential to the development of the pancreas and to the maintenance of the differentiated state of the adult exocrine pancreas. It comprises a dimer of P48/PTF1a (a pancreas and neural restricted basic helix-loop-helix [bHLH] protein) and a class A bHLH protein, together with a third protein that we show can be either the mammalian Suppressor of Hairless (RBP-J) or its paralogue, RBP-L. In mature acinar cells, PTF1 exclusively contains the RBP-L isoform and is bound to the promoters of acinar specific genes. P48 interacts with the RBP subunit primarily through two short conserved tryptophan-containing motifs, similar to the motif of the Notch intracellular domain (NotchIC) that interacts with RBP-J. The transcriptional activities of the J and L forms of PTF1 are independent of Notch signaling, because P48 occupies the NotchIC docking site on RBP-J and RBP-L does not bind the NotchIC. Mutations that delete one or both of the RBP-interacting motifs of P48 eliminate RBP-binding and are associated with a human genetic disorder characterized by pancreatic and cerebellar agenesis, which indicates that the association of P48 and RBPs is required for proper embryonic development. The presence of related peptide motifs in other transcription factors indicates a broader Notch-independent function for RBPJ/SU(H).

An Endocrine-Exocrine Switch in the Activity of the Pancreatic Homeodomain Protein PDX1 through Formation of a Trimeric Complex with PBX1b and MRG1 (MEIS2)

ABSTRACT 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 β-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, β-cell lines are devoid of PBX1b and MRG1, so that a trimeric complex does not form, and the β-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 β-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.

Pancreatic-Duodenal Homeobox 1 Regulates Expression of Liver Receptor Homolog 1 during Pancreas Development

ABSTRACT Liver receptor homolog 1 (LRH-1) and pancreatic-duodenal homeobox 1 (PDX-1) are coexpressed in the pancreas during mouse embryonic development. Analysis of the regulatory region of the human LRH-1 gene demonstrated the presence of three functional binding sites for PDX-1. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis showed that PDX-1 bound to the LRH-1 promoter, both in cultured cells in vitro and during pancreatic development in vivo. Retroviral expression of PDX-1 in pancreatic cells induced the transcription of LRH-1, whereas reduced PDX-1 levels by RNA interference attenuated its expression. Consistent with direct regulation of LRH-1 expression by PDX-1, PDX-1−/− mice expressed smaller amounts of LRH-1 mRNA in the embryonic pancreas. Taken together, our data indicate that PDX-1 controls LRH-1 expression and identify LRH-1 as a novel downstream target in the PDX-1 regulatory cascade governing pancreatic development, differentiation, and function.

Transcriptional Autoregulation Controls Pancreatic Ptf1a Expression during Development and Adulthood

ABSTRACT The basic helix-loop-helix (bHLH) transcription factor PTF1a is critical to the development of the embryonic pancreas. It is required early for the formation of the undifferentiated tubular epithelium of the nascent pancreatic rudiment and then becomes restricted to the differentiating acinar cells, where it directs the transcriptional activation of the secretory digestive enzyme genes. Here we report that the complex temporal and spatial expression of Ptf1a is controlled by at least three separable gene-flanking regions. A 14.8-kb control domain immediately downstream of the last Ptf1a exon is highly conserved among mammals and directs expression in the dorsal part of the spinal cord but has very little activity in the embryonic or neonatal pancreas. A 13.4-kb proximal promoter domain initiates limited expression in cells that begin the acinar differentiation program. The activity of the proximal promoter domain is complemented by an adjacent 2.3-kb autoregulatory enhancer that is able to activate a heterologous minimal promoter with high-level penetrance in the pancreases of transgenic mice. During embryonic development, the enhancer initiates expression in the early precursor epithelium and then superinduces expression in acinar cells at the onset of their development. The enhancer contains two evolutionarily conserved binding sites for the active form of PTF1a, a trimeric complex composed of PTF1a, one of the common bHLH E proteins, and either RBPJ or RBPJL. The two sites are essential for acinar cell-specific transcription in transfected cell lines and mice. In mature acinar cells, the enhancer and PTF1a establish an autoregulatory loop that reinforces and maintains Ptf1a expression. Indeed, the trimeric PTF1 complex forms dual autoregulatory loops with the Ptf1a and Rbpjl genes that may maintain the stable phenotype of pancreatic acinar cells.

LRH-1 and PTF1-L coregulate an exocrine pancreas-specific transcriptional network for digestive function

We have determined the cistrome and transcriptome for the nuclear receptor liver receptor homolog-1 (LRH-1) in exocrine pancreas. Chromatin immunoprecipitation (ChIP)-seq and RNA-seq analyses reveal that LRH-1 directly induces expression of genes encoding digestive enzymes and secretory and mitochondrial proteins. LRH-1 cooperates with the pancreas transcription factor 1-L complex (PTF1-L) in regulating exocrine pancreas-specific gene expression. Elimination of LRH-1 in adult mice reduced the concentration of several lipases and proteases in pancreatic fluid and impaired pancreatic fluid secretion in response to cholecystokinin. Thus, LRH-1 is a key regulator of the exocrine pancreas-specific transcriptional network required for the production and secretion of pancreatic fluid.

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.

Replacement of Rbpj With Rbpjl in the PTF1 Complex Controls the Final Maturation of Pancreatic Acinar Cells

BACKGROUND & AIMS The mature pancreatic acinar cell is dedicated to the production of very large amounts of digestive enzymes. The early stages of pancreatic development require the Rbpj form of the trimeric Pancreas Transcription Factor 1 complex (PTF1-J). As acinar development commences, Rbpjl gradually replaces Rbpj; in the mature pancreas, PTF1 contains Rbpjl (PTF1-L). We investigated whether PTF1-L controls the expression of genes that complete the final stage of acinar differentiation. METHODS We analyzed acinar development and transcription in mice with disrupted Rbpjl (Rbpjl(ko/ko) mice). We performed comprehensive analyses of the messenger RNA population and PTF1 target genes in pancreatic acinar cells from these and wild-type mice. RESULTS In Rbpjl(ko/ko) mice, acinar differentiation was incomplete and characterized by decreased expression (as much as 99%) of genes that encode digestive enzymes or proteins of regulated exocytosis and mitochondrial metabolism. Whereas PTF1-L bound regulatory sites of genes in normal adult pancreatic cells, the embryonic form (PTF1-J) persisted in the absence of Rbpjl and replaced PTF1-L; the extent of replacement determined gene expression levels. Loss of PTF1-L reduced expression (>2-fold) of only about 50 genes, 90% of which were direct targets of PTF1-L. The magnitude of the effects on individual digestive enzyme genes correlated with the developmental timing of gene activation. Absence of Rbpjl increased pancreatic expression of liver-restricted messenger RNA. CONCLUSIONS Replacement of Rbpj by Rbpjl in the PTF1 complex drives acinar differentiation by maximizing secretory protein synthesis, stimulating mitochondrial metabolism and cytoplasmic creatine-phosphate energy stores, completing the packaging and secretory apparatus, and maintaining acinar-cell homeostasis.

DNA sequence of a plasmid-encoded dihydrofolate reductase

SummaryThe sequence of the methotrexate-resistant dihydrofolate reductase (DHFR) gene borne by the plasmid R-388 was determined. The gene was subcloned and mapped by an in vitro mutagenesis method involving insertion of synthetic oligonucleotide decamers encoding the BamHI recognition site. Sites of insertion that destroyed the methotrexate resistance fell in two regions separated by 300 bp within a 1.2 kb fragment. One of these regions encodes a 78 amino acid polypeptide homologous to another drug-resistant DHFR. The second region essential for DHFR expression appears to be the promoter of the DHFR gene.

The acinar differentiation determinant PTF1A inhibits initiation of pancreatic ductal adenocarcinoma

Understanding the initiation and progression of pancreatic ductal adenocarcinoma (PDAC) may provide therapeutic strategies for this deadly disease. Recently, we and others made the surprising finding that PDAC and its preinvasive precursors, pancreatic intraepithelial neoplasia (PanIN), arise via reprogramming of mature acinar cells. We therefore hypothesized that the master regulator of acinar differentiation, PTF1A, could play a central role in suppressing PDAC initiation. In this study, we demonstrate that PTF1A expression is lost in both mouse and human PanINs, and that this downregulation is functionally imperative in mice for acinar reprogramming by oncogenic KRAS. Loss of Ptf1a alone is sufficient to induce acinar-to-ductal metaplasia, potentiate inflammation, and induce a KRAS-permissive, PDAC-like gene expression profile. As a result, Ptf1a-deficient acinar cells are dramatically sensitized to KRAS transformation, and reduced Ptf1a greatly accelerates development of invasive PDAC. Together, these data indicate that cell differentiation regulators constitute a new tumor suppressive mechanism in the pancreas. DOI: http://dx.doi.org/10.7554/eLife.07125.001