Replacement of Rbpj with Rbpjl in the PTF1 complex controls the final maturation of pancreatic acinar cells

Abstract

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.

Figure 1

PTF1 regulation of acinar development. A. Initiation: The acinar lineage is initiated prior to the induction of Rbpjl. Ptf1a, Rbpj and a common E-protein form PTF1-J, which auto-activates Ptf1a transcription. Maturation: The early PTF1-J target-genes include Rbpjl as well as some of the secretory enzyme genes. The Rbpjl form of the PTF1-complex, PTF1-L, begins to accumulate and is more effective than PTF1-J on most of the secretory enzyme genes. PTF1-L establishes dual auto-activation loops that sustain high transcription rates of Ptf1a and Rbpjl. Maintenance: In the mature acinar pancreas, PTF1-L is the sole form, is sustained at high levels by autoregulation, and maintains a maximal acinar phenotype. B. In the absence of Rbpjl, PTF1-J persists, but is less effective on genes for secretory proteins and other select acinar proteins.

Figure 2

Engineered inactivation of Rbpjl. A. The structure and disruption of the mouse Rbpjl gene. Black and grey boxes indicate coding exons; white, 5’ and 3’ untranslated regions. A central Rbpjl gene region (grey exons) was exchanged for an nlacZ linked to a bGH 3’ untranslated region and polyA signal. B. rtPCR detection of the long or short forms of Rbpjl mRNA. C. Southern hybridization with NcoI-cleaved genomic DNA showed the loss of wild type (10-Kb) and gain of lacZ (4.1-Kb) alleles. D. The deletion of Rbpjl exons 7, 8 and 9 was confirmed by PCR amplification analysis. E. Q-rtPCR analysis of Matrilin4 mRNA from adult and E17.5 pancreases. The levels of Matn4 mRNA are relative to wild type adult pancreas levels.

Figure 3

Effects of Rbpjl inactivation. A. The pancreases of Rbpjl^ko/ko mice at 8-weeks or 8-months of age were one-third smaller than those of age-matched Rbpjl^+/ko mice. B. RNA and DNA content of newborn pancreases. The numbers of mice analyzed for each genotype are indicated (*p<0.05; **p<0.01). Error bars are SEMs.

Figure 4

Changes in pancreatic gene expression. A. Rbpjl inactivation decreases the levels of the mRNAs encoding the acinar digestive enzymes. The mRNA levels for E17.5 pancreas were quantified by Q-rtPCR and are expressed relative to the level of the mRNA in normal E17.5 pancreas (*P<0.05; **p<0.01; ***p<0.001). Error bars are SDs. The black bars are positioned at the known developmental age of the appearance of the mRNA in embryonic rat pancreas . The midpoint of the first decade of mRNA accumulation was taken as a measure of the time of appearance. The remaining mRNAs (grey bars) are placed relative to the known mRNAs according to their levels in Rbpjl^ko/ko pancreas, in prediction of the timing of their appearance. B. Results of the genome-wide analysis of pancreatic mRNAs for Rbpjl^ko/ko and Rbpjl^+/+ embryos at E17.5 by RNA-Seq. Grey bars indicate mRNAs measured by Q-rtPCR as well in panel A.

Figure 5

The absence of Rbpjl partly depletes PTF1a from target promoters (A) and leads to detectable Rbpj (B) on the same promoters in adult pancreatic chromatin (*P<0.05, **P<0.01). in (input) is the result using DNA from chromatin without immunoprecipitation and represents no enrichment. The amounts of Ptf1a and Rbpj on these promoters were unchanged between Rbpjl^+/+ and Rbpjl^+/ko mice, and neither Rbpjl nor β-galactosidase was detected on chromatin from Rbpjl^ko/ko mice (data not shown). Fisher’s method for combining the probabilities of multiple tests of a hypothesis provides a p value <0.001 that Ptf1a occupancy does not decrease (panel A) and a p value <0.01 that Rbpj occupancy does not increase (panel B) on PTF1 target genes in Rbpjl^ko/ko chromatin. Error bars are SEMs.

Figure 6

Chip-Seq and RNA-Seq results for examples of (A) a secretory enzyme (Ela1), (B) a secretory packaging protein (Zg16), and (C) a metabolic enzyme (Gatm). Shown for each, from top to bottom: ChIP-Seq for input DNA, Ptf1a-binding, and Rbpjl-binding; RNA-Seq (grey highlight) for E17.5d pancreas from Rbpjl^+/+ and Rbpjl^ko/ko embryos; the exon organization for the Ref-Seq mRNA; and the mammalian conservation from BLAT . The sequences of the PTF1 binding site within the peaks of Ptf1a and Rbpjl binding are given (capital letters, E- and TC-boxes). Scale bars indicate the peak height for the sequence tags. D. Mitochondrial metabolic pathways linking the functions of Gls2, Gatm, Tdh, Gcat and Bdh2.