As an initial step to identify transcriptional regulatory regions, we surveyed the nucleotide sequence of the mouse Ptf1a gene 15 kb upstream and downstream of the transcriptional start site for evolutionarily conserved areas (Fig. ). A highly conserved region (the 5′ enhancer) lies between −15.6 and −13.4 kb relative to the transcriptional start site, whereas the rest of the 5′-end-proximal flanking region (the promoter region) is relatively unconserved. The Ptf1a coding region is followed by a region of extensive conservation (the 3′ control region) that begins after the 3′ untranslated region of the Ptf1a mRNA and continues for 12.4 kb. More than one-third of this 3′ flanking sequence is highly conserved among species from zebrafish to mice.
FIG. 1. The flanking regions of the Ptf1a gene contain domains highly conserved among vertebrates. (A) Regions of the Ptf1a gene with phylogenetic conservation. (Top line) Schematic of the Ptf1a gene and approximately 15 kb of 5′ and 3′ flanking (more ...)
To determine whether Ptf1a
transcription may be controlled by the PTF1 trimeric complex, we searched the 30 kb of the gene and flanking regions for PTF1 binding sites conserved among vertebrates. Only two conserved PTF1 binding sites were found, and both were in the 5′ enhancer region (Fig. ). Each of these potential binding sites conforms to the known consensus sequence (5
), with an E box and the TC box separated by one helical turn of DNA. In the interaction of the PTF1 complex with DNA, the E protein-PTF1a dimer binds the E box while RBPJ or RBPJL binds the TC box (4
). The 5′ enhancer together with the promoter region was able to direct the expression of a reporter gene in pancreatic AR4-2J acinar cells but not in nonpancreatic HEK-293 cells (Fig. ). Despite the absence of discernible PTF1 binding sites, both the promoter region alone and the 3′ control region also were active in the pancreatic cells but not in the nonpancreatic cells. Thus, all three regions have potential pancreatic transcriptional activity.
FIG. 2. The 5′ enhancer, the promoter region, and the 3′ control region are active in pancreatic acinar cell lines, but only the 5′ enhancer is responsive to PTF1. (A) The 5′ enhancer together with the promoter region, the promoter (more ...)
To determine whether the PTF1 complex controls the transcriptional activities of the three control regions, 293 cells were transfected with expression plasmids for the PTF1 subunits along with reporter genes linked to the Ptf1a-flanking sequences (Fig. ). The 5′ enhancer region together with the promoter region was activated 2.5-fold by the ectopic expression of PTF1a. The addition of the other components of the complex (HEB, RBPJ, and RBPJL) did not increase the activity further, likely due to the presence of endogenous RBPJ and E proteins in 293 cells (data not shown). The promoter and 3′ control regions, however, were unresponsive to the PTF1 subunits.
The 5′ enhancer alone induced the expression of a reporter gene with a minimal promoter in the AR4-2J and 266-6 pancreatic acinar cell lines but not in 293 cells (Fig. ). To determine whether this activity depended on the presence of the two PTF1 binding sites, the TC box of each site was inactivated by three contiguous base changes (Fig. ). For each of the sites, the mutation of the TC box blocked the binding of the PTF1 complex in EMSAs (Fig. , lane 15). Nearly all of the enhancer activity in transfected acinar cells was lost when the proximal site was altered, about two-thirds of the activity was lost when the distal site was altered, and the activity was reduced to background levels when both sites were altered (Fig. ). In a similar fashion, the mutation of the E box associated with each TC box eliminated PTF1 complex binding in EMSAs (data not shown) and transcriptional activity in both acinar cell lines (Fig. ). In this transfection assay, therefore, both the bHLH binding and the RBPJ binding parts of the bipartite PTF1 sites were required for complete enhancer activity.
FIG. 3. The trimeric PTF1 complex binds PTF1 sites in EMSAs and in vivo. (A) Complexes formed on the proximal or distal PTF1 sites of the 5′ enhancer. Antibodies against PTF1a (αPTF1a), RBPJ, or RBPJL were tested for their ability to supershift (more ...)
To confirm that the 5′ enhancer is activated by the binding of PTF1 to the conserved PTF1 binding sites, reporter genes directed by either the 5′ enhancer or the 5′ enhancer with both TC boxes mutated were introduced into 293 cells, along with expression plasmids for the PTF1 subunits (Fig. ). The activity of the enhancer was dependent on the coexpression of PTF1a and the presence of an intact TC box. This requirement for a TC box also confirms that PTF1a is acting as part of a complete trimeric complex of HEB-PTF1a-RBPJ or HEB-PTF1a-RBPJL, because an HEB-PTF1a dimer can bind to an E box in the absence of a TC box but this heterodimer is transcriptionally ineffective (4
Trimeric PTF1 complexes formed with in vitro-synthesized subunits can bind to oligonucleotides matching the sequence of either the proximal or distal PTF1 binding site of the 5′ enhancer (Fig. ). The subunit compositions of the complexes were confirmed by the ability of antibodies against PTF1a, RBPJ, or RBPJL to supershift the EMSA bands. For both the HEB-PTF1a-RBPJ and HEB-PTF1a-RBPJL complexes, the in vitro-synthesized proteins bound the proximal binding site slightly more effectively than the distal binding site (Fig. , compare lanes 3 and 7). When nuclear extract from adult mouse pancreas tissue was incubated with either the proximal or distal PTF1 site, a trimeric PTF1 complex also formed (Fig. , lane 11). The results of antibody supershifts showed that for both sites, most if not all of the complex contained RBPJL (Fig. , compare lanes 13 and 14), even though RBPJ was also present in the nuclear extract (Fig. , lane 11, band 1) (4
). Therefore, in adult pancreatic acini, the predominant form of PTF1 capable of binding the PTF1 sites in the enhancer contains RBPJL rather than RBPJ.
To determine whether both sites are bound by the PTF1 complex in acinar nuclei, we performed ChIP of cross-linked, sheared chromatin from adult mouse pancreas tissue with antibodies specific for PTF1a, RBPJL, and RBPJ (Fig. ). The region containing the proximal PTF1 site was enriched 6.2- and 3.2-fold in chromatin immunoprecipitated with anti-PTF1a and anti-RBPJL, respectively. No enrichment was seen with anti-RBPJ, which is consistent with the results for PTF1 sites in genes for several pancreatic enzymes that also are bound by the RBPJL form exclusively (4
). The distal PTF1 site of the 5′ enhancer was enriched only slightly with antibodies to PTF1a and not enriched with antibodies specific for either RBPJ or RBPJL. Thus, the distal site is less important than the proximal site for activity in transfected acinar cells (Fig. ), binds the PTF1 complex to a lesser extent than the proximal site does (Fig. ), and is not occupied by a complete complex in vivo.
lacZ reporter transgenes were created with the 5′ enhancer, the promoter region, and the 3′ control region to examine the role of each region during pancreatic development. Founder embryos were collected at 17.5 days postcoitum (E17.5) and stained for β-galactosidase activity. When the transgene comprised the 5′ enhancer plus the promoter region, β-galactosidase was detectable in most pancreatic acinar cells of nearly all transgenic embryos (10 of 12) (Fig. ). The β-galactosidase activity was restricted to the acinar cells (Fig. ), where PTF1a is normally present at this stage, and a few scattered islet cells. The promoter region alone also directed acinar cell-specific expression, but only in about one-third of transgenic embryos and then only in a small minority of acinar cells (Fig. ). The 5′ enhancer was sufficient to direct acinar cell-specific β-galactosidase activity, although with lower penetrance (10 of 22 embryos) and fewer positive pancreatic cells than were obtained with the 5′ enhancer in combination with the promoter region (Fig. ). The dependence of the activity of the 5′ enhancer on the PTF1 binding sites was confirmed when mutations of both TC boxes were introduced into the transgene. None of the 24 transgenic embryos bearing the 5′ enhancer with TC box mutations had detectable β-galactosidase in the pancreas (Fig. ). The 3′ control region also directed pancreatic expression, but in very few and widely scattered cells (Fig. ).
FIG. 4. PTF1 sites in the Ptf1a promoter augment transcription in developing acinar cells. Ptf1a regions were ligated upstream of a lacZ reporter and introduced into fertilized mouse eggs. Embryos derived from the implanted oocytes were sacrificed at E17.5 and (more ...)
FIG. 5. Regions of the Ptf1a 5′ flanking sequence direct acinar cell-specific expression at E17.5 but no pancreatic expression at E10.5. (A to C) Transgenic embryos at E17.5 were fixed and stained to detect β-galactosidase (Fig. (more ...)
None of the lacZ
-based transgenes with Ptf1a
-flanking regions were active in early pancreatic development, prior to the appearance of acinar cells (Fig. ). At E10.5, the transgenes directed by the 5′ enhancer and the promoter region, the promoter region alone, the 5′ enhancer alone, or the 3′ control region failed to be expressed in the pancreas, although each was active in the spinal cord, a known site of Ptf1a
). The endogenous Ptf1a
locus is active in both the pancreas and the spinal cord at this developmental stage (Fig. ).
Expression of the transgenic β-galactosidase reporter was first detected in a few scattered cells at E13.5 (Fig. and Table ) and in many more cells, often in distinct acinar rosettes, by E14.5 (Fig. ). As development progresses, a greater fraction of transgenic embryos and acinar cells activate the enhancer. Cells detected by immunofluorescence analysis for β-galactosidase (Fig. ) were also positive for PTF1a (Fig. ). Many of the β-galactosidase-containing cells at E13.5 also had the early acinar marker CPA1 (Fig. ). At E14.5, emerging acinar cells were much more prominent and most β-galactosidase-positive cells were associated with CPA1-positive acini (Fig. , inset). Like PTF1a, transgenic β-galactosidase was excluded from the glucagon-containing cells, which are the prevalent endocrine cells at this developmental stage (see, e.g., Fig. ), and from the insulin-containing cells (data not shown). These results indicate that the 5′ enhancer is active in cells beginning the acinar developmental program.
FIG. 6. Activation of the isolated 5′ enhancer at the onset of acinar cell development. (A) Scattered cells of an E13.5 embryo stained for β-galactosidase (β-gal) are present near the periphery of the pancreatic epithelium (outlined) lying (more ...)
Summary of the activity of the 5′ enhancer during embryonic pancreatic development
To investigate more thoroughly the activity of the enhancer during early development, we examined the expression of the 5′ enhancer driving an EGFP reporter in embryos of two independently derived transgenic lines (rather than founder embryos). In contrast to the lacZ reporter construct in founder embryos prior to E13.5, which lacked detectable expression, this construct was active at E10.5 (Fig. ) and E12.5 (Fig. ), prior to the emergence of acinar cells during the secondary transition. At E10.5, EGFP fluorescence was present in the epithelial cells of the ventral and dorsal buds. At E12.5, the activity of the enhancer increased around the periphery of the epithelium (Fig. ), in accordance with the accumulation of PTF1a protein in these proacinar crescents. At E14.5, enhancer activity was localized exclusively to PTF1a+ proacinar cells (Fig. ). Nonepithelial cells and the epithelial cells of nontransgenic embryos had no fluorescence over the background level. Expression levels in the precursor epithelium at E10.5 and E12.5 were much lower than those in nascent acinar cells at E14.5 (Fig. ), which may explain the absence of detectable activity of the lacZ-based enhancer transgene. In contrast to the construct comprising the 5′ enhancer and the green fluorescent protein reporter gene, which was active at E10.5, the 3′ control region with a fluorescent protein reporter gene was inactive at this stage in three transgenic lines, although fluorescence was intense in the neural tube and later in sparse cells of the E13.5 pancreas (data not shown).
FIG. 7. Activity of the 5′ enhancer in the epithelium of the early pancreatic buds. EGFP fluorescence is shown in green; PTF1a immunofluorescence is shown in red. (A) Expression of the 5′ enhancer transgene-EGFP reporter in the dorsal (dP) and (more ...)
The expression patterns for the transgenic enhancer-EGFP gene construct and the endogenous PTF1a detected by immunofluorescence were not congruent: the relative intensities of EGFP and anti-PTF1a fluorescence did not coincide for many of the expressing cells. Although the majority of the epithelial cells expressed both, there were cells with a high level of one but little or none of the other (Fig. ). The discrepancies may be due to the absence of control sequences outside the 2.3-kb enhancer fragment. As observed for the expression of the endogenous Ptf1a locus, our results show that the 5′ enhancer is active in the early pancreatic epithelium, albeit at low levels, and then superinduced in cells committed to the acinar fate.
To determine whether the activity of the 5′ enhancer in the precursor epithelium requires the early trimeric PTF1-J complex, we examined the expression of the EGFP gene-based enhancer transgene in homozygous Ptf1a
) embryos. The tryptophan-to-alanine substitution at position 298 eliminates the early developmental functions of PTF1a (14
) because the altered PTF1a cannot bind and recruit RBPJ into the trimeric complex effectively (4
). The effects of the W298A mutation on pancreatic development are identical to those of the Ptf1a
null mutation (14
): the pancreatic evaginations of the endoderm initiate, but the subsequent early growth and morphogenesis of the epithelium are curtailed beginning at about E10.5, and the secondary transition with its formation of islet and acinar tissues does not occur. At E10.5, the 5′ enhancer was inactive in the pancreatic epithelia of homozygous Ptf1a
) embryos (Fig. ), even though the endogenous Ptf1a
) locus was active and the W298A protein was present (Fig. ). Therefore, because this early activity of the enhancer requires the interaction between PTF1a and RBPJ, it must be due to autoregulation. Because the initial induction of Ptf1a
gene transcription cannot depend on preexisting PTF1a protein, the autoregulation of the enhancer must represent a maintenance function that follows an initial activation event. Furthermore, because the endogenous locus is active at E10.5 in Ptf1a
) pancreas tissue, but the isolated enhancer is not, critical regulatory information for this initial activation of Ptf1a
transcription must reside outside the 2.3-kb enhancer fragment.