Abstract
BackgroundPancreas organogenesis is the result of well-orchestrated and balanced activities of transcription factors. The homeobox transcription factor PDX-1 plays a crucial role in the development and function of the pancreas, both in the maintenance of progenitor cells and in determination and maintenance of differentiated endocrine cells. However, the activity of homeobox transcription factors requires coordination with co-factors, such as PBX and MEIS proteins. PBX and MEIS proteins belong to the family of three amino acid loop extension (TALE) homeodomain proteins. In a previous study we found that PDX-1 negatively regulates the transcriptional activity of the ductal specific keratin 19 (Krt19). In this study, we investigate the role of different domains of PDX-1 and elucidate the functional interplay of PDX-1 and MEIS1 necessary for Krt19 regulation.Methodology/Principal FindingsHere, we demonstrate that PDX-1 exerts a dual manner of regulation of Krt19 transcriptional activity. Deletion studies highlight that the NH2-terminus of PDX-1 is functionally relevant for the down-regulation of Krt19, as it is required for DNA binding of PDX-1 to the Krt19 promoter. Moreover, this effect occurs independently of PBX. Second, we provide insight on how PDX-1 regulates the Hox co-factor MEIS1 post-transcriptionally. We find specific binding of MEIS1 and MEIS2 to the Krt19 promoter using IP-EMSA, and siRNA mediated silencing of Meis1, but not Meis2, reduces transcriptional activation of Krt19 in primary pancreatic ductal cells. Over-expression of PDX-1 leads to a decreased level of MEIS1 protein, and this decrease is prevented by inhibition of the proteasome.Conclusions/SignificanceTaken together, our data provide evidence for a dual mechanism of how PDX-1 negatively regulates Krt19 ductal specific gene expression. These findings imply that transcription factors may efficiently regulate target gene expression through diverse, non-redundant mechanisms.
Highlights
The pancreas is a multifunctional organ that comprises an endocrine compartment, which regulates glucose homeostasis mainly through insulin secretion and is responsible for the secretion of other hormones, and an exocrine compartment, where acinar cells produce digestive enzymes that are secreted into the intestine via a network of pancreatic ducts
To characterize the PDX-1 domains required for keratin 19 (Krt19) repression we generated PDX-1 mutants with deletions of the NH2- terminus (D1–37, D1–109 and D1–144), deletions of the COOH-terminus (D260–284 and D235–284) and an internal mutation of the amino acid sequence FPWMK to AAGGQ, thereby abrogating possible interaction of PDX-1 with PBX proteins in the latter PDX-1 mutant (Fig. 1A)
Since homeobox proteins are capable of interacting with members of the three amino acid loop extension (TALE) homeodomain protein family, we evaluated for possible protein-protein interactions between PDX-1 and MEIS1a by co-transfection of HEK 293T cells
Summary
The pancreas is a multifunctional organ that comprises an endocrine compartment, which regulates glucose homeostasis mainly through insulin secretion and is responsible for the secretion of other hormones, and an exocrine compartment, where acinar cells produce digestive enzymes that are secreted into the intestine via a network of pancreatic ducts. The Pancreatic and Duodenal homeobox protein PDX-1 is critical for pancreatic development It is expressed in the foregut endoderm at E8.5, prior to the onset of bud formation and embryonic deletion of PDX-1 results in pancreatic agenesis [5]. In addition to its crucial functions during development, PDX-1 is required for accurate endocrine function by regulating endocrine gene expression [6] and b-cell survival in the adult [7]. The homeobox transcription factor PDX-1 plays a crucial role in the development and function of the pancreas, both in the maintenance of progenitor cells and in determination and maintenance of differentiated endocrine cells. The activity of homeobox transcription factors requires coordination with co-factors, such as PBX and MEIS proteins. In a previous study we found that PDX-1 negatively regulates the transcriptional activity of the ductal specific keratin 19 (Krt). We investigate the role of different domains of PDX-1 and elucidate the functional interplay of PDX-1 and MEIS1 necessary for Krt regulation
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