Identification of PCIF1, a POZ domain protein that inhibits PDX-1 (MODY4) transcriptional activity.

Hox factors are evolutionarily conserved homeodomain-containing transcription factors that are critical for the determination of the body plan during early development and for the regulation of key differentiation-specific genes that determine cellular specificity. Proper spatial and temporal expression of Hox factors is required to orchestrate anterior-posterior segmentation, limb development, and organogenesis. Hox proteins regulate gene transcription by a well-coordinated and tightly regulated balance of gene activation and repression. In particular, the addition and refinement of repression domains have contributed to the evolutionary diversification of body segmentation (15, 37). Pancreatic-duodenal homeobox 1 (PDX-1; also known as IPF-1, IDX-1, STF-1, Xlhbox8, GSF, and IUF-1) is a Hox-type homeodomain protein encoded by a gene in the ParaHox cluster that is expressed in the region of the developing foregut that later becomes the duodenum, distal stomach, and pancreas (5, 24, 28, 29, 32, 46). As development proceeds, high-level PDX-1 expression is eventually restricted to the hormone-producing β and δ cells of the fully developed endocrine pancreas, whereas lower levels of expression are detected in certain ductal cells, the majority of exocrine acinar cells, and epithelial cells and Brunner's glands in the distal stomach and proximal duodenum. In the differentiated endocrine pancreas, PDX-1 regulates the transcription of important target genes, including the insulin, somatostatin, and glucose transporter 2 genes (reviewed in reference 14). Gene dosage has a marked influence on the phenotype of Pdx-1 mutations. Homozygous disruption of the Pdx-1 gene results in pancreatic agenesis in both mice and humans (23, 39), whereas heterozygous truncation and missense Pdx-1 (Ipf-1) mutations are associated with both early (MODY4)- and late-onset forms of type 2 diabetes in humans (18, 26, 40). Heterozygous Pdx1+/− mice exhibit glucose intolerance, impaired secretagogue-induced insulin secretion in vivo, abnormal islet architecture, increased islet apoptosis, and, in older mice, decreased β cell mass (4, 12, 22). Mice in which Pdx-1 is specifically inactivated in islet β cells by Cre-LoxP recombination develop diabetes with decreased insulin and Glut 2 expression and an increased proportion of α cells in the islets (1). Recent mouse models allowing conditional regulation of Pdx-1 expression also demonstrate the key role of proper levels of Pdx-1 expression throughout embryonic development and adult life (19, 43). PDX-1 is a 283-amino-acid (aa) protein with a central homeodomain flanked by proline-rich N- and C-terminal domains (24, 29). A transactivation domain maps to the N terminus in the context of insulin and somatostatin gene transcription, and deletion of the N terminus results in a dominant negative PDX-1 protein (25, 33). The functional synergy of PDX-1 with E box proteins, such as E12 and E47, to activate insulin gene transcription is mediated by the N terminus (33), and glucose augments PDX-1 transactivation via the N terminus as well (35). The coactivator CBP physically interacts with the N terminus (2). PDX-1 interacts with the homeoprotein PBX1 through a conserved pentapeptide motif, FPWMK, located N terminal to the homeodomain; this interaction is important for the proper proliferation and expansion of all of the differentiated cell types of the pancreas (13). Depending on the alternatively spliced isoform of PBX1 that is involved, corepressors may be recruited to the PDX-1/PBX1 heterodimer complex (2). The PDX-1 homeodomain contains the DNA binding domain as well as an atypical nuclear localization signal (30). Human mutations in the C-terminal region of PDX-1 are associated with the development of type 2 diabetes mellitus, emphasizing the functional importance of this region in vivo (18, 44). However, in contrast to the function of the N terminus and the homeodomain, that of the well-conserved C terminus is poorly understood. The available data support both repressive and activating roles for these sequences. C-terminal truncation of PDX-1 is associated with increased synergy with E47 on the insulin promoter (33). The PDX-1 C terminus also confers inhibition in the context of fusion to the heterologous Gal4 DNA binding domain (34, 38). In contrast, on the somatostatin TAAT1 enhancer, the PDX-1 C terminus may also contain a cryptic transactivation domain (25, 33). To gain insight into the role of the PDX-1 C terminus, we investigated it as a protein-protein interaction domain by yeast two-hybrid analysis. We identified a nuclear POZ domain protein that we named PCIF1 (PDX-1 C-terminal interacting factor). PCIF1 is a nuclear protein that interacts with PDX-1 both in vitro and in vivo and inhibits PDX-1 transactivation. In the adult pancreas, PCIF1 is expressed in the insulin-producing β cells and in a subset of exocrine acinar and duct cells. PCIF1 is the first protein identified to interact with the C-terminal domain of PDX-1, and it is the first partner that represses PDX-1 transactivation. The coexpression of PCIF1 with PDX-1 in the pancreas suggests that modulation of PDX-1 function by PCIF1 may be required for normal pancreas development and/or differentiation.