Section 2: ␤-Cell : Functional Aspects Regulation of pdx-1 Expression Danielle Melloul, Sonya Marshak, and Erol Cerasi

The homeodomain-containing factor pan- tion of the endoderm, it is crucial for the development creatic duodenal homeobox 1 (PDX-1) plays a key role of endocrine and exocrine cell types (2,6). Differentiation in pancreas development and in ␤-cell function. Up- and maintenance of the ␤-cell phenotype also require stream sequences of the gene up to about ؊6 kb show PDX-1. In mice, ␤-cell–selective disruption of pdx-1 led to islet-specific activity in transgenic mice. Attempts to the development of diabetes with increasing age and was identify functional regulatory elements involved in the associated with reduced and GLUT2 expression controlled expression of the pdx-1 gene led to the (7). Indeed, mice heterozygous for pdx-1 were found to be ␤ identification of distinct distal -cell–specific enhanc- glucose intolerant (7,8). In transgenic mice expressing ers in human and rat genes. Three additional sequences, ؅ an antisense ribozyme specific for mouse pdx-1 in the conserved between the mouse and the human 5 -flank- ␤ ing regions, two of which are also found in the chicken -cells, the expression of the endogenous gene was de- gene, conferred ␤-cell–specific expression on a reporter creased and followed by impaired glucose tolerance and gene, albeit to different extents. A number of transcrip- elevated glycated hemoglobin levels (9). Impaired expres- tion factors binding to and modulating the transcrip- sion of PDX-1 as a consequence of hyperglycemia or tional activity of the regulatory elements were increased lipid concentrations (10) is associated with identified, such as hepatocyte nuclear factor (HNF)-3␤, diabetes. HNF-1␣, SP1/3, and, interestingly, PDX-1 itself. A fourth In humans, a subpopulation of type 2 diabetes is mono- conserved region was localized to the proximal pro- genic and carries in genes important for normal moter around an E-box motif and was found to bind ␤-cell function. Heterozygous individuals carrying one of members of the upstream stimulatory factor (USF) the mutant genes develop a form of maturity-onset diabe- family of transcription factors. We postulate that dis- ruption of pdx-1 cis-acting regulatory sequences and/or tes of the young (MODY). MODY4 has been linked to mutations or functional impairment of transcription heterozygosity for mutations in pdx-1 (11,12). Other mo- factors controlling the expression of the gene can lead nogenic forms of MODY have been associated with muta- to diabetes. Diabetes 51 (Suppl. 3):S320–S325, 2002 tions in genes coding for transcription factors hepatocyte nuclear factor (HNF)-1␣, HNF-1␤, HNF-4␣, and Beta2 (13), most of which will be described below as regulators of pdx-1 gene transcription. Together, these data indicate ancreatic duodenal homeobox 1 (PDX-1) is an that PDX-1 has a dosage-dependent regulatory effect on orphan homeodomain that plays an im- the expression of ␤-cell–specific genes and therefore as- portant role in pancreas development. It is ini- sists in the maintenance of euglycemia. As a consequence, Ptially detected on embryonic day 8.5 in the part of mutations or functional impairment of other transcription the dorsal and ventral primitive gut epithelium that later factors that control the expression of the pdx-1 gene in the develops into the pancreas. A high expression is main- ␤-cell could result in additional subtypes of MODY or be tained in most epithelial cells of the pancreatic bud until candidates for susceptibility to diabetes. Because PDX-1 day embryonic day 10.5 and then decreases to later appears to play such a central role in ␤-cell differentiation ␤ reappear predominantly in the differentiated -cell. Tar- and function, as well as in pancreatic regeneration (14), geted inactivation of this gene in the mouse (1,2) as well as understanding the molecular basis of its regulation and its its in humans (3) result in agenesis of the maintained expression in the ␤-cell will enable the identi- pancreas. In the mouse model, malformations in areas fication of factors that govern these processes. within the duodenum and absence of Brunner’s glands were observed (1,2,4,5). Although pdx-1 does not appear to be required for pancreatic determina- STRUCTURE OF THE pdx-1 GENE The of the pdx-1 gene comprises two . The first encodes for the NH2-terminal region of From the Department of Endocrinology and Metabolism, Hadassah University PDX-1, and the second encodes for the homeodomain and Hospital, Jerusalem, Israel. Address correspondence and reprint requests to Dr. Danielle Melloul, COOH-terminal domain. The human, mouse, and rat genes Department of Endocrinology, Hadassah University Hospital, P.O. Box 12 000, are localized on chromosomes 13 (15,16), 5 (17), and 12 Jerusalem 91120. E-mail: [email protected]. Received for publication 16 April 2002 and accepted in revised form 8 May (18), respectively. Although the activation domain of 2002. PDX-1 is contained within the NH2-terminal domain, its HNF, hepatocyte nuclear factor; MODY, maturity-onset diabetes of the homeodomain is involved in DNA binding; both are in- young; PDX-1, pancreatic duodenal homeobox 1. The symposium and the publication of this article have been made possible volved in protein–protein interactions (19–25). The pdx-1 by an unrestricted educational grant from Servier, Paris. gene is TATA-less; thus, it utilizes three principal tran-

S320 DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002 D. MELLOUL, S. MARSHAK, AND E. CERASI scription initiation sites (17), followed by a short 5Ј proximal duodenum and pyloric glands of the distal stom- untranslated sequence of ϳ100 nucleotides. ach and coincided with the expression of pdx-1 mRNA. Occasionally, ectopic activity was observed in exocrine tissue of the adult pancreas, submucosal layer of the REGULATION OF pdx-1 EXPRESSION duodenum, and even in the spleen (28). Distal rat and human pdx-1 elements. To Failure of the pancreas to develop in both humans and understand the mechanisms that control the expression of mice lacking PDX-1, as well as the dosage-dependent PDX-1 during pancreas development and in the adult effect of PDX-1 on the expression of ␤-cell–specific genes ␤-cell, the pdx-1 gene from different species was mapped. (and on the maintenance of euglycemia), led to the as- Regulatory regions lying upstream from the transcription sumption that sequences conserved between the two start sites are under characterization in transgenic mice as species could be essential for its transcriptional control. A well as in cultured cells in several laboratories. A genomic striking divergence at the nucleotide level was observed ϳ Ј fragment containing 6.5 kb of the 5 -flanking rat pdx-1 between the two species with the exception of four ␤ sequence was sufficient to target -galactosidase expres- regions that showed significant (94, 81, 73, and 78%) sion to pancreatic islets and duodenal cells in transgenic similarity. In addition to the conserved proximal mice (17). A longer fragment containing the coding region Ј sequence (20), three short highly homologous regions and the 3 -flanking sequences of the gene restored the were found between Ϫ2.81 and Ϫ1.67 kb of the human and development of all pancreatic lineages and corrected Ϫ Ϫ between Ϫ2.7 and Ϫ1.8 kb of the mouse pdx-1 gene (Fig. glucose intolerance in pdx-1 / animals (24). In tran- 1). These regions were designated PH1, PH2, and PH3 for siently transfected ␤-cells, the fragment extending from PDX-1 homologous regions 1–3 (22) or areas I, II, and III, Ϫ6.2 to ϩ68 linked to a showed 20- to 100-fold higher activity than that in non-islet cells. Using as determined by Gerrish et al. (31). In transient transfec- ␤ tion experiments, each of the conserved sequences was deletion analyses, the -cell–specific regulated expression ␤ of the rat sequence appeared to require a distal enhancer able to confer -cell–specific activity on a heterologous element located between the Ϫ6.2- and Ϫ5.67-kb region of promoter; however, it was done to different extents. the gene. This element was shown to bind the endodermal PH1/areaI and PH2/areaII showed the highest preferential ␤ ␤ factors HNF-3␤ and Beta2, which act cooperatively to induction in -cell versus non– -cell activity (22,31). An induce PDX-1 expression. Furthermore, glucocorticoids interesting observation was the absence of the PH2/areaII Ј reduced pdx-1 gene expression by interfering with domain in the chicken 5 -flanking region (31), suggesting HNF-3␤ activity on the islet enhancer (26). that the regulation of pdx-1 expression in birds may differ To characterize the regulatory elements and potential from that in rodents and humans. transcription factors necessary for the expression of hu- Attempting to identify factors that regulate the tran- man pdx-1 in ␤-cells, a series of 5Ј and 3Ј deletion scriptional activity of the conserved domains, DNase I fragments of a 7-kb sequence of the 5Ј-flanking region of footprinting analyses, gel electrophoretic mobility shift the gene, fused to a reporter gene, was tested. By transient assays and mutational studies led to the identification of transfections in ␤-cells and non–␤-cells, a ␤-cell–specific several transcription factors (Fig. 1). PH1/areaI and PH2/ distal enhancer element located between Ϫ3.7 and Ϫ3.45 areaII sequences bind and are transactivated by HNF-3␤. kb was delineated. This enhancer fragment strongly stim- Although mutations in the HNF-3␤ binding site within the ulated reporter gene activity in all ␤-cell lines tested and PH2/areaII sequence did not modify its transcriptional was much less active in non–␤-cells, including glucagon- activity, in PH1/areaI, it had a profound effect. Interest- producing cells, and in acinar and hepatic cells. No se- ingly, the PH1/areaI enhancer element was reported to quence similarity was revealed between the enhancer bind the PDX-1 itself both in vitro (22) sequence and the available mouse or rat pdx-1 genomic and in vivo (32), suggesting a possible autoregulatory loop sequences. DNase I footprinting analysis revealed two as a mechanism for PDX-1 to control its own expression. protected regions: one binding the transcription factors The involvement of HNF-1␣ in regulating PH1/areaI was SP1 and SP3 and another binding HNF-3␤ and HNF-1␣. also determined (32). It was further shown that the PDX-1 Similar to the rat distal enhancer, these factors act in protein binds HNF-3␤, and all three transcription factors concert to regulate the transcriptional activity of the pdx-1 appear to act cooperatively to regulate transcription. Iden- gene (27). tification of factors binding and regulating conserved Human and mouse pdx-1 conserved regulatory ele- sequence PH3 is the focus of ongoing studies. ments. In vivo characterization of the mouse pdx-1 se- Thus, from the studies on the binding and the cooper- quences was independently initiated using a fragment of ativity between the different factors acting in concert to ϳ4.5 kb upstream of the initiation start site (28–30). The control the transcriptional activity of pdx-1 regulatory transgene driven by these sequences was shown to ap- elements, it emerges that at least some aspects of the proximately recapitulate the endogenous expression pat- expression of the gene rely on the transcription factor tern. An extended fragment containing the coding region, HNF-3␤. Indeed, its absence in mouse embryonic stem 3kbofthe3Ј-flanking region, and 6.2 kb of upstream cells had a profound effect on pdx-1 gene expression (29). sequences was able to completely rescue the apancreatic HNF-3␤ is a member of the forkhead/winged helix family pdx-1 null phenotype and ultimately restore glucose ho- of transcription factors and is essential for endodermal meostasis. Moreover, the sequences sufficient for appro- cell lineages (33,34). It is structurally related to histone H5, priate developmental and islet-specific expression were which can alter the nucleosomal structure and thus prime located within ϳ4.5 kb of 5Ј-flanking DNA (30). The target genes for expression by opening the chromatin ␤-galactosidase was detected in Brunner’s glands of the structure and providing promoter access to other tran-

DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002 S321 pdx-1 GENE EXPRESSION

FIG. 1. Organization of mouse, human, and rat pdx-1 enhancer/promoter regions. cis-acting regulatory elements of the pdx-1 5؅-flanking sequences are boxed. Conserved sequences between human, mouse, and rat pdx-1 genes are indicated as dark boxes and nonconserved enhancer elements as empty boxes. A: DNA fragments of the mouse pdx-1 gene used in transgenic mice experiments in their expression patterns are depicted (28–30). B: Transcription factors binding to the regulatory elements are indicated above each box. PH1, PH2, and PH3 equals PDX-1 homology regions 1, 2, and 3, respectively. scription factors (35,36). Because HNF-3␤ is not restricted determination for the glut-2 gene (41). The in vivo studies to ␤-cells, the selective transcription of PDX-1 is likely to on HNF-1␣ knockout mice did not give an unequivocal rely on the combination of additional factors, among them answer regarding the importance of this transcription HNF-1␣, SP1, PDX-1 itself, and possibly other unidentified factor for pdx-1 gene expression. Shih et al. (42) reported factors. that in HNF-1␣ null animals’ pdx-1 mRNA levels were In addition, the distal human enhancer element and the significantly decreased (42); however, little or no effect on PH1/areaI domain bind the HNF-1 members of transcrip- its expression was obtained by Parrizas et al. (43). Fur- tion factors. HNF-1␣ and HNF-1␤ homeoproteins are ca- thermore, no reduction in PDX-1 protein was observed in pable of binding the HNF-1 site as homodimers or transgenic mice with selective expression of a dominant- heterodimers (37,38). Transient transfection experiments negative form of HNF-1␣ in pancreatic ␤-cells (44). in fibroblasts demonstrated that both HNF-3␤ and HNF-1␣ Mutations in the HNF-1␣ gene represent the most fre- independently activate the distal human enhancer and, quent form of MODY (45). The observed diabetic pheno- when cotransfected, act in a synergistic manner. However, type was first suggested to be the result of impaired HNF-1␤ did not affect enhancer-driven transcription sepa- binding of HNF-1␣ to the insulin promoter, thus causing rately or in combination with HNF-3␤. Although high decreased transcriptional activity of the gene. However, levels of HNF-1␤ mRNA are observed at 6–7.5 days of binding of HNF-1␣ to the flanking AT sequences (FLAT)-F gestation (39), HNF-1␣ is expressed at a later developmen- element of the insulin gene appears to be unique to the rat tal stage (40) and appears to be the predominant form insulin I gene because this AT-rich motif is not conserved present in adult ␤-cells (27). We found that the relative among insulin promoter sequences. In fact, HNF-1␣ is a abundance of HNF-1␣ and HNF-␤ differ in various weak transactivator of the human insulin gene; we there- pancreatic cell lines, suggesting that differences in HNF-1 fore favor the explanation that the impact on insulin gene subtype ratios may be one of the factors contributing to expression is indirect, via its effect on pdx-1 gene tran- tissue-specific expression of the pdx-1 gene. The relative scription. Impaired pancreatic function in MODY3 is also abundance of the two major HNF-1 species was recently the result of defective transcription of genes involved in suggested to be a mechanism for expression pattern ␤-cell glucose sensing and glucose metabolism (42).

S322 DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002 D. MELLOUL, S. MARSHAK, AND E. CERASI

FIG. 2. The proximal promoter of the mouse, human, and rat pdx-1 gene. Sequence homology between the proximal region of the TATA-less rat, mouse, and human pdx-1 genes. The sequences corresponding to the transcription start sites (S1–S3) are highly conserved and indicated as determined for the rat gene (17).

The in vivo importance of the conserved domains was tional regulation of the rat pdx-1 gene was reported to rely addressed in transgenic mice (28–30). Fragment XbaI– in part on a proximal promoter sequence containing an XhoI, spanning the sequence between Ϫ4.3 and Ϫ1.88 kb E-box motif located at Ϫ104. Pursuing our search for of the mouse pdx-1 gene, which includes both PH1/areaI functional regulatory elements in the human pdx-1 5Ј- and PH2/areaII domains, directed the transgene expres- flanking region, deletion analysis of the proximal promoter ‘sion to pancreatic islets but not to any other cell popula- was performed, and a ␤-cell–specific tion in which PDX-1 is normally expressed. However, a between Ϫ160 and Ϫ100 bp was identified. Comparison of smaller region (PstI–BstEII) that still contained PH1/areaI this region between the promoters of insulin genes from and PH2/areaII sequences was active in the islets as well different origins showed a relatively high degree of homol- as in the pyloric sphincter and the common bile duct. ogy (78%), with greater heterogeneity further upstream. Furthermore, within the pancreas, expression of the re- DNAse I footprinting, using the conserved proximal pro- porter gene driven by the PstI–BstEII fragment was found moter of the human gene and ␤-cell extracts, revealed a in the majority of insulin-, glucagon-, and somatostatin- specific protected region around the conserved E-box producing cells; therefore, this fragment was considered motif (CACGTG) (17). This site predominantly binds a to be an endocrine-specific enhancer. Furthermore, the complex containing the transcription factor USF (17). fragment (XhoI–BglII) containing PH3/areaIII drove the Mutations abolishing its binding impaired the activity of reporter expression to clusters of insulin-producing cells, the pdx-1 promoter. Expression of a dominant-negative whereas it was almost inactive in glucagon- and soma- form of USF-2 in ␤-cells reduced both the pdx-1 promoter tostatin-positive cells of the neonatal pancreas. However, activity as well as PDX-1 mRNA and protein levels. This this ␤-cell–specific activity was transient and lost in adult led to a reduction of PDX-1 binding to the insulin promoter pancreases (30). Although the reason for the silencing of and consequently a dramatic decrease in insulin gene this transgene is not clear, it may be proposed that the expression (46). Thus, USF1 and USF2 interactions with XhoI–BglII region plays a specific role in immature ␤-cells the E-box sequence in the pdx-1 promoter appear to or provides critical developmental cues for the initiation of contribute to the preferential expression of the gene in the mature ␤-cell lineage. Altogether, these data suggest ␤-cells. that the conserved domains confer islet-specific, and to a certain extent ␤-cell–specific, transcription both in vivo and in vitro. However, separately or in combination, these DISCUSSION elements cannot faithfully recapitulate the expression of Taken together, these results suggest that the transcrip- endogenous pdx-1, thus implicating that additional regu- tional stimulation of pdx-1 in ␤-cells is mediated by a latory elements must be involved in this process. Muta- unique combination of protein–protein interactions and tions or deletions of important regulatory sequences in the that separate modules in the gene can be active at a given context of the endogenous pdx-1 gene will help assess stage by binding a specific set of transcription factors. their critical importance in the regulated expression of the Indeed, the transcription factors HNF-3␤, HNF-1␣, HNF- gene. 1␤, SP1/3, USF1/2, and PDX-1 itself regulate the expres- sion of the pdx-1 gene. Most of these factors have been THE PROXIMAL PROMOTER OF THE pdx-1 GENE previously shown, mainly by knockout experiments in As mentioned earlier, the pdx-1 gene is TATA-less, and mice, to be important developmental regulators. PDX-1 is although the sequences corresponding to the transcription a key factor with multiple functions both during develop- start sites among the rat, mouse, and human genes are ment and in the differentiated ␤-cell. In the adult, its role in highly conserved (17) (Fig. 2), great heterogeneity is regulating islet-specific genes and, most importantly, in revealed further upstream. The ␤-cell–specific transcrip- mediating the glucose effect on insulin gene transcription

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