Brad A. Amendt

The Molecular Basis of Rieger Syndrome ANALYSIS OF PITX2 HOMEODOMAIN PROTEIN ACTIVITIES

Rieger syndrome is an autosomal-dominant developmental disorder that includes glaucoma and mild craniofacial dysmorphism in humans. Mutations in the Pitx2 homeobox gene have been linked to Rieger syndrome. We have characterized wild type and mutant Pitx2 activities using electrophoretic mobility shift assays, protein binding, and transient transfection assays. Pitx2 preferentially binds the bicoid homeodomain binding site and transactivates reporter genes containing this site. The combination of Pitx2 and another homeodomain protein, Pit-1, yielded a synergistic 55-fold activation of the prolactin promoter in transfection assays. Addition of Pit-1 increased Pitx2 binding to the bicoidelement in electrophoretic mobility shift assays. Furthermore, we demonstrate specific binding of Pit-1 to Pitx2 in vitro. Thus, wild type Pitx2 DNA binding activity is modulated by protein-protein interactions. We next studied two Rieger mutants. A threonine to proline mutation (T68P) in the second helix of the homeodomain retained DNA binding activity with the same apparentK D and only about a 2-fold reduction in theB max. However, this mutant did not transactivate reporter genes containing the bicoid site. The mutant Pitx2 protein binds Pit-1, but there was no detectable synergism on the prolactin promoter. A second mutation (L54Q) in a highly conserved residue in helix 1 of the homeodomain yielded an unstable protein. Our results provide insights into the potential mechanisms underlying the developmental defects in Rieger syndrome.

The Pitx2 protein in mouse development

The Rieger syndrome, an autosomal dominant disorder involving ocular, dental, and umbilical defects is caused by mutations in PITX2, a Bicoid‐type homeobox protein. Mouse Pitx2 mRNA is expressed in eye, tooth and umbilicus consistent with the human Riegers phenotype. Moreover, Pitx2 is involved in the Nodal/Sonic hedgehog pathway that determines left/right polarity. In this report we demonstrate a 32‐kDa polypeptide on Western blots of nuclear extracts from a rat pituitary cell line, using a Pitx2 specific antibody (designated P2R10). We describe also for the first time expression of the Pitx2 protein in mouse. Pitx2 protein immunostaining was detectable during the development of the eye, tooth, umbilicus, and also in the pituitary, heart, gut, and limb. We demonstrate for the first time directly that Pitx2 is asymmetrically expressed in early heart, gut, and lung development. Dev Dyn;218:195–200.

Multifunctional Role of the Pitx2 Homeodomain Protein C-Terminal Tail

ABSTRACT Pitx2 is a newly described bicoid-like homeodomain transcription factor that is defective in Rieger syndrome and shows a striking leftward developmental asymmetry. We have previously shown that Pitx2 (also called Ptx2 and RIEG) transactivates a reporter gene containing abicoid enhancer and synergistically transactivates the prolactin promoter in the presence of the POU homeodomain protein Pit-1. In this report, we focused on the C-terminal region which is mutated in some Rieger patients and contains a highly conserved 14-amino-acid element. Deletion analysis of Pitx2 revealed that the C-terminal 39-amino-acid tail represses DNA binding activity and is required for Pitx2-Pit-1 interaction and Pit-1 synergism. Pit-1 interaction with the Pitx2 C terminus masks the inhibitory effect and promotes increased DNA binding activity. Interestingly, cotransfection of an expression vector encoding the C-terminal 39 amino acids of Pitx2 specifically inhibits Pitx2 transactivation activity. In contrast, the C-terminal 39-amino-acid peptide interacts with Pitx2 to increase its DNA binding activity. These data suggest that the C-terminal tail intrinsically inhibits the Pitx2 protein and that this inhibition can be overcome by interaction with other transcription factors to allow activation during development.

Rieger syndrome: a clinical, molecular, and biochemical analysis.

Abstract. Rieger syndrome (RIEG 1; MIM 180500) is an autosomal dominant disorder of morphogenesis. It is a phenotypically heterogeneous disorder characterized by malformations of the eyes, teeth, and umbilicus. RIEG belongs to the Axenfeld-Rieger group of anomalies, which includes Axenfeld anomaly and Rieger anomaly (or Rieger eye malformation), which display ocular features only. Recently, mutations in the homeodomain transcription factor, PITX2, have been shown to be associated with Rieger syndrome. This review discusses the clinical manifestations of Rieger syndrome and how they correlate with the current molecular and biochemical studies on this human disorder.

PITX2, β-catenin and LEF-1 interact to synergistically regulate the LEF-1 promoter

PITX2, β-catenin and lymphoid enhancer factor (LEF-1) are required for the inductive formation of several epithelial-derived organs, including teeth. Lef-1 is expressed in the dental epithelium after Pitx2, and both factors have overlapping expression patterns in the tooth bud and cap stages. Our analysis of Pitx2–/– mutant mice showed reduced Lef-1 expression in facial tissues by RT-PCR and quantitative RT-PCR. Consistent with these results we show that the human 2.5 kb LEF-1 promoter is activated by PITX2. Furthermore, the LEF-1 promoter is differentially activated by PITX2 isoforms, which are co-expressed in dental epithelium. The 2.5 kb LEF-1 promoter contains two regions that act to inhibit its transcription in concert with PITX2. The proximal region contains a Wnt-responsive element (WRE) that attenuates PITX2 activation. LEF-1 cannot autoregulate LEF-1 expression; however co-transfection of PITX2 and LEF-1 result in a synergistic activation of the 2.5 kb LEF-1 promoter. LEF-1 specifically interacts with the PITX2 C-terminal tail. Deletion of a distal 800 bp segment of the LEF-1 promoter resulted in enhanced PITX2 activation, and increased synergistic activation in the presence of LEF-1. Furthermore, β-catenin in combination with PITX2 synergistically activates the LEF-1 promoter and this activation is independent of the Wnt-responsive element. β-catenin directly interacts with PITX2 to synergistically regulate LEF-1 expression. We show a new mechanism where LEF-1 expression is regulated through PITX2, LEF-1 and β-catenin direct physical interactions. LEF-1 and β-catenin interactions with PITX2 provide new mechanisms for the regulation of PITX2 transcriptional activity.

Deletion of G protein-coupled receptor 48 leads to ocular anterior segment dysgenesis (ASD) through down-regulation of Pitx2

The development of the anterior segment of the mammalian eye is critical for normal ocular function, whereas abnormal development can cause glaucoma, a leading cause of blindness in the world. We report that orphan G protein-coupled receptor 48 (Gpr48/LGR4) plays an important role in the development of the anterior segment structure. Disruption of Gpr48 causes a wide spectrum of anterior segment dysgenesis (ASD), including microphthalmia, iris hypoplasia, irdiocorneal angle malformation, cornea dysgenesis, and cataract. Detailed analyses reveal that defective iris myogenesis and ocular extracellular matrix homeostasis are detected at early postnatal stages of eye development, whereas ganglion cell loss, inner nuclear layer thinness, and early onset of glaucoma were detected in 6-month-old Gpr48−/− mice. To determine the molecular mechanism of ASD caused by the deletion of Gpr48, we performed gene expression analyses and revealed dramatic down-regulation of Pitx2 in homozygous knockout mice. In vitro studies with the constitutively active Gpr48 mutant receptor demonstrate that Pitx2 is a direct target of the Gpr48-mediated cAMP-CREB signaling pathway in regulating anterior segment development, suggesting a role of Gpr48 as a potential therapeutic target of ASD.

Short-Chain Acyl-Coenzyme A Dehydrogenase Deficiency in Mice

ABSTRACT: A murine model for short-chain acyl-coenzyme A dehydrogenase (SCAD) deficiency has been identified and characterized in BALB/cByJ mice. These mice have undetectable SCAD activity, severe organic aciduria; excreting ethylmalonic and methylsuccinic acids and Nbutyrylglycine, and develop a fatty liver upon fasting or dietary fat challenge. The mutant mice develop hypoglycemia after an 18-h fast, and have elevated urinary and muscle butyrylcarnitine concentrations. Most of these findings parallel those of human disorders associated with SCAD deficiency and other β-oxidation defects. This mouse model presents important opportunities to investigate the biology of mammalian fatty acid metabolism and the related human diseases.

MicroRNAs Play a Critical Role in Tooth Development

MicroRNAs are known to regulate gene function in many tissues and organs, but their expression and function, if any, in tooth development are elusive. We sought to identify them by microRNA screening analyses and reveal their overall roles by inactivating Dicer1 in the dental epithelium and mesenchyme. Discrete sets of microRNAs are expressed in molars compared with incisors as well as epithelium compared with mesenchyme. Conditional knockout (cKO) of Dicer1 (mature microRNAs) in the dental epithelium of the Pitx2-Cre mouse results in multiple and branched enamel-free incisors and cuspless molars, and change in incisor patterning and in incisor and molar size and shape. Analyses of differentiating dental epithelial markers reveal a defect in ameloblast differentiation. Conversely, the cervical loop (stem cell niche) is expanded in Dicer1 cKO. These results demonstrate that tooth development is tightly controlled by microRNAs and that specific microRNAs regulate tooth epithelial stem cell differentiation.

PITX2 and β-catenin Interactions Regulate Lef-1 Isoform Expression.

ABSTRACT Lef-1 and PITX2 function in the Wnt signaling pathway by recruiting and interacting with β-catenin to activate target genes. Chromatin immunoprecipitation (ChIP) assays identified the Lef-1 promoter as a PITX2 downstream target. Transgenic mice expressing LacZ driven by the 2.5-kb LEF-1 promoter demonstrated expression in the tooth epithelium correlated with endogenous Lef-1 FL epithelial expression. PITX2 isoforms regulate the LEF-1 promoter, and β-catenin synergistically enhanced activation of the LEF-1 promoter in combination with PITX2 and Lef-1 isoforms. PITX2 enhances endogenous expression of the full-length β-catenin-dependent Lef-1 isoform (Lef-1 FL) while decreasing expression of the N-terminally truncated β-catenin-independent isoform. Our research revealed a novel interaction between PITX2, Lef-1, and β-catenin in which the Lef-1 β-catenin binding domain is dispensable for its interaction with PITX2. PITX2 interacts with two sites within the Lef-1 protein. Furthermore, β-catenin interacts with the PITX2 homeodomain and Lef-1 interacts with the PITX2 C-terminal tail. Lef-1 and β-catenin interact simultaneously and independently with PITX2 through two different sites to regulate PITX2 transcriptional activity. These data support a role for PITX2 in cell proliferation, migration, and cell division through differential Lef-1 isoform expression and interactions with Lef-1 and β-catenin.

MicroRNAs Regulate Pituitary Development, and MicroRNA 26b Specifically Targets Lymphoid Enhancer Factor 1 (Lef-1), Which Modulates Pituitary Transcription Factor 1 (Pit-1) Expression

To understand the role of microRNAs (miRNAs) in pituitary development, a group of pituitary-specific miRNAs were identified, and Dicer1 was then conditionally knocked out using the Pitx2-Cre mouse, resulting in the loss of mature miRNAs in the anterior pituitary. The Pitx2-Cre/Dicer1 mutant mice demonstrate growth retardation, and the pituitaries are hypoplastic with an abnormal branching of the anterior lobe, revealing a role for microRNAs in pituitary development. Growth hormone, prolactin, and thyroid-stimulating hormone β-subunit expression were decreased in the Dicer1 mutant mouse, whereas proopiomelanocortin and luteinizing hormone β-subunit expression were normal in the mutant pituitary. Further analyses revealed decreased Pit-1 and increased Lef-1 expression in the mutant mouse pituitary, consistent with the repression of the Pit-1 promoter by Lef-1. Lef-1 directly targets and represses the Pit-1 promoter. miRNA-26b (miR-26b) was identified as targeting Lef-1 expression, and miR-26b represses Lef-1 in pituitary and non-pituitary cell lines. Furthermore, miR-26b up-regulates Pit-1 and growth hormone expression by attenuating Lef-1 expression in GH3 cells. This study demonstrates that microRNAs are critical for anterior pituitary development and that miR-26b regulates Pit-1 expression by inhibiting Lef-1 expression and may promote Pit-1 lineage differentiation during pituitary development.