BIOLOGICAL OVERVIEW

The term pangolin refers to a long tailed, sticky-tongued tropical Old World (Asia and Africa) mammal; it feeds chiefly on ants. The pangolin's body is covered with large, flat, imbricated horny scales; it somewhat resembles the New World armadillo in terms of its feeding habits and its employment of a curled up, hedgehog-like defensive posture. For these reasons, the gene pangolin is most appropriately named, calling to mind both Armadillo and Hedgehog, two proteins discussed below in connection with Pangolin function.

Pangolin is one of Drosophila's more recently sequenced segment polarity genes. The discovery of the involvement of an HMG-domain in vertebrate Wnt signaling (Wnts are homologs of Wingless) encouraged a search for a similar HMG-domain protein in Drosophila. The vertebrate HMG-domain proteins Lef-1 and XTcf-3 have been shown to physically interact with ß-catenin, a homolog of Drosophila Armadillo. HMG-domain proteins are presumptive transcription factors; evidence from Xenopus suggests that during Wnt signaling the HMG protein interacting with ß-catenin ( XTcf-3) is transported as a complex into the nucleus, where it can act as a transcription factor (Behrens, 1996, Huber, 1996 Molenaar, 1996). Both ß-catenin and Armadillo serve as downstream components of the Wnt/Wingless signal transduction pathway; this pathway is involved in vertebrate mesoderm formation, neural differentiation and limb patterning, and in Drosophila, is involved in similar functions but is best known as a segment polarity gene.

Isolated as a homolog of the vertebrate HMG-domain proteins described above, Pangolin is able to physically interact with vertebrate ß-catenin. Mutations of pan cause a segment polarity phenotype similar to that of wingless mutation (Brunner, 1997). In an unexpected association, Pangolin is found immediately adjacent to the gene cubitus interruptus, a zinc finger transcription factor that targets wingless, decapentaplegic and patched (Brunner, 1997). The two genes have a head-to-head orientation (van de Wetering, 1997). CI acts in the hedgehog pathway.

Genetic studies show that pangolin acts to transduce the wingless signal. A constitutively active form of Armadillo was expressed in wild type flies resulting in a naked cuticle phenotype. Denticles normally positioned in the anterior portion of each segment are replaced by naked cuticle. In pan mutants, no naked cuticle due to active Armadillo overexpression is observed, thus showing that in the absence of PAN, ARM activity is blocked and cannot cause a biological Wingless response in vivo (Brunner, 1997).

One mode of action for Wingless and Wnts is suggested by the observation that the LEF-1/ß-catenin complex binds to an E-cadherin promoter fragment. Dring primitive streak formation in mouse embryos, embryonic ectodermal cells, which represent a true epithelial cell layer, give rise to mesoderm. During primitive streak formation, some ectodermal cells lose E-cadherin expression and express LEF-1. It is tempting to speculate that a complex of LEF-1 and ß-catenin is involved in down-regulating E-cadherin, resulting in an altered cell adhesion accompanying a cell fate transformation (Huber, 1996).

GENE STRUCTURE

The pan gene is located just upstream of and distal to cubitus interruptusand is transcribed in the opposite direction. It is a curious coincidence that pan and ci are adjacent genes, as ci encodes a transcription factor that is essential for transducing all examples of hedgehog signaling, whereas the present evidence suggests an equivalent role for PAN in wingless signal transduction (Brunner, 1997).

PROTEIN STRUCTURE

Amino Acids - 751

Structural Domains

The HMG-domain shows 88% identical residues with murine Lef-1. The N-terminal region of PAN is also conserved between PAN, Lef-1 and XTcf-3 (Brunner, 1997). Three regions of conservation are noted. First is the N-terminus, which in XTcf-3 and LEF-1 constitutes the ß-catenin interaction domain. Second is the high mobility group (HMG) box DNA-binding domain. Infrequently, an alternative exon encoding the second part of the HMG box is encountered. The alternative protein is termed dTCF-B. Third, a small region directly C-terminal to the HMG box is conserved among dTCF, TCF-1 and C. elegans pop-1 (van de Wetering, 1997).

A mammalian T cell factor 1 (TCF-1)-like protein from Drosophila, encoded by the pangolin (pan) locus consists of a DNA binding domain similar to that of other high mobility group proteins and a protein-protein interaction domain that binds beta-catenin (Armadillo in Drosophila) but it lacks a transcriptional activation domain. The pan locus spans approximately 50 kb and the mRNA results from the splicing of 13 exons. The open reading frame of pan is 2253 bp, encoding a putative 751 amino acid protein. The overall sequence identity, at the protein level, with murine TCF-1 and LEF-1 is 48% and 54% respectively. This identity is comparable to that seen between murine TCF-1 and LEF-1, which is 54%. The translation start site is at 414 bp. The HMG box is situated in the middle of the open reading frame, between nucleotide positions 1229 and 1453. This is followed by a stretch of 50 amino acids bearing striking similarity to human and mouse TCF-1 with almost 70% identity. This highly basic region just C-terminal to the HMG domain is conserved in TCF-1 and C. elegans POP-1 but severely truncated in LEF-1. This basic region is much less highly conserved compared with human and mouse LEF-1. There is a remarkable conservation of the exon/intron boundaries between the human and D. melanogaster genes, suggesting that they share a common ancestor. Chromosomal in situ hybridization locates pan to the base of chromosome 4, near the cubitus interruptus locus. pan and ci are separated by approximately 10 kb and are transcribed in opposite directions. Restriction map and sequence analyses confirm their close proximity. The small fourth chromosome undergoes little or no recombination and was previously reported to lack DNA polymorphisms; however, there are two DNA polymorphisms occurring in three combinations within the pan locus, demonstrating the presence of synonymous substitutions and the past occurrence of recombination. Evidence is presented suggesting that the protein encoded by pan is more similar to mammalian TCF-1 and Caenorhabditis elegans POP-1 than to mammalian LEF-1 (Dooijes, 1998).

date revised: 10 February 98 

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