Sequence Analysis, Identification of Evolutionary Conserved Motifs and Expression Analysis of Murine tcof1 Provide Further Evidence for a Potential Function for the Gene and Its Human Homologue, TCOF1

Jill Dixon 0 1 3 Karine Hovanes 0 1 2 3 Rita Shiang 0 1 2 3 Michael J. Dixon 0 1 3 0 College of Medicine, University of California , Irvine, CA 92717 , USA 1 University of Manchester , Oxford Road, Manchester M13 9PT , UK 2 Department of Biological Chemistry 3 School of Biological Sciences and Departments of Dental Medicine and Surgery , 3.239, Stopford Building - The gene mutated in Treacher Collins syndrome, an autosomal dominant disorder of facial development, has recently been cloned. While the function of the predicted protein, Treacle, is unknown, it has been shown to share a number of features with the highly phosphorylated nucleolar phosphoproteins, which play a role in nucleolarcytoplasmic transport. In the current study, the murine homologue of the Treacher Collins syndrome gene has been isolated and shown to encode a low complexity, serine/alanine-rich protein of 133 kDa. Interspecies comparison indicates that the proteins display 61.5% identity, with the level of conservation being greatest in the regions of acidic/basic amino acid repeats and nuclear localization signals. These features are shared with the nucleolar phosphoproteins. Confirmation that the gene isolated in the current study is orthologous with the Treacher Collins syndrome gene was provided by the demonstration that it mapped to central mouse chromosome 18 in a conserved syntenic region with human chromosome 5q21q33. Expression analysis in the mouse indicated that the gene was expressed in a wide variety of embryonic and adult tissues. Peak levels of expression in the developing embryo were observed at the edges of the neural folds immediately prior to fusion, and also in the developing branchial arches at the times of critical morphogenetic events. These observations support a role for the gene in the development of the craniofacial complex and provide further evidence that the gene encodes a protein which may be involved in nucleolar-cytoplasmic transport. Treacher Collins syndrome (TCS) is a congenital disorder of craniofacial development which occurs with an incidence of ~ 1/50 000 live births (1,2). The clinical features of TCS, which are usually bilaterally symmetrical in nature (3), include: (i) abnormalities of the external ears which are frequently associated with atresia of the ear canals and anomalies of the middle ear ossicles. Bilateral conductive hearing loss is therefore a common feature of TCS (4); (ii) hypoplasia of the facial bones, particularly the mandible and zygomatic complex; (iii) lateral downward slanting of the palpebral fissures with colobomas of the lower eyelids and a lack of eyelashes medial to the defect; (iv) cleft palate (1,5). However, a high degree of inter- and intra-familial phenotypic variability is observed in TCS (6,7). Moreover, while TCS is inherited in an autosomal dominant fashion, 60% of cases arise without a previous family history, presumably as the result of a de novo mutation (8). These combined facts create diagnostic and genetic counselling difficulties. The TCS locus was initially mapped to human chromosome 5q31q34 (9) and the gene, TCOF1, was subsequently identified using positional cloning strategies (10). TCOF1 contains an open reading frame of 4233 bp, which encodes the low complexity, serine/alanine-rich, 144 kDa protein, Treacle. The identification of all 26 exons of TCOF1 has permitted the identification of >50 largely family-specific mutations which result in the introduction of a premature termination codon into Treacle (1012; unpublished data). The identification of the gene, and delineation of the wide mutational spectrum, have failed to reveal the exact biochemical nature of the disorder, as, to date, database comparisons have only revealed weak, but significant, homology to a family of highly phosphorylated proteins, the nucleolar phosphoproteins (13). Nevertheless, a series of repeated units have been identified within the gene and these have been shown to map onto individual exons. The function of the repeating units is unclear, however, each unit contains a number of potential sites for casein kinase II phosphorylation, suggesting that phosphorylation is important for the correct function of the protein. The homology between the nucleolar phosphoproteins and TCOF1 also appears to be greatest at these motifs (13). Moreover, a number of potential nuclear localization signals have been noted towards the 3 end of the coding sequence of both the nucleolar phosphoproteins and Treacle. On the basis that the tissues affected by TCS are derived from the first and second branchial arches, which in turn have a significant contribution from the neural crest, it has been proposed that the disorder may be the result of a defect in neural crest cell migration, improper cellular differentiation during development (14,15) or an abnormality of the extracellular matrix (16). Additionally, phenocopies of TCS have been produced in mice following acute maternal exposure to 13-cisretinoic acid at 9.09.5 days post-fertilization (17), suggesting that the disorder may result from abnormal development of the first and second branchial arch ectodermal placodes. Whatever the underlying mechanism of the disorder, it is evident that this gene plays a crucial role in the formation of the craniofacial complex during early embryonic development. In the current investigation, we have isolated the murine homologue of TCOF1 which has allowed us to make a preliminary study of the spatio-temporal distribution of the gene during embryonic development. Comparison of the human and mouse cDNA sequences has allowed us to identify evolutionary conserved regions of the gene which are likely to be important for its function. The results of these analyses provide further support for the hypothesis that Treacle may play a role in nucleolarcytoplasmic transport. Elucidation of the murine cDNA sequence Zooblot analysis performed using a human TCOF1 cDNA clone (10) showed that the gene is highly conserved in genomic DNA extracted from dog, pig, sheep, cow and monkey. The intensity of the bands observed in murine and chicken DNA was weaker, suggesting a lower level of evolutionary conservation in the genomes of these animals (Fig. 1). In order to isolate a full-length murine tcof1 cDNA clone, the combined techniques of cDNA library screening and rapid amplification of cDNA ends (RACE) were employed. Initially, a mouse embryonic day 10 (E10) cDNA library was screened at reduced stringency with a human TCOF1 cDNA clone (10). Fourteen clones were identified, of which eight were purified. The longest of these clones, designated E10-1C, was sequenced in its entirety and found to contain a single open reading frame of 1991 bp encompassing the entire clone. As this clone did not contain a translation initiation signal, a polyadenylation signal or a poly A tail, the ends of the remaining clones were sequenced, but were found not to extend the sequence in either a 5 or 3 directio (...truncated)