Vol. 48 No. 1/2001 221–226 QUARTERLY

Lymphoid enhancer-binding factor-1 (LEF-1), a member of the high mobility group (HMG) family of proteins, regulates expression of T-cell receptor-alpha gene and is one of the key regulatory molecules in the epithelial-mesenchymal interactions during embryonic development. Among others, LEF-1 regulates expression of cytokeratin genes involved in formation of hair follicles and the gene encoding the cell-adhesion molecule E-cadherin. Transcription factor LEF-1, which acts as a dimer, binds beta-catenin and is involved in signal transduction by the wnt pathway. We have cloned and sequenced a novel isoform of human LEF-1 gene transcript. This isoform encodes a truncated protein devoid of HMG domain and nuclear localization signal but retaining beta-catenin binding domain. This isoform might either act in a dominant-negative manner by interfering with native LEF-1, or might bind beta-catenin in the cytosol, which would result in attenuation of the signals transmitted by the LEF-beta-catenin pathway.

sharp bend in the double helix, thus facilitating the binding of other transcription factors to the adjacent DNA sequences in order to modulate transcription [3].It has been shown that LEF-1 plays a critical role in organogenesis requiring concerted interaction between cells of epithelial and mesenchymal origin [4] and is expressed in neural crest, mesencephalon, tooth buds, hair follicles and eccrine sweat glands [2].It has been evidenced that LEF-1 interacts with b-catenin to form a ternary complex with DNA, suggesting that this interaction might provide a molecular mechanism of signal transduction by cell-adhesion molecules or components of the wnt pathway [5].Zhou et al. [6] have demonstrated that genes involved in hair development in mice contain Lef-1 binding motif and that Lef-1 can bind to these sequences when expressed in hair follicles.The studies on Lef-1-deficient mice have revealed that normal development of tooth buds requires transient expression of Lef-1 gene in the dental epithelium [7].These observations have implicated a functional importance of Lef-1 in hair and tooth development.
Kere et al. [8] discovered LEF-1 binding motif (HK-1) within the regulatory region of EDA gene, whose protein product is involved in the development of tooth buds, hair follicles, and sweat glands [8,9].Localization of the HK-1 motif suggested that this gene is an important target for the regulatory signals carried by LEF-1 during epithelial morphogenesis [8].
In order to investigate a potential role of LEF-1 in the regulation of transcription of EDA gene, we have cloned and sequenced cDNA encoding a long form of the human LEF-1 (AF198532) corresponding to the protein described by Waterman et al. [10].In this study we describe a novel isoform of LEF-1 gene transcript which encodes a protein devoid of the HMG domain but retaining the b-catenin binding domain.

MATERIALS AND METHODS
Lymphocytes of a healthy individual who did not exhibit any abnormalities of teeth, hair and sweat glands, were isolated from peripheral blood by centrifugation in a Ficol density gradient and were washed twice in phosphate-buffered saline.The lymphocytes were cultured in RPMI 1640 medium supplemented with L-glutamine, 10% foetal calf serum and 1% antibiotic antimycotic mixture, and were stimulated with lectin-phytohemagglutinin (10 mg/ml).Three days later the medium was changed and the cells were treated with interleukin-2 (100 U/ml) from that time onwards.All reagents used for cell culture were obtained from Sigma (U.S.A.).After 11 days of culture, total RNA was isolated from samples containing approximately 1 ´10 6 cells [11].LEF-1 cDNA was obtained by reverse transcription of polyA + RNA followed by nested PCR (RT-PCR) with the use of the forward primer F: 5¢-TAC TTA GGT ACC TGC CCC AAC TTT CCG GAG-3¢.This primer corresponded to nucleotides 991-1008 of the mouse Lef-1 mRNA and contained a KpnI restriction site (underlined).The reverse primer: R: 5¢-GGG GTT TCA ACA AGC TTC CAT CTC CAG AAG-3¢, whose sequence corresponded to nucleotides 2246-2275 of mouse Lef-1 mRNA, contained a restriction site for HindIII (underlined).
Total RNA was also extracted from hair follicles of the same individual, as well as from human peripheral blood lymphocytes, chondrocytes, keratinocytes, umbilical cord and ovary [11].The isolated polyA + RNA was reverse-transcribed and the specific cDNA fragments were amplified (RT-PCR).The following primers were used for amplification: forward (CAC TGT CAG TCG ACA CTT), complementary to exon 6 and reverse (TGC TCC TTT CTC TGT TCA), complementary to exon 9 of LEF-1 gene.

RESULTS AND DISCUSSION
The full length cDNA encoding the isoform of human LEF-1 gene transcript was amplified with the use of primers designed for its murine counterpart, since at the time of the initial experiments the sequence of introns of human gene was unknown.During selection of the clones, in addition to the long isoform of LEF-1 gene transcript, we identified a novel isoform which differed from the short and the long forms, as well as from transcript isoforms described by Hovanes et al. [12] but corresponded to proteins described earlier by Waterman et al. [10].The nucleotide sequence of cDNA, corresponding to the novel isoform of LEF-1 gene transcript (Fig. 2) was almost identical to cDNA sequence of the long form of human LEF-1, except an 80 nt insertion, and contained in-frame stop codon.
calization signal and the entire HMG domain, but retained b-catenin binding domain (Fig. 3B).The 80 nt insertion was localized between the sequences corresponding to exons 7 and 8 (Fig. 4A).Comparison of the 80 nt insertion with a partial sequence of human chromosome 4, deposited in GenBank (AC021524), revealed a 100% homology with part of the sequence of intron 7.This suggested that the novel isoform of LEF-1 gene transcript might arise due to selection of an alternative splice site localized in intron 7 (Fig. 4A).
In order to substantiate this observation, we searched for this isoform in a number of cells and tissues.The novel isoform was detected in hair follicles, but not in freshly isolated lymphocytes, cultured keratinocytes, or chondrocytes, nor in the umbilical cord and ovary (Fig. 4B).Our findings suggest that the novel isoform is specifically expressed in hair follicles.It was inferred from our results that the expression of the novel isoform in cultured lymphocytes was illegitimate, since it was absent from freshly isolated peripheral blood lymphocytes.
Recently, Hovanes et al. [12] demonstrated that LEF-1 gene encodes multiple isoforms of the transcript, which are due to alternative splicing.The isoform described herein encodes a truncated protein devoid of HMG domain and nuclear localization signal, but retaining the region responsible for interaction with b-catenin.The function of this protein is unknown.We postulate that it might either act in a dominant-negative manner by interfering with native LEF-1 as suggested by Behrens et al. [5], or it might bind b-catenin in the cytosol which results in attenuation of the signals transmitted by the wnt pathway in hair follicles.

Figure 1 .
Figure 1.Identification of the lymphoid enhancerbinding factor-1 (LEF-1) cDNA cloned from human cultured lymphocytes.The cDNA was amplified by PCR using primers specific for mouse Lef-1.The amplification product was resolved by electrophoresis in 1% agarose gel and visualized by ethidium bromide staining.

Figure 2 .Figure 3 .Figure 4 .
Figure 2. Sequence alignment of cDNA corresponding to the novel isoform of LEF-1 gene transcript.The upper sequence represents the novel isoform, whereas the lower sequence the long isoform of LEF-1 gene transcript.The 80 nt insertion is underlined.The sequence corresponding to stop codon is printed in bold.