Lymphoid Enhancer-Binding Factor 1

Description

LEF1 is a nuclear protein that is expressed in pre-B and T cells. It binds to a functionally important site in the T-cell receptor-alpha (TCRA; see 186880) enhancer and confers maximal enhancer activity. LEF1 belongs to a family of regulatory proteins that share homology with high mobility group protein-1 (HMG1; 163905) (Waterman et al., 1991; van Genderen et al., 1994).

Cloning and Expression

By sequencing tryptic peptides of TCF1-alpha purified from Jurkat human T-cell nuclear extracts, followed by PCR and screening a Jurkat cDNA library, Waterman et al. (1991) cloned 2 TCF1-alpha splice variants. The deduced full-length protein contains 399 amino acids and has a calculated molecular mass of 44.2 kD. The C-terminal half of TCF1-alpha contains a serine- and threonine-rich segment, a 68-amino acid domain that shares similarity with a conserved region of HMG and nonhistone chromosomal proteins, and a putative nuclear localization signal. The shorter TCF1-alpha variant encodes a protein with an in-frame 28-amino acid deletion within the serine- and threonine-rich segment. Northern blot analysis detected an abundant 3.4-kb transcript and a minor 2.3-kb transcript in mouse and human T-cell lines and in mouse thymus, but not in B-cell lines, a macrophage cell line, or in other mouse tissues examined. SDS-PAGE of purified human TCF1-alpha showed 3 distinct protein bands, and the major TCF1-alpha protein had an apparent molecular mass of 55 kD.

By screening a human genomic PAC library and testis, melanoma, and fetal brain cDNA libraries, Hovanes et al. (2000) identified several LEF1 splice variants. The most common transcript encodes the 399-amino acid protein, which contains a conserved N-terminal beta-catenin (CTNNB1; 116806)-binding domain and a C-terminal HMG-type DNA-binding/bending domain. Another variant encodes a 413-amino acid protein that differs at the C terminus. Use of alternatively spliced sequences in intron 3, which the authors called exons 3a and 3b, introduces in-frame stop codons, producing proteins of 16 and 18 kD that lack the HMG-like DNA-binding domain and nuclear localization signal. These proteins are predicted to be cytoplasmic and capable of interacting with beta-catenin.

Gene Function

Using footprint, mobility-shift, and Southwestern blot experiments, Waterman et al. (1991) showed that recombinant full-length TCF1-alpha and its isolated HMG-like domain bound to the DNA sequence 5-prime-GGCACCCTTTGAA-3-prime in the TCRA enhancer, most likely as a monomer. Following expression in HeLa cells, TCF1-alpha activated expression of a reporter gene containing the TCF1-alpha-binding motif.

Zhou et al. (1995) reported that several hair keratin genes (see KRTHA1; 601077) possess consensus LEF1-binding motifs located in similar positions relative to their TATA box. They demonstrated that LEF1 binds to hair keratin promoters in vitro, and that LEF1 is present during skin development in ectoderm that will express these promoters as the cells differentiate. In addition, they showed that mouse Lef1 mRNA is present in pluripotent ectoderm, and that it is upregulated in a highly restricted pattern just before the formation of underlying mesenchymal condensates and the commitment of overlying ectodermal cells to invaginate and become hair follicles.

Hovanes et al. (2000) transfected human and other mammalian cell lines with a reporter gene construct containing the LEF1 promoter region and observed highest LEF1 expression in mature human T- and B-cell lines. Since the LEF1 gene is not expressed in B lymphocytes, Hovanes et al. (2000) proposed that expression of LEF1 is normally silenced in B cells by elements not present in the promoter fragment tested.

Constitutive activation of the Wnt (see 164975) signaling pathway is a root cause of many colon cancers. Activation of this pathway is caused by genetic mutations that stabilize the beta-catenin protein (CTNNB1; 116806), allowing it to accumulate in the nucleus and form complexes with other members of the lymphoid enhancer factor and T-cell factor family of transcription factors (referred to collectively as LEF/TCFs; see TCF4, 602272) to activate transcription of target genes. Hovanes et al. (2001) reported that LEF1 is a target gene ectopically activated in colon cancer. The pattern of this ectopic expression is unusual because it derives from selective activation of a promoter for a full-length LEF1 isoform that binds beta-catenin, but not a second, intronic promoter that drives expression of a dominant-negative isoform. Beta-catenin/TCF complexes can activate the promoter for full-length LEF1, indicating that in cancer high levels of these complexes misregulate transcription to favor a positive feedback loop for Wnt signaling by inducing selective expression of full-length, beta-catenin-sensitive forms of LEF/TCFs. The significance of the findings was discussed by de Lau and Clevers (2001).

Merrill et al. (2001) showed that Lef1 and Tcf3 (604652) controlled differentiation of multipotent stem cells in mouse skin. Lef1 required Wnt signaling and stabilized beta-catenin to express hair-specific keratins and control hair differentiation. In contrast, Tcf3 acted independently of its beta-catenin-interacting domain to suppress features of epidermal differentiation, in which Tcf3 was normally shut off, and promote features of the follicle outer root sheath and multipotent stem cells. Lef1 lacking its beta-catenin-binding domain suppressed hair differentiation and supported sebocyte differentiation.

Yasumoto et al. (2002) found that functional cooperation between LEF1 and an MITF (156845) isoform, MITF-M, in several mammalian cell lines resulted in synergistic transactivation of the DCT (191275) promoter, an early melanoblast marker. Beta-catenin was required for efficient transactivation, but was dispensable for the interaction between MITF-M and LEF1.

The morphogenesis of organs as diverse as lungs, teeth, and hair follicles is initiated by a downgrowth from a layer of epithelial stem cells. During follicular morphogenesis, stem cells form this bud structure by changing their polarity and cell-cell contact. Jamora et al. (2003) showed that this process is achieved through simultaneous receipt of 2 external signals: a WNT protein (WNT3A; 606359) to stabilize beta-catenin, and a bone morphogenetic protein inhibitor (Noggin; 602991) to produce Lef1. Beta-catenin binds to and activates Lef1 transcription complexes that appear to act uncharacteristically by downregulating the gene encoding E-cadherin (192090), an important component of polarity and intercellular adhesion. When either signal is missing, functional Lef1 complexes are not made, and E-cadherin downregulation and follicle morphogenesis are impaired. In Drosophila, E-cadherin can influence the plane of cell division and cytoskeletal dynamics. Consistent with this notion, Jamora et al. (2003) showed that forced elevation of E-cadherin levels block invagination and follicle production. Jamora et al. (2003) concluded that their findings reveal an intricate molecular program that links 2 extracellular signaling pathways to the formation of a nuclear transcription factor that acts on target genes to remodel cellular junctions and permit follicle formation.

Hematopoietic stem cells (HSCs) have the ability to renew themselves and to give rise to all lineages of the blood. Reya et al. (2003) showed that the WNT signaling pathway has an important role in this process. Overexpression of activated beta-catenin expands the pool of HSCs in long-term cultures by both phenotype and function. Furthermore, HSCs in their normal microenvironment activate a LEF1/TCF reporter, which indicates that HSCs respond to WNT signaling in vivo. To demonstrate the physiologic significance of this pathway for HSC proliferation, Reya et al. (2003) showed that the ectopic expression of axin (603816) or a frizzled (603408) ligand-binding domain, inhibitors of the WNT signaling pathway, led to inhibition of HSC growth in vitro and reduced reconstitution in vivo. Furthermore, activation of WNT signaling in HSCs induced increased expression of HOXB4 (142965) and NOTCH1 (190198), genes previously implicated in self-renewal of HSCs. Reya et al. (2003) concluded that the WNT signaling pathway is critical for normal HSC homeostasis in vitro and in vivo, and provide insight into a potential molecular hierarchy of regulation of HSC development.

Epithelial mesenchymal transformation (EMT) of the medial edge epithelial seam creates palatal confluence. Nawshad and Hay (2003) showed that Tgfb3 (190230) brought about palatal seam EMT in mice by stimulating expression of Lef1 in medial edge epithelial cells. Tgfb3 activated Lef1 in the absence of beta-catenin via nuclear phospho-Smad2 (601366) and Smad4 (600993).

EDAR (604095) plays a key role in ectodermal differentiation via activation of the NF-kappa-B (see 164011) pathway. Using transfected human embryonic kidney cells and fibroblasts from mouse embryos defective in NF-kappa-B pathway components, Shindo and Chaudhary (2004) showed that EDAR signaling repressed LEF1-beta-catenin-dependent transcription independent of its stimulatory effect on NF-kappa-B activity. In addition, EDAR with an anhidrotic ectodermal dysplasia (129490)-associated mutation exhibited defects in both NF-kappa-B activation and LEF1/beta-catenin repression. Since LEF1/beta-catenin controls expression of EDA (300451), the results suggested negative feedback regulation of the EDA-EDAR axis.

Galceran et al. (2004) found that mouse Lef1 bound multiple sites in the Dll1 (606582) promoter in vitro and in vivo, and mutation of the Lef1 sites impaired expression of a reporter transgene in the presomitic mesoderm of embryonic mice.

Skokowa et al. (2006) found significantly decreased or absent LEF1 expression in arrested promyelocytes from patients with congenital neutropenia (see 202700). LEF1 decrease resulted in defective expression of downstream target genes, including CCND1 (168461), MYC (190080), and BIRC5 (603352). Promyelocytes from healthy individuals showed highest LEF1 expression. Reconstitution of LEF1 in early hematopoietic progenitors from 2 individuals with congenital neutropenia resulted in the differentiation of these progenitors into mature granulocytes. Competitive binding and chromatin immunoprecipitation (ChIP) assays showed that LEF1 directly bound to and regulated the transcription factor CEBPA (116897). The findings indicated that LEF1 plays a role in granulopoiesis.

TCF/LEF proteins form transcriptional units with CTNNB1 in the Wnt signaling pathway during embryogenesis and tumor formation. Yamada et al. (2006) had previously reported that NLK (609476) negatively regulates Wnt signaling via phosphorylation of TCF/LEF proteins. By yeast 2-hybrid and coimmunoprecipitation analyses, they found that Xenopus and human NARF (RNF138; 616319) interacted with NLK. NARF ubiquitinated TCF4 (TCF7L2; 602228) and LEF1, but not NLK, in a dose-dependent manner. Inclusion of wildtype NLK, but not kinase-dead NLK, enhanced the interaction between NARF and TCF4 or LEF1. NLK facilitated NARF-dependent ubiquitination of TCF4 and LEF1 and enhanced proteasome-mediated degradation of TCF4 and LEF1. Reporter gene assays confirmed that NARF inhibited TCF/LEF-dependent activation of a Wnt-responsive element. Expression of Narf in Xenopus embryos inhibited Ctnnb1-dependent secondary axis formation, and knockdown of NARF in HeLa cells enhanced WNT3A-dependent gene expression. Yamada et al. (2006) concluded that NARF is an NLK-associated negative regulator of Wnt signaling that ubiquitinates phosphorylated TCF/LEF proteins, targeting them for degradation.

Gattinoni et al. (2009) reported that induction of Wnt/beta-catenin signaling by inhibitors of Gsk3b (605004) or by Wnt3a arrested mouse Cd8 (see 186910)-positive T-cell development into effector T cells capable of cytotoxicity or Ifng (147570) production. Instead, Wnt signaling promoted expression of Tcf7 (189908) and Lef1 and generation of self-renewing multipotent Cd8-positive memory stem cells capable of proliferation and antitumor activity. Gattinoni et al. (2009) concluded that Wnt signaling has a key role in maintaining the self-renewing stem cell-like properties of mature memory CD8-positive T cells.

Using RT-PCR and flow cytometric analysis, Zhao et al. (2010) demonstrated that mouse Tcf7 and Lef1 were highly expressed in naive T cells, downregulated in effector T cells, and upregulated in memory T cells. Memory Cd8-positive T cells expressing the p45 Tcf7 isoform and beta-catenin had enhanced Il2 (147680) production capacity and enhanced effector capacity to clear Listeria monocytogenes. Zhao et al. (2010) concluded that constitutive activation of the Wnt pathway favors memory CD8 T-cell formation during immunization, resulting in enhanced immunity upon a second encounter with the same pathogen.

Using a genetic approach, Driessens et al. (2010) found no evidence that the beta-catenin pathway regulates T-cell memory phenotype, in contrast with the findings of Gattinoni et al. (2009). The findings of Driessens et al. (2010) suggested that the generation of Cd8-positive memory stem cells observed by Gattinoni et al. (2009) with the use of Gsk3b inhibitors was not a consequence of activation of the beta-catenin pathway, but was rather due activation of another Gsk3b-dependent pathway. In a reply, Gattinoni et al. (2010) noted that others, including Zhao et al. (2010) and Jeannet et al. (2010), had also identified Wnt and beta-catenin as crucial factors in postthymic Cd8-positive T-cell differentiation and memory development. Using Western blot analysis, Gattinoni et al. (2010) showed that addition of Wnt3a or Gsk3b inhibitor stabilized beta-catenin in primed Cd8-positive mouse T cells.

Gene Structure

Hovanes et al. (2000) determined that the LEF1 gene spans at least 52 kb and contains 12 exons. In addition, 2 alternative exons (exons 3a and 3b) appear to be located in intron 3. The 5-prime UTR is highly GC rich and contains 4 major alternative start sites, the first of which falls within an initiator-like consensus sequence. The promoter region contains no TATA box, but it has SP1 (189906)-binding sites, a GAGA site, and an E box.

Molecular Genetics

In a genomewide analysis of leukemic cells from 242 pediatric acute lymphocytic leukemia (ALL; 613065) patients using high resolution, single-nucleotide polymorphism (SNP) arrays and genomic DNA sequencing, Mullighan et al. (2007) identified mutations in genes encoding principal regulators of B-lymphocyte development and differentiation in 40% of B-progenitor ALL cases. Deletions were detected in LEF1, IKZF1 (603023), IKZF3 (606221), TCF3 (147141), and EBF1 (164343). The PAX5 (167414) gene was the most frequent target of somatic mutation, being altered in 31.7% of cases.

Biochemical Features

Love et al. (1995) reported the solution structure for a complex of the LEF1 HMG domain and basic region with its DNA-binding site. They found that LEF1 binding occurs in the minor groove through its HMG domain. It creates a sharp bend in the DNA that facilitates the binding of other transcription factors to adjacent sequences.

Mapping

By Southern blot analysis of DNA from panels of interspecies somatic cell hybrids, Milatovich et al. (1991) assigned LEF1 to 4cen-q31.2. They further refined the assignment to 4q23-q25 by in situ hybridization. The corresponding gene was assigned to distal mouse chromosome 3 by the study of recombinant inbred strains.

Animal Model

Lef1 is a sequence-specific DNA-binding protein that is expressed in pre-B and T lymphocytes of adult mice, and in the neural crest, mesencephalon, tooth germs, whisker follicles, and other sites during mouse embryogenesis. Van Genderen et al. (1994) generated mice carrying a homozygous germline mutation in the Lef1 gene that eliminated Lef1 protein expression and caused postnatal lethality. The mutant mice lacked teeth, mammary glands, whiskers, and hair, although they developed rudimentary hair follicles. The Lef1-deficient mice also lacked the mesencephalic nucleus of the trigeminal nerve, the only neural crest-derived neuronal populations. The mutant mice showed no obvious defects in lymphoid cell populations at birth. Van Genderen et al. (1994) suggested that Lef1 plays an essential role in the formation of several organs and structures that require inductive tissue interactions.

To test whether LEF1 patterning might be functionally important for hair patterning and morphogenesis, Zhou et al. (1995) used transgenic technology to alter the patterning and timing of human LEF1 expression over the surface ectoderm of mice. Striking abnormalities arose in the positioning and orientation of hair follicles, leaving a marked disruption of this normally uniform patterning. Moreover, elevated levels of LEF1 in the lip furrow epithelium of developing transgenic mice triggered these cells to invaginate, sometimes leading to the inappropriate adoption of hair follicle and tooth cell fates.

Kratochwil et al. (2002) investigated LEF1 function in inductive signaling during tooth development and concluded that FGF4 (164980) is a direct target of Lef1 and Wnt signaling. They observed that developmentally arrested tooth rudiments in Lef1 null mice (Van Genderen et al., 1994) failed to express Fgf4, Shh (600725), and Bmp4 (112262). Kratochwil et al. (2002) generated mice carrying a mutation to eliminate the interaction of LEF1 with CTNNB1, and concluded that the role of LEF1 in tooth development is dependent on its interaction with CTNNB1 and Wnt signaling. They showed that beads soaked with recombinant FGF4 protein induced the delayed expression of Shh in the epithelium and could fully overcome the developmental arrest of Lef1-deficient tooth germs. Using a chemical inhibitor of FGF signaling, they were able to mimic the arrest of tooth development seen in Lef1-deficient mice. Kratochwil et al. (2002) hypothesized that the sole function of LEF1 in odontogenesis may be to activate Fgf4 and to connect the Wnt and FGF signaling pathways at a specific developmental step.

By inserting the bacterial beta-galactosidase gene in-frame into the exon encoding the DNA-binding domain of Lef1, Galceran et al. (2004) created mice expressing a truncated form of Lef1 that could interact with beta-catenin but could not bind DNA. Homozygous mutant mice died perinatally. The vertebral column and the rib cage were severely malformed, suggesting that Lef1 is involved in the generation and patterning of paraxial mesoderm. Mutant embryos showed fusion of somites, defects in the rostrocaudal patterning of somites, and a lack or misrouting of neural crest-derived spinal nerves. They also showed abnormal expression of developmentally regulated transcription factors.