The Lymphoid Enhancer Factor (LEF)/TCF family of High Mobility Group (HMG) box transcription factors function in a developmental signaling pathway driven by Wnt proteins, secreted ligands that direct cell fate, polarity and growth changes in receptive cells. Wnt ligand recognition by transmembrane receptors at the cell surface initiates a signaling cascade that stabilizes the normally labile armadillo repeat protein β-catenin and re-directs it to the nucleus. In the nucleus, any of the four mammalian LEF/TCF proteins can bind tightly to Wnt-triggered nuclear β-catenin protein and through their single HMG DNA binding domains, tether the potent transcription activating domain in β-catenin to Wnt target genes. In the absence of Wnt signaling, some of the LEF/TCF factors have been shown to engage in active gene silencing through recruitment of co-repressors (1
). Much of the current work is focused on the function of specific LEF/TCF proteins in Wnt signaling, but little is directed towards understanding how their full complement of alternative isoforms and their expression patterns influence signaling.
During embryogenesis Wnt signaling occurs at myriad sites of tissue differentiation and this is reflected in the pattern of expression of LEF/TCF proteins. Embryonic expression patterns for each of the four known mammalian LEF/TCF proteins (LEF-1, TCF-1, TCF-3 and TCF-4) show distinct but broad, overlapping distributions (5
). Each of the LEF/TCF factors have been genetically inactivated in mice, and the phenotypes that result from these knock-outs are unique for each factor (8
). Only a subset of the tissues that express an embryonic LEF/TCF are missing or damaged in the knock-out mice, suggesting a certain level of expression and functional redundancy among LEF/TCF proteins. Redundancy has been shown experimentally in LEF-1/TCF-1 double knock-out mice which die during embryogenesis with multiple, fatal deficiencies (12
). However, LEF/TCF proteins are not entirely redundant. For example, both LEF-1 and TCF-1 are expressed to high levels in developing T lymphocytes in the thymus, but only TCF-1 knock-out mice have defects in T cell differentiation (10
). Recently, transcription co-repressors have been shown to bind to TCF-1 and Xenopus
TCF-3 and silence transcription in the absence of Wnt signaling (2
). One of these co-repressors, CtBP, binds to the C-terminal tail of xTCF-3, a region that is encoded by alternative splicing and is most homologous to the C-terminus of TCF-1E and TCF-4E (although the CtBP binding site is present in TCF-4E and not TCF-1E) (2
). We show in this manuscript that although this C-terminal ‘E’ region has been found to be a common gene product for all of the TCF genes, the LEF-1
gene is unique in that it cannot encode a similar sequence. Thus, the genetic locus for each family member may encode a unique subset of isoforms that are required at specific sites of differentiation.
Hours after birth, the broad patterns of LEF/TCF expression disappear and only very restricted patterns of LEF/TCF mRNA can be detected by northern analysis of mouse tissues (5
). For example, both LEF-1 and TCF-1 mRNA are easily detected in the thymus, but very little mRNA can be detected in any other tissue (13
). It is important to note that this type of northern data can be misleading; expression of LEF/TCFs has been detected by RT–PCR or in situ
hybridization of tissues that appear negative by northern analysis. Such tissues include skin, hair follicles, intestine, colon and testes. These tissues are continually replenished by waves of differentiating cells that derive from a small population of mitotically active stem cells. With the exception of the thymus, which is loaded with mitotically active differentiating lymphocytes, stem cells comprise a small fraction of each tissue and are not easily detected by northern analysis. LEF/TCF expression has been detected in some of these small populations. The apparent unifying pattern of LEF-1 expression is that expression is silenced or dramatically down-regulated when cells reach a non-cycling, differentiated state. The best examples are from studies of differentiating B and T lymphocytes and cells at the base of hair follicles. In these tissues, LEF-1 expression can no longer be detected in mature B lymphocytes or in keratin secreting cells of the hair shaft, and only at very low levels in mature, immunocompetent T lymphocytes (7
In contrast to these low levels of expression, moderate to high levels of LEF-1 are frequently detected in tumors, even in cancerous cells from tissues that are normally negative for LEF-1 expression (18
; K.Hovanes and M.L.Waterman, unpublished observations). Either these cancers derive from a tiny fraction of cells that normally express LEF-1, or its expression is activated in the transformation process. Such is the case for LEF-1 expression in transformed cell lines derived from mature T and B lymphocytes, cells that normally do not express much LEF-1 mRNA. LEF-1 expression has also been detected frequently in melanomas and colon cancer (18
). A frequent problem in colon cancer and melanomas involves ectopic and constitutive activation of the Wnt pathway due to genetic mutations that lead to a de-regulation of β-catenin. Thus, β-catenin becomes an abundant and constitutively available co-activator in the nucleus and if a LEF/TCF factor is present, inappropriate activation of gene targets and cell transformation is possible. Defining the mechanisms that drive this aberrant LEF-1 expression is important for understanding how the cancers initiate and how they progress to malignancy.
Here we describe the characterization of the LEF-1 gene, its multiple isoforms, its promoter and its preferential activity in lymphocytes. We find that the structure of the human LEF-1 gene shows remarkable conservation with the human TCF-1 gene, but that a few striking differences set it apart from its family members.