Hans Clevers

Identification and cloning of TCF-1, a T lymphocyte-specific transcription factor containing a sequence-specific HMG box.

CD3‐epsilon expression is controlled by a downstream T lymphocyte‐specific enhancer element. We report the identification of a T cell‐specific transcription factor, TCF‐1, binding to this element. The multimerized recognition motif of TCF‐1 constituted a T cell‐specific enhancer. Subsequent cloning of TCF‐1 identified three splice alternatives. TCF‐1 contained a single DNA‐binding HMG box most closely related to similar boxes in the putative mammalian sex‐determining gene SRY and in the Schizosaccharomyces pombe Mc mating type gene. TCF‐1 mRNA was expressed uniquely in T lymphocytes. Upon cotransfection into non‐T cells, TCF‐1 could transactivate through its cognate motif. These results identify TCF‐1 as a T cell‐specific transcription factor, which might play a role in the establishment of the mature T cell phenotype.

Sox-4, an Sry-like HMG box protein, is a transcriptional activator in lymphocytes.

Previous studies in lymphocytes have described two DNA‐binding HMG box proteins, TCF‐1 and LEF‐1, with affinity for the A/TA/TCAAAG motif found in several T cell‐specific enhancers. Evaluation of cotransfection experiments in non‐T cells and the observed inactivity of an AACAAAG concatamer in the TCF‐1/LEF‐1‐expressing T cell line BW5147, led us to conclude that these two proteins did not mediate the observed enhancer effect. We therefore searched for additional HMG box proteins. By a PCR‐aided strategy, we cloned Sox‐4, a gene with homology to the HMG box region of the sex determining gene SRY. Sox‐4 was expressed in T and pre‐B lymphocyte lines and in the murine thymus. Significantly, BW5147 T cells did not express Sox‐4. Recombinant Sox‐4 bound with high affinity (Kd 3 × 10(−11) M) to the minor groove of the AACAAAG motif, most likely contacting all seven base pairs. In contrast with observations on TCF‐1 and LEF‐1, cotransfection with Sox‐4 unveiled a transactivating capacity, which mapped to its serine‐rich C terminus. This region remained functional upon grafting onto a GAL4 DNA‐binding domain. Sox‐4 is thus the first ‘classical’ transcription factor in the Sox gene family with separable DNA‐binding and transactivation domains. Our observations indicate that a detailed understanding of T cell‐specific gene control must integrate the concerted activity of at least three tissue‐specific HMG box genes.

TCF: Lady Justice casting the final verdict on the outcome of Wnt signalling.

Abstract The Wnt signalling cascade plays an important role during embryonic patterning and cell fate determination and is highly conserved throughout evolution. Factors of the TCF/LEF HMG domain family (Tcfs) are the downstream effectors of this signal transduction pathway. Upon Wnt signalling, a cascade is initiated that results in the translocation of βcatenin to the nucleus, where it interacts with Tcf to generate a transcriptionally active complex. This bipartite transcription factor is targeted to the upstream regulatory regions of Tcf target genes. In the absence of Wnt signals, βcatenin is degraded in the cytoplasm via the ubiquitinproteasome pathway. Several proteins are instrumental in achieving this tight regulation of βcatenin levels in the cell, including adenomatous polyposis coli (APC), GSK3 β, and Axin/Conductin. Deregulation of the Wnt signalling pathway is implicated in several forms of cancer, such as colon carcinoma and melanoma. This deregulation is achieved via mutation of APC, βcatenin or Axin, resulting in elevated βcatenin levels and the presence of constitutively active Tcfβcatenin complexes in the nucleus. The accompanying inappropriate activation of target genes is considered to be a critical, early event in this carcinogenesis. In addition to regulating βcatenin levels, normal healthy cells have evolved a second level of regulation, by manipulating the activity of the Tcf proteins themselves. In the absence of Wnt signalling, Tcf complexes with several transcriptional repressor proteins ensuring active repression of Tcf target genes. In this review the dual role of Tcf proteins in the Wnt signalling cascade will be discussed.

Sequence-specific interaction of the HMG box proteins TCF-1 and SRY occurs within the minor groove of a Watson-Crick double helix.

The high mobility group I (HMG) box is proposed to mediate DNA binding in a novel group of transcription‐regulating proteins. Two of these, the proteins encoded by the T cell‐specific TCF‐1 and the mammalian sex‐determining gene SRY, carry a single HMG box with specificity for the heptamer motif A/T A/T C A A A G. We have now analysed the mode of interaction of the HMG boxes of TCF‐1 and SRY with this motif. Methylation interference footprinting revealed that both HMG boxes contacted adenines on both strands in the minor groove, whereas no major groove guanine contacts were discerned. Diethylpyrocarbonate (DEPC) carbethoxylation interference footprinting of TCF‐1 indicated the absence of major groove contacts on positions 5, 6 and 7 of the motif. Carbethoxylation interference was observed, however, on positions 2, 3 and 4 and to a lesser extent on position 1 in the major groove. Combined T‐‐‐‐C and A‐‐‐‐I substitution, which changes the surface of the major groove but leaves the minor groove intact, did not interfere with sequence‐specific binding by TCF‐1 and SRY. These observations indicate that recognition of the heptamer motif by the HMG boxes of the distantly related TCF‐1 and SRY proteins predominantly occurs through nucleotide contacts in the minor groove.

Identification of APC2, a homologue of the adenomatous polyposis coli tumour suppressor

The adenomatous polyposis coli (APC) tumour-suppressor protein controls the Wnt signalling pathway by forming a complex with glycogen synthase kinase 3beta (GSK-3beta), axin/conductin and betacatenin. Complex formation induces the rapid degradation of betacatenin. In colon carcinoma cells, loss of APC leads to the accumulation of betacatenin in the nucleus, where it binds to and activates the Tcf-4 transcription factor (reviewed in [1] [2]). Here, we report the identification and genomic structure of APC homologues. Mammalian APC2, which closely resembles APC in overall domain structure, was functionally analyzed and shown to contain two SAMP domains, both of which are required for binding to conductin. Like APC, APC2 regulates the formation of active betacatenin-Tcf complexes, as demonstrated using transient transcriptional activation assays in APC -/- colon carcinoma cells. Human APC2 maps to chromosome 19p13.3. APC and APC2 may therefore have comparable functions in development and cancer.

Very Long-term Self-renewal of Small Intestine, Colon, and Hair Follicles from Cycling Lgr5+ve Stem Cells

The intestinal epithelium and the hair follicle represent examples of rapidly self-renewing tissue in adult mammals. We have recently identified a novel stem cell gene Lgr5 expressed in multiple adult tissues. At the bottoms of crypts in small intestine and colon as well as in hair follicles, Lgr5 marks cycling cells with stem cell properties (Barker et al. 2007; Jaks et al. 2008). Using an inducible Lgr5-Cre knockin allele in conjunction with the Rosa26-LacZ Cre reporter strain, long-term lineage-tracing experiments were performed in adult mice. The Lgr5(+ve) crypt-based cell generated all epithelial lineages during a 14-month period, implying that it represents the stem cell of the small intestine and colon. Similarly, lineage tracing during a 14-month period revealed that Lgr5(+ve) cells located in the bulge of the hair follicle sustained multiple rounds of hair growth. These observations support the counterintuitive notion that Lgr5(+ve) cells are actively cycling, yet represent long-term stem cells of these adult, self-renewing tissues.

Human Intestinal Tissue with Adult Stem Cell Properties Derived from Pluripotent Stem Cells

Summary Genetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many applications are limited due to the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. Here, using an endogenous LGR5-GFP reporter, we derived adult stem cells from hPSCs that gave rise to functional human intestinal tissue comprising all major cell types of the intestine. Histological and functional analyses revealed that such human organoid cultures could be derived with high purity and with a composition and morphology similar to those of cultures obtained from human biopsies. Importantly, hPSC-derived organoids responded to the canonical signaling pathways that control self-renewal and differentiation in the adult human intestinal stem cell compartment. This adult stem cell system provides a platform for studying human intestinal disease in vitro using genetically engineered hPSCs.

Functional redundancy between Apc and Apc2 regulates tissue homeostasis and prevents tumorigenesis in murine mammary epithelium

Aberrant Wnt signaling within breast cancer is associated with poor prognosis, but regulation of this pathway in breast tissue remains poorly understood and the consequences of immediate or long-term dysregulation remain elusive. The exact contribution of the Wnt-regulating proteins adenomatous polyposis coli (APC) and APC2 in the pathogenesis of human breast cancer are ill-defined, but our analysis of publically available array data sets indicates that tumors with concomitant low expression of both proteins occurs more frequently in the ‘triple negative’ phenotype, which is a subtype of breast cancer with particularly poor prognosis. We have used mouse transgenics to delete Apc and/or Apc2 from mouse mammary epithelium to elucidate the significance of these proteins in mammary homeostasis and delineate their influences on Wnt signaling and tumorigenesis. Loss of either protein alone failed to affect Wnt signaling levels or tissue homeostasis. Strikingly, concomitant loss led to local disruption of β-catenin status, disruption in epithelial integrity, cohesion and polarity, increased cell division and a distinctive form of ductal hyperplasia with ‘squamoid’ ghost cell nodules in young animals. Upon aging, the development of Wnt activated mammary carcinomas with squamous differentiation was accompanied by a significantly reduced survival. This novel Wnt-driven mammary tumor model highlights the importance of functional redundancies existing between the Apc proteins both in normal homeostasis and in tumorigenesis.

PS21 - 103. Multipotent pancreatic progenitor cells in 3D culture of human pancreatic ductal tissue: potential relevance for beta cell therapy in Type 1 diabetes

An attractive regenerative medicine approach towards β-cell replacement therapy is the derivation of insulinsecreting cells from stem/progenitor cells. We applied a novel 3-dimensional (3D) matrigel-based culture system to generate human pancreatic tissue (‘pancreatic organoids’) in vitro as a potential source for human β-cells.