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H Clevers

Publications and source records attributed to H Clevers.

At least 91 records · Page 5Linked to original sources
PubMed1996

Selectively impaired development of intestinal T cell receptor gamma delta+ cells and liver CD4+ NK1+ T cell receptor alpha beta+ cells in T cell factor-1-deficient mice.

T cell factor-1 (Tcf-1) is a transcription factor that binds to a sequence motif present in several T cell-specific enhancer elements. In Tcf-1-deficient (Tcf-1-/-) mice, thymocyte development is partially blocked at the transition from the CD4-8+ immature single-positive stage to the CD4+8+ double-positive stage, resulting in a marked decrease of mature peripheral T cells in lymph node and spleen. We report here that the development of most intestinal TCR gamma delta+ cells and liver CD4+ NK1.1+TCR alpha beta+ (NK1+T) cells, which are believed to be of extrathymic origin, is selectively impaired in Tcf-1-/- mice. In contrast, thymic and thymus-derived (splenic) TCR gamma delta+ cells are present in normal numbers in Tcf-1-/- mice, as are other T cell subsets in intestine and liver. Collectively, our data suggest that Tcf-1 is differentially required for the development of some extrathymic T cell subsets, including intestinal TCR gamma delta+ cells and liver CD4+ NK1+T cells.

Animals↗
PubMed1996

Extensive alternative splicing and dual promoter usage generate Tcf-1 protein isoforms with differential transcription control properties.

Previously, we reported the isolation of cDNA clones representing four alternative splice forms of TCF-1, a T-cell-specific transcription factor. In the present study, Western blotting (immunoblotting) yielded a multitude of TCF-1 proteins ranging from 25-55 kDa, a pattern not simply explained from the known splice alternatives. Subsequent cDNA cloning, PCR amplification, and analysis by rapid amplification of 5' cDNA ends revealed (i) the presence of an alternative upstream promoter, which extended the known N terminus by 116 amino acids, (ii) the presence of four alternative exons, and (iii) the existence of a second reading frame in the last exon encoding an extended C terminus. Inclusion of the extended N terminus into the originally reported protein resulted in a striking similarity to the lymphoid factor Lef-1. Several of the TCF-1 isoforms, although less potent, mimicked Lef-1 in transactivating transcription through the T-cell receptor alpha-chain (TCR-alpha) enhancer. These data provide a molecular basis for the complexity of the expressed TCF-1 proteins and establish the existence of functional differences between these isoforms. Furthermore, the functional redundancy between Tcf-1 and Lef-1 explains the apparently normal TCR-alpha expression in single Tcf-1 or Lef-1 knockout mice despite the firm in vitro evidence for the importance of the Tcf/Lef site in the TCR-alpha enhancer.

Alternative Splicing↗
PubMed1995

Solution structure of the sequence-specific HMG box of the lymphocyte transcriptional activator Sox-4.

Two groups of HMG box proteins are distinguished. Proteins in the first group contain multiple HMG boxes, are non-sequence-specific, and recognize structural features as found in cruciform DNA and cross-over DNA. The abundant chromosomal protein HMG-1 belongs to this subgroup. Proteins in the second group carry a single HMG box with affinity for the minor groove of the heptamer motif AACAAAG or variations thereof. A solution structure for the non-sequence-specific C-terminal HMG box of HMG-1 has recently been proposed. Now, we report the solution structure of the sequence-specific HMG-box of the SRY-related protein Sox-4. NMR analysis demonstrated the presence of three alpha-helices (Val10-Gln22, Glu30-Leu41 and Phe50-Tyr65) connected by loop regions (Ser23-Ala49 and Leu42-Pro49). Helices I and II are positioned in an antiparallel mode and form one arm of the HMG box. Helix III is less rigid, makes an average angle of about 90 degrees with helices I and II, and constitutes the other arm of the molecule. As in HMG1B, the overall structure of the Sox-4 HMG box is L-shaped and is maintained by a cluster of conserved, mainly aromatic residues.

Amino Acid Sequence↗
PubMed1995

An assay for the identification of antigens recognized by cytotoxic T cells, based on transient transfection of COS cells.

The studies reported here describe methodology permitting the direct identification of antigens recognized by cytotoxic T lymphocytes. We demonstrated that bovine alloreactive CTL can detect a bovine MHC molecule transiently expressed in a COS cell population in a standard microcytotoxicity assay. We then showed that alloreactive CTL can detect cells expressing the bovine class I MHC molecule in a population of cells transfected with the plasmid containing the corresponding gene plus 100-fold as many plasmids containing an irrelevant gene. In addition, the transiently transfected COS cells can specifically restimulate CTL as detected by a standard microcytotoxicity assay using the target cell line. Overall, the results suggest that COS cells could be employed for the direct screening of an antigen or antigen gene library by immune CTL.

Animals↗
PubMed1995

The human TCF-1 gene encodes a nuclear DNA-binding protein uniquely expressed in normal and neoplastic T-lineage lymphocytes.

The TCF-1 gene encodes a putative transcription factor with affinity for a sequence motif occurring in a number of T-cell enhancers. TCF-1 mRNA was originally found to be expressed in a T cell-specific fashion within a set of human and mouse cell lines. In contrast, expression reportedly occurs in multiple nonlymphoid tissues during murine embryogenesis. We have now raised a monoclonal antibody to document expression and biochemistry of the human TCF-1 protein. As expected, the TCF-1 protein was detectable only in cell lines of T lineage. Its expression was always restricted to the nucleus. Immunohistochemistry on a panel of human tissues revealed that the TCF-1 protein was found exclusively in thymocytes and in CD3+ T cells in peripheral lymphoid tissues. Western blotting yielded a set of bands ranging from 25 kD to 55 kD, resulting from extensive alternative splicing. The TCF-1 protein was detectable in all samples of a set of 22 T-cell malignancies of various stages of maturation, but was absent from a large number of other hematologic neoplasms. These observations imply a T cell-specific function for TCF-1, a notion corroborated by recent observations on Tcf-1 knock-out mice. In addition, these results indicate that nuclear TCF-1 expression can serve as a pan-T-lineage marker in the diagnosis of lymphoid malignancies.

Amino Acid Sequence↗
PubMed1995

The human high mobility group (HMG)-box transcription factor TCF-1: novel isoforms due to alternative splicing and usage of a new exon IXA.

The C-terminal peptide sequences of the human lymphocyte-specific high mobility group (HMG)-box transcription factor TCF-1 are determined by alternative splice mechanisms affecting the exons VIII to X. Here we report, in addition to four splice forms described previously (TCF-1A, B, C, D), the identification of three novel transcripts designated TCF-1E, F, G. Cloning and sequencing of the novel cDNAs revealed (i) joining of the exons VIII and IX to an internal exon X splice acceptor site resulting in a new open reading frame (ORF) of 99 amino acids derived from exon X sequences, (ii) the identification of an additional functional splice acceptor site within exon X, and (iii) a new 81-nucleotide insertion between exon VIII and exon X sequences in a novel transcript form. Genomic cloning and sequence analysis of this transcribed segment of 81 basepairs revealed that it was bordered by canonical splice consensus sites and located in a distance of some 400 bp from both the exons IX and X. It was therefore termed exon IXA. Novel ORFs were generated as a consequence of these alternative splice mechanisms resulting in TCF-1 gene products with significantly different C-terminal peptide sequences, which are prone to selective protein-protein interactions or transactivating functions.

Alternative Splicing↗
PubMed1995

An HMG-box-containing T-cell factor required for thymocyte differentiation.

Two candidate genes for controlling thymocyte differentiation, T-cell factor-1 (Tcf-1) and lymphoid enhancer-binding factor (Lef-1), encode closely related DNA-binding HMG-box proteins. Their expression pattern is complex and largely overlapping during embryogenesis, yet restricted to lymphocytes postnatally. Here we generate two independent germline mutations in Tcf-1 and find that thymocyte development in (otherwise normal) mutant mice is blocked at the transition from the CD8+, immature single-positive to the CD4+/CD8+ double-positive stage. In contrast to wild-type mice, most of the immature single-positive cells in the mutants are not in the cell cycle and the number of immunocompetent T cells in peripheral lymphoid organs is reduced. We conclude that Tcf-1 controls an essential step in thymocyte differentiation.

Animals↗
PubMed1995

Circumvention of tolerance for the nuclear T cell protein TCF-1 by immunization of TCF-1 knock-out mice.

Molecular events that underlie the well-defined phenotypic changes of the differentiating thymocyte are poorly understood. A candidate gene to control thymocyte differentiation, T cell factor-1 (TCF-1)* encodes a DNA-binding protein. Its mRNA expression pattern is complex during embryogenesis, yet restricted to lymphocytes postnatally. Expression studies on TCF-1 protein have been hampered by the difficulty to raise antibodies due to extreme evolutionary conservation. TCF-1 knock-out mice, generated recently in our laboratory, have strongly decreased numbers of thymocytes, but are otherwise normal. We have used these mice to generate anti-TCF-1 antibodies. By immunization with a recombinant fusion protein, we show that TCF-1 knock-out mice readily yield antiserum titers against human and mouse TCF-1 protein. Wild-type littermates remain unresponsive to TCF-1 while they mount a high-titer antibody response to the fusion protein, Maltose Binding Protein (MBP). Subsequently, TCF-1-specific hybridomas could be prepared from the spleens of immunized knock-out mice. This study illustrates the almost complete tolerance of mice for human TCF-1 and demonstrates that this tolerance is readily broken by gene knock-out. Furthermore, the usefulness of knock-out mice for the generation of monoclonal antibodies against the gene product of interest is underscored.

Animals↗
PubMed1994

Large deletion of the peroxisomal acyl-CoA oxidase gene in pseudoneonatal adrenoleukodystrophy.

We have cloned the cDNA encoding human peroxisomal acyl-CoA oxidase, the first enzyme in the peroxisomal beta-oxidation of very long chain fatty acids. Its nucleotide sequence was found to be highly homologous (85%) to the rat cDNA counterpart. An 88% homology between rat and human was found in the COOH-terminal end of the cDNA which includes the Ser-Lys-Leu peroxisomal targeting signal common to many peroxisomal proteins. The gene spans approximately 30-40 kb and is poorly polymorphic. Southern blot analyses were performed in two previously reported siblings with an isolated peroxisomal acyl-CoA oxidase deficiency (pseudoneonatal adrenoleukodystrophy). A deletion of at least 17 kb, starting down-stream from exon 2 and extending beyond the 3' end of the gene, was observed in the two patients. These observations provide a molecular basis for the observed acyl-CoA oxidase deficiency in our family. In addition, our study will enable the characterization of the genetic defect in unrelated families with suspected acyl-CoA oxidase disorders.

Acyl-CoA Oxidase↗
PubMed1993

The Schizosaccharomyces pombe mating-type gene mat-Mc encodes a sequence-specific DNA-binding high mobility group box protein.

The Schizosaccharomyces pombe gene mat-Mc plays a determinative role in the sexual differentiation of the fission yeast. The mat-Mc protein has been suggested to belong to a novel family of so-called high mobility group (HMG) box proteins, characterized by homology to high mobility group-1 and -2 proteins. Several HMG box proteins, including the mammalian sex-determining gene product SRY and the lymphoid transcription factors TCF-1 and LEF-1, have been shown to bind to DNA in a sequence-specific fashion. To analyze possible DNA-binding properties of mat-Mc, we have cloned and expressed its putative HMG box in Escherichia coli. Gel retardation analysis revealed that the mat-Mc HMG box recognizes the AACAAAG heptamer in a sequence-specific fashion. Combined T-->C and A-->I substitutions on both strands of the AACAAAG heptamer, which change the surface of the major groove while leaving the minor groove intact, did not interfere with sequence-specific binding of mat-Mc. Methylation interference analysis confirmed that the mat-Mc HMG box contacts adenine residues in the minor groove. By using a circular permutation assay, the mat-Mc HMG box was observed to bend DNA. These results indicate that mat-Mc is indeed a member of the HMG box family with DNA-binding characteristics assigned earlier to other members of this novel transcription factor family.

Amino Acid Sequence↗
PubMed1993

The sequence-specific high mobility group 1 box of TCF-1 adopts a predominantly alpha-helical conformation in solution.

The High Mobility Group (HMG) 1 box is a protein motif that mediates DNA binding in a novel family of transcription-regulating proteins. Several members of this family, including the lymphoid-specific proteins TCF-1 and LEF-1 and the mammalian sex-determining factor SRY, carry a single HMG box with affinity for the minor groove of the heptamer motif AACAAAG or variations thereof. To initiate studies on the structural characteristics of the TCF-1 HMG box, we have expressed the 87-amino acid HMG box in milligram quantities in Escherichia coli and purified the soluble peptide to > 95% homogeneity. The peptide bound DNA with the same specificity as the complete protein and was capable of inducing DNA bending. Circular dichroism (CD) analysis revealed the TCF-1 HMG box to adopt an approximately 60% alpha-helix/40% random coil conformation in solution. In the presence of an equimolar amount of double-stranded DNA containing the cognate motif, the CD spectrum changed significantly, implying the induction of a structural modification upon DNA/protein association.

Amino Acid Sequence↗
PubMed1993

HB4 antibody recognizes a carbohydrate structure on lymphocyte surface proteins related to HB6, CDw75, and CD76 antigens.

Cells regulate the specificity of the carbohydrate chains on their membrane-bound glycoconjugates by differential expression of glycosyltransferases. In lymphocytes, beta-galactoside alpha 2,6-sialyltransferase is reportedly involved in the generation of epitopes recognized by HB6, CDw75, and CD76 mAb. The HB4 mAb binds to an Ag present on subpopulations of B and NK cells. We now show that this Ag represents another member of a set of neuraminidase-sensitive, alpha 2,6-sialyltransferase-generated sugar Ag. Transient expression of a cDNA encoding this enzyme in COS cells generated a minor population of HB4+ cells that was completely contained within the HB6+ COS cell population. Using various proteinases and an inhibitor of N-linked carbohydrate processing, we show both epitopes to represent components of N-glycosylated membrane proteins. Remarkably, porcine thyroglobulin, an alpha 2,6-NeuAc+ glycoprotein, is specifically recognized by both mAb. These data underline a close relationship between HB4 and HB6 epitopes and imply further that both mAb react with oligosaccharide chains irrespective of the carrier molecule nature. Thus, the terminal sugar residue sialic acid plays a pivotal role in at least four distinct epitopes that are expressed differentially in immune cells. This may point at an important role for these epitopes in biologic recognition.

Animals↗
PubMed1993

Ancestry and diversity of the HMG box superfamily.

The HMG box is a novel type of DNA-binding domain found in a diverse group of proteins. The HMG box superfamily comprises a.o. the High Mobility Group proteins HMG1 and HMG2, the nucleolar transcription factor UBF, the lymphoid transcription factors TCF-1 and LEF-1, the fungal mating-type genes mat-Mc and MATA1, and the mammalian sex-determining gene SRY. The superfamily dates back to at least 1,000 million years ago, as its members appear in animals, plants and yeast. Alignment of all known HMG boxes defined an unusually loose consensus sequence. We constructed phylogenetic trees connecting the members of the HMG box superfamily in order to understand their evolution. This analysis led us to distinguish two subfamilies: one comprising proteins with a single sequence-specific HMG box, the other encompassing relatively non sequence-specific DNA-binding proteins with multiple HMG boxes. By studying the extent of diversification of the superfamily, we found that the speed of evolution was very different within the various groups of HMG-box containing factors. Comparison of the evolution of the two boxes of ABF2 and of mtTF1 implied different diversification models for these two proteins. Finally, we provide a tree for the highly complex group of SRY-like ('Sox' genes), clustering at least 40 different loci that rapidly diverged in various animal lineages.

Amino Acid Sequence↗
PubMed1993

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 x 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.

Amino Acid Sequence↗
PubMed1993

Differential expression of the HMG box factors TCF-1 and LEF-1 during murine embryogenesis.

The recent identification of a number of T lymphocyte-specific enhancers has allowed the cloning of several novel transcription factors. Two of these, TCF-1 and LEF-1, contain a virtually identical DNA-binding domain of the High Mobility Group (HMG-1) box type. TCF-1 and LEF-1 originate from a recent gene duplication event as evidenced by comparison with the chicken homologue, chTCF. We have now analyzed the differential expression of these two transcription factors. In a panel of lymphoid cell lines, TCF-1 was exclusively expressed in the T cell lineage. In contrast, LEF-1 mRNA was detected at equivalent levels in pro- and pre-B cells and in all T lineage cells. In situ hybridization on murine embryos revealed that TCF-1 and LEF-1 were widely expressed at day 7.5 of gestation. At later stages, the expression patterns were complex and only partially overlapping. The expression of TCF-1 and LEF-1 coincided until day 10.5, when mRNAs were detected in limb buds, neural crest, pharyngeal arches and nasal process. At later time points (day 13.5 to 14.5), sites of overlapping expression included lung, the urogenital system, tooth buds, thymus and choroid plexus. Unique expression sites for TCF-1 included Reichert's membrane and trophectoderm-derived cells, the ribs and thoracic prevertebrae, craniofacial structures, the adrenal gland and meninges. Unique LEF-1 expression was observed in the tail prevertebrae, brain and inner ear. Postnatally, expression of both genes could only be detected in lymphoid tissues. These observations suggest that TCF-1 and LEF-1 exert differential functions during murine embryogenesis.

Adrenal Glands↗
PubMed1993

HMG box proteins in early T-cell differentiation.

The central theme of this review is the molecular basis for commitment of cells to the T-cell lineage. Principles of transcriptional regulation are illustrated by two examples; the role of GATA-1 during erythroid differentiation and the function of MyoD-like proteins in myogenesis. Several regulatory proteins have been described in the T-cell lineage. Here, we focus attention on the HMG box family of DNA binding proteins. This recently defined family can be divided in two subfamilies: the HMG/UBF and the TCF/SOX group. The first group contains at least two HMG boxes and binds DNA non-specifically, while the other group of proteins has one HMG box and interacts with DNA sequence-specifically. Characteristics of the most prominent members of both subfamilies will be discussed. In particular, we will address the role of HMG box proteins in controlling the expression of T-cell specific proteins during differentiation.

Amino Acid Sequence↗
PubMed1992

Heterodimeric complex formation with CD8 and TCR by bispecific antibody sustains paracrine IL-2-dependent growth of CD3+ CD8+ T cells.

During physiologic activation of mature CD8+ T cells, TCR and CD8 bind to the same Ag-complexed MHC class I molecule. Thereby, close proximity is induced between CD8 and the TCR/CD3 complex. During this engagement, CD8 may deliver TCR-independent signals via its associated protein tyrosine kinase, p56lck. We studied the potential biologic effects of close association between CD8 and TCR/CD3 complexes by using a bispecific antibody (bsAb) directed against both TCR and CD8 molecules. This hybrid hybridoma (quadroma)-produced bsAb binds as a monomeric molecule to CD3+ CD8+ but not CD3+ CD4+ T cells. The bsAb proved capable of inducing the cytotoxic effector function of cloned CD3+ CD8+ T cells but not of CD3+ CD4+ T cells. When the bsAb was presented to resting T cells by monocytes, proliferation of the CD3+ CD4+ but not the CD3+ CD8+ subset of T lymphocytes was induced. Parental anti-TCR antibody induced vigorous growth of cells of both subsets. Essentially identical results were obtained when bsAb was presented in an immobilized fashion. The unresponsiveness of the CD3+ CD8+ T cells with respect to mitogenesis could be restored by exogenous rIL-2. The data suggest that bsAb-induced activation differs from activation by monospecific anti-TCR antibody. The former appears to more closely mimic physiologic Ag-induced signaling, because it leads to a similar paracrine IL-2-dependent growth pattern. The bsAb may, therefore, be instrumental in studying T cell signaling pathways, in particular the role of CD8-associated p56lck therein.

Antigens, Differentiation, T-Lymphocyte↗