Erythroid-specific inhibition of the tal-1 intragenic promoter is due to binding of a repressor to a novel silencer.

The basic helix-loop-helix tal-1 gene plays a key role in hematopoiesis, and its expression is tightly controlled through alternative promoters and complex interactions of cis-acting regulatory elements. tal-1 is not expressed in normal T cells, but its transcription is constitutive in a large proportion of human T cell leukemias. We have previously described a downstream initiation of tal-1 transcription specifically associated with a subset of T cell leukemias that leads to the production of NH(2)-truncated TAL-1 proteins. In this study, we characterize the human promoter (promoter IV), embedded within a GC-rich region in exon IV, responsible for this transcriptional activity. The restriction of promoter IV usage is assured by a novel silencer element in the 3'-untranslated region of the human gene that represses its activity in erythroid but not in T cells. The silencer activity is mediated through binding of a tissue-specific nuclear factor to a novel protein recognition motif (designated tal-RE) in the silencer. Mutation of a single residue within the tal-RE abolishes both specific protein binding and silencing activity. Altogether, our results demonstrate that the tal-1 promoter IV is actively repressed in cells of the erythro-megakaryocytic lineage and that this repression is released in leukemic T cells, resulting in the expression of the tal-1 truncated transcript.

Anti-apoptotic activity of p53 maps to the COOH-terminal domain and is retained in a highly oncogenic natural mutant.

The tumour suppressor p53 plays a complex role in the regulation of apoptosis. High levels of wild type p53 potentiate the apoptotic response, while physiological range, low levels of the protein have an anti-apoptotic activity in serum starved immortalized fibroblasts. Here we report that primary fibroblast-like cells that show normal growth control are also efficiently protected from apoptosis by the endogenous p53 activity. The capacity to inhibit apoptosis is not restricted to the wild type protein: the R-->H175 p53 mutant fully retains the anti-apoptotic activity of the wild type p53, providing a possible explanation for its high oncogenicity. Using a series of point and deletion mutants of p53 under the control of tetracycline-regulated promoter we show that certain mutants, like the wild type, protect cells at low levels but lead to apoptosis when overexpressed. This latter effect is lost upon deletion of a proline-rich domain in the NH2 part of the protein. The anti-apoptotic activity can be mapped to the extreme carboxy-terminal part of the protein and is therefore independent of other well characterized p53 activities. Our results add a new level of complexity to the network of interactions mediated by p53 in normal physiology and pathology.

Physical interaction of the bHLH LYL1 protein and NF-kappaB1 p105.

The LYL1 gene was first identified upon the molecular characterization of the t(7;9)(q35;p13) translocation associated with some human T-cell acute leukemias (T-ALLs). In adult tissues, LYL1 expression is restricted to hematopoietic cells with the notable exclusion of the T cell lineage. LYL1 encodes a basic helix-loop-helix (bHLH) protein highly related to TAL-1, whose activation is also associated with a high proportion of human T-ALLs. A yeast two-hybrid system was used to identify proteins that specifically interact with LYL1 and might mediate its activities. We found that p105, the precursor of NF-kappaB1 p50, was the major LYL1-interacting protein in this system. The association between LYL1 and p105 was confirmed both in vitro and in vivo in mammalian cells. Biochemical studies indicated that the interaction was mediated by the bHLH motif of LYL1 and the ankyrin-like motifs of p105. Ectopic expression of LYL1 in a human T cell line caused a significant decrease in NF-kappaB-dependent transcription, associated with a reduced level of NF-kappaB1 proteins.

An immunohistochemical study of TAL-1 protein expression in leukaemias and lymphomas with a novel monoclonal antibody, 2TL 242.

Fifty formalin fixed, paraffin-embedded cases of T-acute lymphoblastic leukaemia (T-ALL) from 12 bone marrow trephines and 38 lymph nodes were stained with a new monoclonal antibody, 2TL 242, raised against recombinant TAL1 protein. The antibody recognizes TAL-1 polypeptides of molecular weight 39 and 41 kD (full length). In addition, a variety of other leukaemias and lymphomas were also stained with 2TL 242. Twenty-four of the 50 cases of T-ALL showed nuclear positivity, ranging from 10 to 90 per cent of leukaemic cells. A positive staining reaction was nuclear and stippled in pattern. Nuclear staining was not seen in any other type of leukaemia or lymphoma. Five cases of follicular lymphoma showed diffuse cytoplasmic staining of variable intensity. Although some background staining is obtained with this antibody, positive nuclear staining is easily distinguishable. This monoclonal antibody has a potential role in primary diagnosis and in the detection of minimal residual disease in T-ALL.

SCL/Tal-1 expression in T-acute lymphoblastic leukemia: an immunohistochemical and genotypic study.

A comparative study of the immunohistochemical (Stem cell leukemia/T-cell acute leukemia [SCL/TAL-1] protein expression) and genotypic (deletions in the SCL/tal-1 gene) findings in T-acute lymphoblastic leukemia (T-ALL) is presented. Formalin-fixed tissue from 50 cases of T-ALL were stained with a novel monoclonal antibody, 2TL 242, which recognizes SCL/TAL-1 protein. Twenty-four cases showed nuclear immunolabeling of leukemic cells. Nuclear positivity was not evident in any other type of leukemia or lymphoma tested with the antibody. Genotypic analysis of 25 cases of T-ALL showed a deletion involving the SCL/tal-1 gene in nine cases. These results suggest that protein expression is not dependent on derangement of the SCL/tal-1 gene, because immunohistochemical detection of the protein was noted in the presence and absence of a tal-d1 deletion.

Loss of TAL-1 protein activity induces premature apoptosis of Jurkat leukemic T cells upon medium depletion.

Transcriptional activation of the tal-1 gene occurs in -30% of patients with T cell Acute Lymphoblastic Leukemia and is therefore likely to be involved in human T cell leukemogenesis. However, the TAL-1 protein functional properties involved in this process have not been assessed so far. We have derived a clonal subline of the Jurkat T cell line which produced solely a mutant truncated form of TAL-1 protein. Sequencing of genomic DNA and cDNAs showed that the only transcribed tal-1 allele of this mutant subline harbored a G nucleotide insertion at codon 270. The resulting frameshift modifies TAL-1 residues 272-278 and creates a stop at codon 279. Although the deletion of the 53 carboxy-terminal residues of the TAL-1 protein did not directly affect the TAL-1 basic helix-loop-helix domain (residues 185-243), it had drastic effects on TAL-1 functional properties, since the mutant subline exhibited a dramatic decrease of protein binding activity to the TAL-1 DNA consensus sequence. Growth curves indicated that the mutant subline exhibited premature apoptosis upon medium depletion or serum reduction when compared with the parental cells. However, no difference between Jurkat and the mutant subline was observed in etoposide- or Fas/APO-1-triggered apoptosis. Stable expression of the mutant TAL-1 protein in Jurkat cells resulted in a phenotype that was similar to that of the mutant Jurkat subline, indicating that the TAL-1 mutant protein behaved like a dominant negative mutant and that the premature apoptosis of the mutant subline upon medium depletion was the consequence of the loss of TAL-1 protein activity.

Expression of TAL-1 proteins in human tissues.

Rearrangement of the tal-1 gene (also known as SCL or TCL-5) occurs in at least 25% of T-cell acute lymphoblastic leukemias (T-ALLs) and results in the aberrant expression of tal-1 mRNA in the neoplastic cells. Also, tal-1 mRNA is constitutively expressed in erythroid precursors and megakaryocytes. This report describes a direct immunocytochemical study of the distribution and localization of TAL-1 protein in normal human tissues and cell lines using four monoclonal antibodies raised against recombinant TAL-1 proteins. One of these reagents recognizes a protein of 41 kD molecular weight in in vitro-translated TAL-1 proteins, two others recognize proteins of 39 and 41 kD molecular weight, and the fourth antibody also recognizes a TAL-1 protein of 22 kD in addition to the 39- and 41-kD proteins. These anti-TAL-1 antibodies label the nuclei of erythroid precursor cells and megakaryocytes in fetal liver and adult bone marrow. The punctate pattern of nuclear labeling suggests that TAL-1 may comprise part of a novel nuclear structure, similar to that recently found for the PML protein. The nuclei of T cell lines known to express mRNA encoding the full-length TAL-1 protein (eg, CCRF-CEM, RPMI 8402, and Jurkat) are also labeled. A study of normal human tissues (including thymus) showed labeling of smooth muscle, some tissue macrophages, and endothelial cells. TAL-1 protein is undetectable in other cell types. These reagents may play an important role in the diagnosis of T-ALL and could also be used in the context of lymphoma diagnosis on routinely fixed material.

Distinct DNase-I hypersensitive sites are associated with TAL-1 transcription in erythroid and T-cell lines.

The tal-1 gene, frequently activated in human T-cell acute lymphoblastic leukemia (T-ALL), is expressed in the erythroid, megakaryocytic, and mast cell lineages during normal hematopoiesis. To gain further insight into the molecular mechanisms that control tal-1 expression, we investigated tal-1 chromatin structure in erythroid/megakaryocytic cell lines and in T-cell lines either with or without tal-1 rearrangements. Tal-1 transcription was shown to be monoallelic in Jurkat, a T-cell line that expresses tal-1 in the absence of apparent genomic alteration of the locus. Methylation studies indicated that the tal-15' GC-rich region behaves like a CpG island, hypomethylated in normal cells, and methylated de novo on transcriptionally inactive alleles in established cell lines. Five major DNase-I hypersensitive sites (HS) were mapped in the tal-1 locus. HS I, IV, and V were exclusively observed in the erythroid/megakaryocytic cell lines that express tal-1 from the promoters 1a and 1b. HS II was weak in hematopoietic cell lines, absent in Hela, and greatly enhanced in Jurkat, suggesting that this region might be implicated in the cis-activation of tal-1 promoter 1b in this cell line. HS III was weak in HEL and Jurkat, and greatly enhanced in DU528, a T-cell line that bears a t (1;14) and initiates tal-1 transcription within exon 4. These results suggest that distinct regulatory elements are associated with the use of the different tal-1 promoters.

GATA-and SP1-binding sites are required for the full activity of the tissue-specific promoter of the tal-1 gene.

The tal-1 gene, which is frequently activated in human T cell acute leukemias (T-ALLs), codes for a protein of the basic helix-loop-helix family (b-HLH) and potentially a transcription factor. In human and murine hematopoiesis tal-1 is expressed during the differentiation of the erythroid, megakaryocytic and mastocytic cell lineages. The expression of tal-1 appears to be comodulated with that of the transcription factor GATA-1 gene, suggesting that the GATA-1 protein may regulate the tal-1 gene activity in these hematopoietic lineages. To get further insights into the molecular mechanisms that control tal-1 expression, we have isolated 5' sequences of the murine gene and compared them to their human counterparts. The 5' flanking sequences from the two genes show several regions of high homology. The alignment of both sequences enabled us to predict that similarly, to the human, the mouse gene contains two alternative first exons (Ia and Ib). Remarkably, in both species, the proximal region of the tissue-specific exon Ia (i.e. gene segment -122 to +1) contains two GATA-motifs (at -65 and -33) and one SP-1 consensus binding site (-59). Mobility shift assays demonstrate that GATA proteins are able to interact with both GATA-motifs in a sequence specific fashion, but with different efficiencies. Moreover transfection studies show that the GATA-1 protein directly mediates tal-1 transcription by interacting with the -122/+1 fragment, defined as a minimal promoter in erythroid cells. Mutagenesis of the promoter establishes that the -33 GATA-binding site present in this fragment is critical for tal-1 expression in erythroid cells, but by itself does not lead to full promoter activity. Indeed, further mutations show that the second -65 GATA-binding site and the binding motif for SP1 (-59) significantly contribute to the overall activity of the proximal tal-1 promoter. Altogether, our data provide evidence that GATA-1 cooperates with the transcription factor SP1 to mediate the erythroid-specific expression of the tal-1 gene.

Characterization of translocation t(1;14)(p32;q11) in a T and in a B acute leukemia.

The TAL1 locus on chromosome band 1p32 is rearranged in 15 to 29% of human T-cell acute lymphoblastic leukemias (T-ALLs). These alterations consist of either a tald submicroscopic deletion (12-26% of T-ALL) or a t(1;14)(p32;q11) chromosomal translocation (3% of childhood T-ALL). Both types of alterations preferentially affect the 5' part of the TAL1 locus. Their main consequence appears to be transcriptional activation of the TAL1 gene. We have characterized two cases of t(1;14)(p32;q11) in ALL. Both affect the TCR delta gene segments at 14q11 and the 5' part of the TAL1 locus at 1p32. The first case represented a 'classical' t(1;14), associated with T-ALL. Its analysis indicates the use of a recombination signal-like sequence localized in the third exon of TAL1 in the translocation process. In the other case, the rearrangement to the D delta region occurred 5' to the TAL1 transcription start sites. This case exhibited a B-lymphoid immunophenotype thus suggesting that the putative oncogenicity of TAL1 activation is not restricted to T-cell malignancies.

Expression of tal-1 and GATA-binding proteins during human hematopoiesis.

Tal-1 rearrangements are associated with nearly 30% of human T acute lymphoblastic leukemia. Tal-1 gene encodes a putative transcription factor with a basic helix-loop-helix domain and is known to be predominantly expressed in hematopoietic cells. We investigated the pattern of tal-1 expression in purified human hematopoietic cells by in situ hybridization and reverse transcriptase polymerase chain reaction analysis. Both methods demonstrated that the tal-1 gene is expressed in megakaryocytes and erythroblasts as well as in basophilic granulocytes. In addition, our results indicate that the tal-1 1A promoter, which contains two consensus GATA-binding sites, is active mainly in these lineages. Because the GATA-1 gene is known to transactivate several genes specific for the erythroid, megakaryocytic, and mastocytic/basophilic lineages, we studied GATA-1 expression in these purified hematopoietic cells. We found that GATA-1 and tal-1 genes are coexpressed in these three lineages. Remarkably, the expression of both genes is downmodulated during erythroid and megakaryocytic terminal maturation. In immature hematopoietic cells, tal-1 and GATA-1 genes are coexpressed in committed progenitors cells (CD34+/CD38(2+)), whereas they are not detectable in the most primitive cells (CD34(2+)/CD38-). In contrast, GATA-2 is strongly expressed in both most primitive and committed progenitors cells, whereas GATA-3 is mostly detected in most primitive ones. Altogether our results strongly suggest that GATA-1 modulates the transcription of tal-1 during the differentiation of the erythroid, megakaryocytic, and basosophilic lineages.

Molecular analysis of a variant 18;22 translocation in a case of lymphocytic lymphoma.

We previously reported a 5' rearrangement of the BCL2 locus in a t(18;22) variant translocation found in a lymphocytic lymphoma. Primary structure analysis of both rearranged chromosomes confirmed the localization of the breakpoint in the so-called vcr region (for variant cluster region) that encompasses Z-DNA stretches 5' of the BCL2 locus, and in between J lambda 1 and C lambda 1 segments on the IGL locus. A 1,027 nucleotide segment from chromosome 22 was repeated on both derivative chromosomes 18q+ and 22q-. This segment contained an octanucleotide that was also present in the normal chromosome 18 close to the breakpoint. As a consequence of the translocation, a normal-sized BCL2 transcript was overexpressed in tumor cells.

A third tal-1 promoter is specifically used in human T cell leukemias.

A common feature of T cell acute lymphoblastic leukemias (T-ALLs) is the presence of structural alteration of the 5' part of the tal-1 locus, localized on chromosomal band 1p32. These alterations consist of either a t(1;14)(p32;q11) chromosomal translocation (3% of T-ALLs) or tald submicroscopic deletion (12-25% additional T-ALLs). We have characterized a case of T-ALL with t(1;14)(p32;q11) in which, unlike the majority of t(1;14), the recombination with the T cell receptor delta elements affected the 3' side of the tal-1 locus. In this case, tal-1 transcription is initiated from a promoter located within the fourth exon similarly to the DU 528 cell line. In a T-ALL bearing a t(1;14) affecting the 5' part of tal-1, two types of tal-1 transcripts were observed, namely those probably initiated from the D delta region juxtaposed to tal-1 by the translocation, and those from the exon 4 promoter. It is interesting that this exon 4 promotion was also found in leukemic T cell lines and T-ALL samples without apparent tal-1 genomic alteration. In contrast, no transcript initiated from the exon 4 promoter was found in T-ALL with tald1 or tald2 deletion. In these cells, tal-1 is expressed via SIL-tal-1 fused transcripts. Finally, this exon 4 initiation was detected neither in normal bone marrow, nor in malignant cells from the erythroid/megakaryocytic lineages. Taken as a whole, these data suggest that the exon 4 promoter is specifically active in T cell lineage.

Two site-specific deletions and t(1;14) translocation restricted to human T-cell acute leukemias disrupt the 5' part of the tal-1 gene.

Analysis of several cases of t(1:14)(p32;q11) translocation present in 3% of T-cell acute leukemias (T-ALL) has revealed the tal-1 gene. This gene encodes a helix-loop-helix protein. It has been found to be expressed in normal bone marrow and in leukemic T-cell and erythroleukemia cell lines, but not in normal T cells. Recently, a site-specific deletion, tald, renamed tald1 in this paper, has been detected in a high proportion of pediatric T-ALL, which arose by a site-specific DNA recombination between tal-1 and a new locus termed SIL. In this study we searched for structural rearrangements within tal-1 in a panel of 134 non-selected leukemic patients (including 66 with T-ALL). Only 6% of patients with T-ALL harbored the tald1 deletion. A second specific deletion termed tald2 was observed in another 6% of T-ALL patients; it involves another site within tal-1 plus the same site as tald1 in the SIL locus. Similarly to tald1 deletion, tald2 junctions harbor structural characteristics that are reminiscent of aberrant recombinase activity. Moreover, we report a detailed analysis of the tal-1 gene structure. Transcription analysis and in vitro translation data are consistent with the differential expression of several TAL-1 protein species containing the HLH motif but differing in their amino terminus. Taken together, our data indicate that t(1;14) translocations and both tald deletions disrupt the 5' part of the tal-1 gene, placing its entire coding sequences under the control of the regulatory elements of the TCR-delta gene or the SIL gene, both of which are normally expressed in T-cell lineage.

Structure and evolutionary origin of the human granzyme H gene.

Among the molecules proposed to be involved in cytotoxic T lymphocyte (CTL), natural killer (NK) and lymphokine activated killer (LAK) cell-mediated lysis are the granzymes, a family of serine proteases stored in the cytoplasmic granules of CTLs, NK and LAK cells. In addition to the granzymes A and B, a third member of this family has been cloned in man and designated granzyme H. We present the complete gene sequence including the 5' promoter region and demonstrate that the granzyme H sequence represents a functional gene expressed in activated T cells. Granzyme H shows the highest degree (greater than 54%) of amino acid sequence homology with granzyme B and cathepsin G and, like these genes, consists of five exons separated by introns at equivalent positions. The evolutionary history of granzyme H has been analyzed by reconstructing an evolutionary tree for granzyme sequences. We provide evidence that interlocus recombination between the ancestral genes of granzyme B and granzyme H occurred about 21 million years ago, leading to a replacement of exon 3, intron 3 and part of exon 4 in human granzyme H by human granzyme B sequences. Our results suggest that the ancestral gene of granzyme H is more closely related to cathepsin G and granzyme B than to the murine granzymes C to G. Thus, granzyme H does not represent a human counterpart of the known murine granzymes A to G. It diverged from cathepsin G before mammalian radiation and should, therefore, exist in other mammalian lineages as well.

c-myc gene expression in a leukemic T-cell line bearing a t(8;14) (q24;q11) translocation.

We have examined the expression of the c-myc protooncogene in human T-cell leukemic KE-37R cells carrying a t(8;14) (q24;q11) translocation. The breakpoint on chromosome 8 is located at 2.2 kb downstream of c-myc exon 3 and the 3' part of the TcR-alpha gene (14q11) has been juxtaposed to c-myc. Our results showed that the steady-state levels of c-myc RNA transcripts were increased and the P1/P2 ratio of c-myc promoter utilization did not change, indicating that preferential utilization of P2 was maintained in the rearranged gene. High levels of electrophoretically normal p64 and 67 c-myc proteins were detected and both products kept their instability. In addition, transcription from promoter P0 was not detectable. Our results suggest that the activation of the gene is likely to result from its juxtaposition to the enhancer element of the TcR-alpha gene located downstream of the Ca region which stimulates constitutive synthesis of normal c-myc transcripts from the rearranged allele.

In vitro differentiation from a pluripotent human CD4+CD8+ thymic cloned cell into four phenotypically distinct subsets.

Human thymic cell differentiation is almost totally unknown. In the present study we developed an in vitro system using human thymic cloned cells to analyze precursor-progeny relationship. We obtained several CD4+CD8+ double positive thymic clones that could give rise after several weeks in culture only to either CD4 or CD8 single positive clones. By contrast we isolated a unique pluripotent thymic double positive clone, termed B12, which differentiated into four phenotypically distinct T cell clones, namely double-positive CD4+CD8+, double-negative CD4-CD8- or either single-positive phenotype. We derived stable subclones representative of each phenotype and we showed by molecular analysis that they expressed the same TCR. Utilization of either CD3 or anticlonotypic mAb revealed that this TCR expressed by the four subclones was functional.

Isolation of a Friend recombinant polytropic virus with a T-cell-restricted leukemogenicity.

Hematopoietic malignant cells of various types were isolated from ICFW mice inoculated as newborn or adult with F-MuLV and grafted into recipient mice. After repeated in vivo cell transplants, several recombinant polytropic viruses (also termed MCF or dualtropic viruses) were isolated from tumors by limiting dilution. Two virus isolates designated RA1-17 and EA1-17 were recovered from the spleen and the omentum, respectively, of the same grafted animal. When inoculated into newborn mice, RA1-17 induced erythroblastosis similar to that induced by other Friend recombinant polytropic viruses. Remarkably, EA1-17 induced T-cell leukemia after a short latency. To our knowledge, this is the first description of a polytropic recombinant virus with a T-cell tropism isolated after inoculation of F-MuLV.

Structural organization of the hCTLA-1 gene encoding human granzyme B.

Cytotoxic T lymphocytes (CTLs) and natural killer/lymphokine-activated cells produce granzymes, a family of serine esterase proteins located in cytoplasmic granules. These might be involved in different cytotoxic pathways. We report the structural organization of the human gene encoding granzyme B (hCTLA-1). A 4.75-kb genomic DNA fragment containing all the sequences of granzyme B-encoding cDNA clones has been sequenced. The gene is composed of five exons and four introns. A comparison with the genomic organization of murine CCP1/CTLA-1 showed very similar structure and a 76% nucleotide homology in the coding sequences. This suggests that both genes may have a common ancestor. No typical regulatory element was detected in the 1160 bp upstream from the ATG start codon. The detection of a second locus related to hCTLA-1 is also described.

Correlation between T cell receptor gamma delta isotypic forms and cytotoxic activity: analysis with human T cell clones and lines.

Three biochemically distinct isotypic forms of the human T cell receptor (TcR) gamma delta structure can be expressed at the cell membrane. This unique variation in structure of TcR, which is due to C gamma gene segments utilization, prompted us to look for isotype-association functional differences. In this regard, we have developed human T cell clones or lines from normal thymus or peripheral blood from several patients. In the present report, we have selected by phenotypic, biochemical, and TcR gene rearrangement analysis representative pairs of IL2-dependent clones or lines for each TcR gamma delta isotypic form. The results showed a lack of correlation between the TcR isotypes and the ability of the cells to proliferate in response to TcR stimulation mediated through the CD3 molecular complexes. By contrast, despite the fact that all of these representative cells exhibit an NK-like activity, as measured by their ability to kill K562, the strongest lytic activity was observed with the cells having the disulfide-bonded form of the receptor. Moreover only those latter cells were able to efficiently kill the LAK-sensitive Daudi cell line.