Chromatin immunoselection defines a TAL-1 target gene
1998; Springer Nature; Volume: 17; Issue: 17 Linguagem: Inglês
10.1093/emboj/17.17.5151
ISSN1460-2075
Autores Tópico(s)RNA Research and Splicing
ResumoArticle1 September 1998free access Chromatin immunoselection defines a TAL-1 target gene Sylvia Cohen-Kaminsky Sylvia Cohen-Kaminsky INSERM, U474, Hématologie Moléculaire, Hôpital Henri Mondor, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France Search for more papers by this author Leïla Maouche-Chrétien Leïla Maouche-Chrétien INSERM, U474, Hématologie Moléculaire, Hôpital Henri Mondor, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France Search for more papers by this author Luigi Vitelli Luigi Vitelli Istituto Superiore di Sanita, Department of Hematology and Oncology, Viale Regina Elena, 299, 00161 Rome, Italy Search for more papers by this author Marie-Antoinette Vinit Marie-Antoinette Vinit INSERM, U474, Hématologie Moléculaire, Hôpital Henri Mondor, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France Search for more papers by this author Isabelle Blanchard Isabelle Blanchard INSERM, U474, Hématologie Moléculaire, Hôpital Henri Mondor, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France Search for more papers by this author Masayugi Yamamoto Masayugi Yamamoto Centre for Tsukuba Advanced Research and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, 305 Japan Search for more papers by this author Cesare Peschle Cesare Peschle Istituto Superiore di Sanita, Department of Hematology and Oncology, Viale Regina Elena, 299, 00161 Rome, Italy T.Jefferson University, Kimmel Cancer Center, BLSB, 233 South 10th Street, Philadelphia, PA, 19107-5541 USA Search for more papers by this author Paul-Henri Roméo Corresponding Author Paul-Henri Roméo INSERM, U474, Hématologie Moléculaire, Hôpital Henri Mondor, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France Search for more papers by this author Sylvia Cohen-Kaminsky Sylvia Cohen-Kaminsky INSERM, U474, Hématologie Moléculaire, Hôpital Henri Mondor, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France Search for more papers by this author Leïla Maouche-Chrétien Leïla Maouche-Chrétien INSERM, U474, Hématologie Moléculaire, Hôpital Henri Mondor, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France Search for more papers by this author Luigi Vitelli Luigi Vitelli Istituto Superiore di Sanita, Department of Hematology and Oncology, Viale Regina Elena, 299, 00161 Rome, Italy Search for more papers by this author Marie-Antoinette Vinit Marie-Antoinette Vinit INSERM, U474, Hématologie Moléculaire, Hôpital Henri Mondor, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France Search for more papers by this author Isabelle Blanchard Isabelle Blanchard INSERM, U474, Hématologie Moléculaire, Hôpital Henri Mondor, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France Search for more papers by this author Masayugi Yamamoto Masayugi Yamamoto Centre for Tsukuba Advanced Research and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, 305 Japan Search for more papers by this author Cesare Peschle Cesare Peschle Istituto Superiore di Sanita, Department of Hematology and Oncology, Viale Regina Elena, 299, 00161 Rome, Italy T.Jefferson University, Kimmel Cancer Center, BLSB, 233 South 10th Street, Philadelphia, PA, 19107-5541 USA Search for more papers by this author Paul-Henri Roméo Corresponding Author Paul-Henri Roméo INSERM, U474, Hématologie Moléculaire, Hôpital Henri Mondor, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France Search for more papers by this author Author Information Sylvia Cohen-Kaminsky1, Leïla Maouche-Chrétien1, Luigi Vitelli2, Marie-Antoinette Vinit1, Isabelle Blanchard1, Masayugi Yamamoto3, Cesare Peschle2,4 and Paul-Henri Roméo 1 1INSERM, U474, Hématologie Moléculaire, Hôpital Henri Mondor, 51 avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France 2Istituto Superiore di Sanita, Department of Hematology and Oncology, Viale Regina Elena, 299, 00161 Rome, Italy 3Centre for Tsukuba Advanced Research and Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, 305 Japan 4T.Jefferson University, Kimmel Cancer Center, BLSB, 233 South 10th Street, Philadelphia, PA, 19107-5541 USA ‡S.Cohen-Kaminsky and L.Maouche-Chrétien contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:5151-5160https://doi.org/10.1093/emboj/17.17.5151 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Despite the major functions of the basic helix–loop–helix transcription factor TAL-1 in hematopoiesis and T-cell leukemogenesis, no TAL-1 target gene has been identified. Using immunoprecipitation of genomic fragments bound to TAL-1 in the chromatin of murine erythro-leukemia (MEL) cells, we found that 10% of the immunoselected fragments contained a CAGATG or a CAGGTG E-box, followed by a GATA site. We studied one of these fragments containing two E-boxes, CAGATG and CAGGTC, followed by a GATA motif, and showed that TAL-1 binds to the CAGGTG E-box with an affinity modulated by the CAGATG or the GATA site, and that the CAGGTG–GATA motif exhibits positive transcriptional activity in MEL but not in HeLa cells. This immunoselected sequence is located within an intron of a new gene co-expressed with TAL-1 in endothelial and erythroid cells, but not expressed in fibroblasts or adult liver where no TAL-1 mRNA was detected. Finally, in vitro differentiation of embryonic stem cells towards the erythro/megakaryocytic pathways showed that the TAL-1 target gene expression followed TAL-1 and GATA-1 expression. These results establish that TAL-1 is likely to activate its target genes through a complex that binds an E-box–GATA motif and define the first gene regulated by TAL-1. Introduction The tal-1 gene (also known as scl or TCL-5), was identified via a chromosomal translocation involving human chromosome 1p32 in T-cell acute lymphocytic leukemia (T-ALL) (Begley et al., 1989; Finger et al., 1989; Bernard et al., 1990; Chen et al., 1990). Further studies have shown that aberrant activation of the tal-1 gene occurs in most patients with T-ALL (Bash et al., 1995) with diverse molecular mechanisms, including chromosomal translocations, interstitial deletions and transactivation without detectable chromosomal alteration. Finally transgenic mice have demonstrated a direct role of TAL-1 in T-cell leukemogenesis (Condorelli et al., 1996; Kelliher et al., 1996; Larson et al., 1996; Aplan et al., 1997). The normal function of TAL-1 is the regulation of hematopoiesis (Green, 1996; Shivdasani and Orkin, 1996) and embryonic angiogenesis (Visvader et al., 1998). Mice homozygous for deletion of the tal-1 gene die in utero by embryonic day 10 as a result of the absence of blood formation (Robb et al., 1995; Shivdasani et al., 1995), indicating that TAL-1 has a critical function very early during hematopoietic differentiation. Furthermore, tal-1 null embryonic stem (ES) cells failed to produce any hematopoietic lineage and to contribute to hematopoiesis in chimeric mice, implying that TAL-1 is also critical for definitive hematopoiesis (Porcher et al., 1996; Robb et al., 1996). Finally, transgenic rescue of hematopoietic defects of tal-1−/− embryos has established a role for TAL-1 in angiogenesis (Visvader et al., 1998). TAL-1 belongs to the bHLH class of transcription factors (Baer, 1993). The bHLH domain allows both the formation of protein dimers and sequence-specific DNA recognition, and bHLH dimers bind to E-box motifs which have the general sequence CANNTG (Murre et al., 1989). TAL-1 proteins cannot bind to DNA as homodimers but are able to bind DNA after dimerization with the ubiquitously expressed E2A gene products, E47 and E12 (Hsu et al., 1991, 1994b). CASTing experiments have shown that the TAL-1/E2A heterodimers bind to the preferred sequence 5′-AACAGATGGT-3′ (Hsu et al., 1994a). Using an artificial reporter gene containing multiple copies of the TAL-1/E2A binding sequence, transcriptional activity of the TAL-1/E47 heterodimer was examined in transiently transfected murine fibroblasts. While the E47 homodimer strongly activated the reporter gene, the TAL-1/E47 heterodimer was much less active, suggesting either a negative or a weak positive regulatory role of TAL-1 (Hsu et al., 1994c). The pivotal functions of TAL-1 may be accomplished by DNA binding and tight co-operation with at least two partners, the LIM-only protein LMO2 and GATA-1. TAL-1 and LMO2 genes are co-expressed during primitive and definitive hematopoiesis (Green et al., 1992; Kallianpur et al., 1994; Robb et al., 1996), are activated in T-ALL (Begley et al., 1989; Boehm et al., 1991; Rabbitts, 1994), and the two proteins are functionally related. Furthermore, like TAL-1, LMO2 is also a T-cell oncogene when expressed as a transgene in mice (Boehm et al., 1991; Royer-Pokora et al., 1991), and LMO2−/− and TAL-1−/− mice display the same phenotype (Warren et al., 1994). The other potential partner of TAL-1 is GATA-1, a transcription factor present in all stages of vertebrate erythropoiesis. GATA-1 binds to the WGATAR motif present in the promoters and enhancers of several erythroid-specific genes (reviewed in Weiss and Orkin, 1995), and exerts positive or negative transactivation effects (Aird et al., 1994; Raich et al., 1995; Weiss and Orkin, 1995; Briegel et al., 1996). An essential role for GATA-1 in erythropoiesis has been established through gene disruption. Indeed GATA-1−/− ES cells fail to contribute to the mature erythroid compartment in chimeric mice (Pevny et al., 1991) while expression of a normal GATA-1 transgene in the mutant ES cells rescues erythroid development (Simon et al., 1992). The absence of erythropoiesis obtained in the TAL-1, LMO2 and GATA-1 null mutations suggests that these three proteins have closely related roles in erythroid differentiation. As LMO2 can bind to both GATA-1 and TAL-1 in erythroid cell lines, it has been postulated that it may act as a physical bridge between TAL-1 and GATA-1 proteins to settle a multimeric complex (Osada et al., 1995). Recent CASTing experiments have defined a CAGGTG-GATA bipartite DNA motif (Wadman et al., 1997) that binds a multimolecular complex including TAL-1, E47, LMO2, GATA-1 and the LIM-domain binding protein Ldb1 (Agulnick et al., 1996; Jurata et al., 1996), and generates a positive transcriptional activity (Wadman et al., 1997). Therefore, co-operation between TAL-1, LMO2 and a member of the GATA family may be involved in the regulation of TAL-1 target genes. Despite its major role in hematopoiesis (Green, 1996; Shivdasani and Orkin, 1996) and leukemogenesis (Aplan et al., 1992a, 1997; Condorelli et al., 1996; Kelliher et al., 1996; Larson et al., 1996), as well as the identification of interacting partners (Valge-Archer et al., 1994; Wadman et al., 1994; Osada et al., 1995), no TAL-1 target gene has been identified so far. Furthermore, the sequence initially defined to bind TAL-1 in vitro (Hsu et al., 1994a) and the newly identified in vitro target (Wadman et al., 1997) have not been found in regulatory regions of genes involved in hematopoiesis regulation. In order to identify in vivo TAL-1 binding sites as well as target genes regulated by TAL-1 protein, we set up a strategy based on immunoselection of DNA–protein complexes in chromatin, and we describe here one of the isolated target sequence that binds TAL-1 and is located within an intron of a new gene whose expression is consistent with TAL-1 regulation. Results Isolation of in vivo TAL-1 target genomic fragments by chromatin immunopurification To identify TAL-1 target sequences, we developed an in vivo DNA–protein immunopurification strategy that enriches for short fragments bound to TAL-1 in chromatin (Figure 1). We chose the murine erythro-leukemia (MEL) cell line for this immunopurification method for two reasons. First, MEL cells have been widely used to study the molecular and cellular mechanisms that regulate terminal erythroid differentiation (Visvader et al., 1991; Aplan et al., 1992b; Green et al., 1992; Murrell et al., 1995) and secondly, TAL-1 has major functions in this cell line (Aplan et al., 1992b). We obtained 450 ng of immunoprecipitated DNA, starting from 3×108 cells, i.e. 0.00025% of the initial DNA, and the average insert size was 500 bp. One fifth of the immunoselected material was cloned and 1000 recombinant clones from the library were ordered, among which 60 were sequenced. As expected from the absence of known TAL-1 target genes, none of the cloned fragments sequenced was recorded in sequence databases but interestingly, a high proportion (5/60, i.e. 8.3%) of potential TAL-1 target sequences contained a GATA motif close to an E box of the CAGATG or the CAGGTG type (Table I). As TAL-1 and GATA-1 are considered to be functionally related in erythroid differentiation (for review see Shivdasani and Orkin, 1996), we reasoned that these fragments would be of the utmost interest as potential TAL-1 targets in MEL cells. We therefore selected one 474 bp fragment, whose sequence is shown in Figure 2, for further studies for the following reasons. (i) It contained two E-boxes in tandem spaced by two nucleotides, one of which is of the CAGATG type and the other of the CAGGTG type. Furthermore, the second E box (CAGGTG) lies 13 bp 5′ from a consensus GATA binding site. (ii) This fragment was located in a genomic region highly sensitive to micrococcal nuclease in MEL cells, indicating an open chromatin configuration around the immunoprecipitated region. The high micrococcal nuclease sensitivity of the immunoprecipitated fragment was assessed by comparison with the CD2 locus which is not transcriptionally activated in MEL cells, and needed 5- to 10-fold more nuclease to be digested (data not shown). Therefore, this fragment was a good candidate as a TAL-1 binding site, and we attempted first to determine if the region containing the two potential TAL-1 binding sites and the GATA motif could be a regulatory region. Figure 1.The in vivo immunoselection strategy used to isolate TAL-1 target sequences. Nuclei were isolated, treated with paraformaldehyde to stabilize in situ DNA–protein interactions, and then partially digested with micrococcal nuclease. The soluble chromatin fraction was loaded on a discontinuous caesium chloride gradient to select DNA–protein complexes and subjected to immunoprecipitation using monoclonal anti-TAL-1 antibodies to enrich for TAL-1-bound chromatin. After dissociation of the nucleoprotein complexes, proteins were analyzed by Western blot, and the DNA fragments cloned and sequenced to select potential TAL-1 in vivo binding sites for further investigations. Download figure Download PowerPoint Figure 2.Sequence of the genomic fragment immunoselected. The sequence of the immunoprecipitated 474 bp DNA fragment is shown. The E-box1 (CAGATG), E-box2 (CAGGTG) and the GATA binding sites are shown in bold and underlined. Download figure Download PowerPoint Table 1. Sequences containing an E-box closely associated to a GATA motif immunoselected in MEL cells CAGATG box CAGGTG box GATA-(4 bp)-CAGATG CAGGTG-(9 bp)-GATA CAGATG-(9 bp)-GATA CAGGTG-(12 bp)-GATA CAGGTG-(13 bp)-GATA The immunoprecipitated fragment binds TAL-1 in vitro We studied the ability of the two E-boxes to bind TAL-1 using MEL nuclear extracts. A complex of similar mobility as the TAL-1/E2A-containing complex was observed when an oligonucleotide containing the two E-boxes was used in electromobility shift assays (EMSAs) (Figure 3, lanes 1 and 3) and supershift experiments using an anti-TAL-1 monoclonal antibody showed that this complex actually contains TAL-1 (Figure 3, lanes 2 and 4). To identify which E box binds TAL-1, we first used oligonucleotides that contained only the CAGATG E-box (Figure 3, lane 5) or the CAGATG E-box followed by a mutated CAGGTG E-box (Figure 3, lane 6). No TAL-1-containing complex could be obtained with these probes, suggesting that TAL-1 binds to the CAGGTG E-box. However, a 20 bp oligonucleotide (the length of the TAL-1/E2A oligonucleotide) that contained the CAGGTG E-box could not efficiently bind TAL-1 (Figure 3, lane 7). As the E-box1–E-box2 oligonucleotide contained two more nucleotides at its 3′-end, we used a 22 bp oligonucleotide that contained only the CAGGTG E-box and the same 3′-end nucleotides, and showed that this oligonucleotide binds TAL-1 (Figure 3, lanes 8 and 9). Finally, we showed that an oligonucleotide containing the CAGGTG E-box and a mutated CAGATG E-box also binds TAL-1 (Figure 3, lanes 10 and 11), demonstrating that, in vitro, TAL-1 can bind the CAGGTG E-box located in the immunoprecipitated fragment. Figure 3.Gel retardation assay using MEL nuclear extracts and wild-type or mutated E-box1–E-box2 oligonucleotides. EMSAs with MEL nuclear extracts were performed using oligonucleotide probes containing the previously described TAL-1/E2A high-affinity binding site (TAL-1/E2A oligonucleotide, lanes 1 and 2), the E-box1–E-box2 sequence (lanes 3 and 4), the E-box1 sequence alone (lane 5), the E-box1 linked to a mutated E-box2 (mutant a, lane 6), the E-box2 oligonucleotide α (lane 7), the E-box2 oligonucleotide β (lanes 8 and 9) or the E-box2 linked to a mutated E-box1 (mutant b, lanes 10 and 11). Supershift experiments performed using anti-TAL-1 monoclonal antibody are shown on lanes 2, 4, 9 and 11. The band corresponding to the TAL-1/E2A heterodimer is indicated by an arrow. Download figure Download PowerPoint The GATA-1 site increases TAL-1 binding affinity We then explored the role of the GATA motif located 13 bp downstream from the CAGGTG E-box. Gel-shift experiments showed that the probe containing the two E-boxes and the GATA site generated numerous complexes (Figure 4A, lane 4). One complex contained TAL-1, and TAL-1 affinity for this composite sequence was higher than for the E-box1–E-box2 probe, and similar to the one observed for the TAL-1 consensus probe (Figure 4A, lanes 2, 3 and 4). We determined whether the GATA binding site can modulate the TAL-1 binding affinity to the CAGGTG E-box. Competition experiments were performed using the TAL-1/E2A oligonucleotide as a probe and increasing amounts of wild-type or mutant E-box1–E-box2–GATA oligonucleotides as competitors. A typical result of such an experiment is shown in Figure 4B, and indicated that the E-box1–E-box2–GATA oligonucleotide (Figure 4B, lane 5) was more efficient in competition than the E-box1–E-box2–mutantGATA oligonucleotide (Figure 4B, lane 3) and less efficient than the mutant E-box1–E-box2–GATA oligonucleotide (Figure 4B, lane 8). Quantification of the TAL-1-containing complex indicated a ratio of 2 between the E-box1–E-box2–GATA and the E-box1–E-box2–mutantGATA oligonucleotides, and a ratio of 1.5 between the mutant E-box1–E-box2–GATA and the E-box1–E-box2–GATA oligonucleotides. Altogether these results showed that TAL-1 binds to the CAGGTG E-box and that the TAL-1 binding affinity can be modulated positively (by the GATA binding site) or negatively (by the CAGATG E-box). Figure 4.The GATA binding site potentiates the DNA binding activity of TAL-1. (A) EMSAs with MEL nuclear extracts were performed using oligonucleotide probes containing the GATA sequence derived from the immunoprecipitated fragment (GATA oligonucleotide, lane 1), the two E-boxes without a downstream GATA site (E-box1–E-box2 oligonucleotide, lane 2), the TAL-1/E2A high-affinity binding site (TAL-1/E2A oligonucleotide, lane 3), and the two E-boxes followed by the GATA site (E-box1–E-box2–GATA oligonucleotide, lane 4). Bands corresponding to GATA-1- and TAL-1-containing complexes are indicated by arrows. (B) EMSAs with MEL nuclear extracts were performed using the TAL-1/E2A oligonucleotide as radioactive probe. A supershift experiment, performed using anti-TAL-1 antibody is shown on lane 2 and competition experiments using E-box1–E-box2–mutantGATA oligonucleotide, E-box2 oligonucleotide α, E-box1–E-box2–GATA, E box1, E-box1–mutantE-box2–GATA and mutantE-box1–E-box2–GATA oligonucleotides are shown respectively on lanes 3–8. The band corresponding to the TAL-1/E2A heterodimer is indicated by an arrow. Download figure Download PowerPoint TAL-1 binds to the in vivo target as a part of a multicomponent complex present only in normal erythroid cells As our observations of MEL cells might not reflect the TAL-1 binding during hematopoiesis, we investigated the binding of nuclear proteins obtained from cultures of purified human hematopoietic progenitor cells (HPCs) in liquid suspension (Condorelli et al., 1995). In this system HPCs are induced to undergo erythroid or granulopoietic determination in the first culture week, and terminal differentiation in the second week. In all experiments, nuclear extracts prepared from cultures at day 9 were used. The E-box1–E-box2–GATA oligonucleotide generated multiple complexes that were detected with nuclear extracts obtained from erythroid cells, and not with nuclear extracts obtained from granulocytic cells, indicating specific erythroid complexes (Figure 5, compare lanes 1 and 2, and lanes 3 and 4). One of the complexes (indicated by an arrow in Figure 5) has a mobility similar to the complex previously described (Wadman et al., 1997) using a CAGGTG type E-box followed by a GATA-site oligonucleotide, and shown to contain LMO2, GATA-1, TAL-1, E2A and Ldb1 (Figure 5, lanes 1 and 3). Interestingly, no TAL-1/E2A complex was observed, indicating that TAL-1 is essentially tied up in the multicomponent complex present in erythroid cells and not in granulocytic cells. Figure 5.A TAL-1-containing multicomponent complex binds the E-box1–E-box2–GATA composite motif in erythroid cells and is unilineage-specific. EMSAs using nuclear extracts from normal erythroid (lanes 1 and 3) or granulopoietic (lanes 2 and 4) cells, were performed with the oligonucleotide probe containing the in vitro consensus TAL-1 binding site followed by a GATA site 9 bp downstream (lanes 1 and 2), or the E-box1–E-box2–GATA oligonucleotide derived from the immunoselected sequence (lanes 3 and 4). The band corresponding to the TAL-1/E2A/LMO2/GATA-1 high-mobility complex is indicated by an arrow and the GATA-1 containing complex is also shown. Download figure Download PowerPoint The in vivo immunoprecipitated TAL-1 target sequence behaves as a cis regulatory acting sequence with positive transcriptional activity To determine if the immunoprecipitated TAL-1 target sequence is a transcriptional regulatory region we used transient transfection experiments. Transcriptional activity of the E-box1–E-box2–GATA sequence cloned 5′ to the minimal promoter from the glycophorin B gene (Rahuel et al., 1992) was studied using the Dual Luciferase™ Reporter Assay System (Figure 6). Contrary to other studies that have used tandems of identical TAL-1/E2A binding sites of the CAGATG type (Hsu et al., 1994c; Nielsen et al., 1996) or of the CAGGTG type followed by a GATA site (Wadman et al., 1997), we chose to work with only one copy of each oligonucleotide, which is closer to the in vivo context. Transcriptional activities of the wild-type or mutated E-box1–E-box2–GATA–glycophorin B (GpB) luciferase reporter constructs were compared in erythrocytic (MEL), erythro/megakaryocytic (HEL) and non-hematopoietic (HeLa) cells. No transcriptional activity was detected in HeLa cells that do not express TAL-1, while similar transcriptional activities were obtained in MEL and HEL cells that express TAL-1 (Figure 6) showing a correlation between the transcriptional activity of this sequence and TAL-1 expression. The wild-type E-box1–E-box2 GATA–GpB construct produced a 2-fold increase of luciferase activity as compared with the GpB minimal construct, and mutation of E-box1 produced a 4-fold increase in luciferase activity indicating that the CAGGTG–GATA motif acts as a positive cis-acting sequence whose function is negatively modulated by E-box1 (Figure 6). Double mutations that abolished both the E-box1 and E-box2, or the E-box1 and GATA binding sites resulted in background transcriptional activity demonstrating a co-operativity between TAL-1 and GATA-1 (Figure 6). Similar results were obtained when oligonucleotides were inserted in opposite orientation indicating an orientation-independent transcriptional activity (data not shown). These data showed that the region containing the two E-boxes and the GATA site from the immunoprecipitated fragment behaves as a cis regulatory acting sequence with a positive transcriptional activity that can be tuned by the repressive activity mediated by E-box1 (CAGATG). Figure 6.The oligomeric TAL-1 containing complex is functional and generates positive transcriptional activity. MEL (open bar) or HeLa (hatched bar) cells were transiently transfected with the Firefly luciferase reporter plasmid constructs indicated together with a Renilla luciferase control reporter (pRL-TK). After 24 h, the cells were harvested, extracts made, and aliquots used for quantification of the luminescent signals from each of the two luciferase reporter enzymes. The values represent the average of three independent experiments using different plasmid preparations for each construct. The values are shown in arbitrary units and (H) indicates the standard error to the mean. Download figure Download PowerPoint The immunoprecipitated sequence is located within an intron of a new gene Using the immunoprecipitated sequence as a probe, we screened a murine genomic library and got several phages that encompassed the probe. One of them, containing a 18 kb insert, was cut with various restriction enzymes and the DNA fragments that overlapped or lay around the immunoprecipitated sequence were used in an exon-trapping assay. We found that a 5 kb NcoI–NcoI fragment (Figure 7A) that started within the immunoprecipitated sequence contained three exons. Neither the nucleotide sequence nor the polypeptide encoded by these exons (Figure 7A) were found in databases, indicating that these exons belong to a new transcription unit. However, the encoded polypeptide displayed a significant homology to the N-terminus of the recently cloned otogelin (Cohen-Salmon et al., 1997) i.e. it contained a cysteine-rich domain that precedes the beginning of a von Willebrand Factor (vWF) D-type domain (Figure 7A). Fine mapping of the three exons defined their genomic organization and located the immunoprecipitated sequence 56 bp 5′ from the first exon identified (Figure 7B). Finally, the sequence of the DNA that flanked the three exons showed intron–exon boundaries that are consistent with the GT-AG rule applied to the splicing point (Figure 7B). Therefore, we conclude that the immunoprecipitated sequence is located in an intron of a new gene. Figure 7.The immunoselected sequence is located in an intron of a new gene. (A) An 18 kb murine genomic DNA that contained the immunoselected sequence (filled bar) was cut with various restriction enzymes and the NcoI–NcoI DNA fragment was cloned into the pSPL3 vector and used in an exon-trapping assay. The sequence of the three exons located in the 5 kb NcoI–NcoI DNA fragment is shown below the partial restriction map of the phage used. Down arrow indicates the end of each exon, right-angled arrow indicates the beginning of a von Willebrand D-type domain. The amino acids conserved between the polypeptide encoded by these three exons and the N-terminus of otogelin are shown in bold. The oligonucleotides used for RT–PCR analysis of the TAL-1 target gene are indicated (right-facing arrow and left-facing arrow). (B) A fine mapping of the three exons found in the 5 kb fragment is shown together with the location of the immunoprecipitated fragment and the sequences of the intron–exon boundaries. Download figure Download PowerPoint The expression of the identified gene is consistent with TAL-1 regulation The transcriptional status of TAL-1 and the target gene we have identified was evaluated by reverse transcription-coupled PCR (RT–PCR). We found a co-expression of these two genes in MEL cells, fetal liver and in a murine stromal cell line, M2-10 B4, that contains endothelial cells (Lemoine et al., 1988) (Figure 8A). On the other hand, the putative TAL-1 target gene was not expressed in NIH 3T3 fibroblastic cell line or in adult liver where indeed no TAL-1 mRNA could be detected (Figure 8A). Finally, we used an in vitro differentiation assay of ES cells towards the erythro/megakaryocytic pathways to study the expression of TAL-1, LMO2, GATA-1 and of the TAL-1-associated transcription unit. Gene expression in these cultures was also assayed by RT–PCR analysis at days 0, 3, 5, 6 and 9. As shown in Figure 8B, the TAL-1 target gene started to be detected at day 6, i.e. 1 day after the appearance of TAL-1, GATA-1 and LMO2 mRNAs. Taken together, these data show that the transcription unit we have identified is expressed in a manner consistent with TAL-1 regulation. Figure 8.The TAL-1 target gene mRNA levels correlate with TAL-1 expression pattern. (A) RT–PCR analysis of total RNA extracted from a stromal cell line that contains endothelial cells (M2-10B4), a fibroblastic cell line (NIH 3T3), the MEL cell line, fetal liver and adult liver were performed. The amount of cDNA used for PCR was normalized by the HPRT PCR and the identity of the TAL-1 and TAL-1 target gene fragment was assessed by hybridization with a specific oligonucleotide (data not shown). The sizes of
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