Identification and Characterization of a Transcriptional Regulator for the lck Proximal Promoter*

Atsuko YamadaDagger §, Satoshi TakakiDagger §||, Fumitaka Hayashi**DaggerDagger, Katia Georgopoulos§§, Roger M. Perlmutter**¶¶, and Kiyoshi TakatsuDagger

From the Dagger  Division of Immunology, Department of Microbiology and Immunology, the Institute of Medical Science, the University of Tokyo, Minato-ku, Tokyo 108-8639, Japan, the ** Department of Immunology, University of Washington, Seattle, Washington 98195, the §§ Cutaneous Biology Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, and ¶¶ AMGEN, Thousand Oaks, California 91320

Received for publication, September 13, 2000, and in revised form, February 19, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The lck gene encodes a protein-tyrosine kinase that plays a key role in signaling mediated through T cell receptor (TCR) and pre-TCR complexes. Transcription of the lck gene is regulated by two independent promoter elements: the proximal and distal promoters. Previous studies employing transgenic mice demonstrated that the sequence between -584 and -240 from the transcription start site in the mouse lck proximal promoter is required for its tissue-specific expression in the thymus. In this study, we demonstrate that a Krüppel-like zinc finger protein, mtbeta (BFCOL1, BERF-1, ZBP-89, ZNF148), previously cloned as a protein that binds to the CD3delta gene enhancer, binds to the -365 to -328 region of the lck proximal promoter. mtbeta is ubiquitously expressed in various cell lines and mouse tissues. Overexpressed mtbeta is more active in T-lineage cells than B-lineage cells for transactivating an artificial promoter consisting of the mtbeta binding site and a TATA box. Activity of the lck proximal promoter was significantly impaired by mutating the mtbeta binding site or by reducing mtbeta protein expression level by using antisense mRNA. Our results indicate that mtbeta activity is regulated in a tissue-specific manner and that mtbeta is a critical transactivator for the lck proximal promoter.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The lck gene encodes a lymphocyte-specific protein-tyrosine kinase, p56lck, a member of the src kinase family (1). It has been demonstrated by co-immunoprecipitation that p56lck associates with the cytoplasmic domains of CD4 and CD8 co-receptors (2) and with the acidic region of the IL-21 receptor beta -chain in T cells (3). By a series of biochemical analysis, it has been shown that p56lck plays a key role in signal transduction mediated through the T cell receptor (TCR) complex in mature T cells (4, 5). It also contributes to signaling through the pre-TCR complex, thereby playing an essential role in thymocyte development. lck-deficient mice and transgenic mice overexpressing a dominant negative form of p56lckck exhibit severe impairment in the expansion of CD4/CD8-double negative immature thymocytes (6, 7). A simple doubling of wild type p56lck expression levels in immature thymocytes in transgenic mice was sufficient to block maturation of thymocytes (8). These findings suggest that the transcriptional control of the lck gene must be tightly regulated to express adequate amounts of p56lck at the right developmental stage during thymopoiesis.

The lck gene is transcribed from two structurally unrelated promoters (9-13). The lck proximal promoter is positioned immediately adjacent to the first coding exon, and is active in the thymus, but is essentially silent in peripheral T cells. The distal promoter is located far 5'-upstream from the proximal promoter and is active during all developmental stages of T-lineage cells. Since the proximal promoter becomes active only at an early developmental stage of T-lymphopoiesis (14, 15), and since the level of p56lck greatly influences thymocyte maturation (6-8), the transcriptional regulators of this promoter play a critical role in the developmental program for T-lineage cells.

The 5'-flanking sequence of the lck proximal promoter that is critical for the thymocyte-specific and developmental stage-specific expression has been defined by transgenic mouse models (16). Transgenic animals bearing truncations in the mouse lck proximal promoter revealed that as little as 584 bases of the 5'-flanking sequence can confer appropriate developmentally regulated expression of heterologous reporter genes. The 5' sequence critical for the promoter activity contains several binding sites for nuclear proteins. Among those nuclear proteins, "B-factor," which binds to the G-rich stretch within the -365 to -328 region was reported as a candidate for the critical transcriptional regulator. B-factor is only found in cells expressing the lck transcript derived from the proximal promoter, namely thymocytes and thymoma cell lines such as LSTRA and EL4 (16).

In this study, we characterized the B-factor and identified an 86-kDa Krüppel-type zinc finger protein, which had been cloned previously as a binding protein to the CD3delta gene enhancer, as a component of the B-factor. The NH2-terminal half of the protein is 90% identical to htbeta , a 49-kDa protein that binds to the human TCR Vbeta 8.1 gene promoter and the TCR alpha  gene silencer (17), indicating that mtbeta is the murine homologue of htbeta , and the reported amino acid sequence of htbeta is a part of its full-length protein. mtbeta is ubiquitously expressed in various cell lines and tissues. We re-evaluated distribution of the B-factor and found it is also expressed in various cell lines and tissues. However, the transcriptional activity of mtbeta measured by reporter constructs carrying the B-factor binding site is observed only in T-lineage cells. The transcription from the lck proximal promoter is greatly impaired by introducing mutations in the B-factor binding site or by expression of mtbeta antisense mRNAs. Our results demonstrate that mtbeta is one of the critical transactivators driving the lck proximal promoter and that its activity is regulated in a tissue-specific manner.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells-- EL4, LSTRA, WEHI231, and BAL17 cells were grown in RPMI 1640 medium supplemented with 8% FCS and 50 µM 2-mercaptoethanol. FDC-P1 and Ba/F3 cells were grown in RPMI 1640 medium supplemented with 8% FCS, 50 µM 2-mercaptoethanol, and 5 units/ml mIL-3. Murine IL-3 was prepared as a culture supernatant of X63Ag8.653 cells transfected with mIL-3 cDNA (18). COS7 cells were grown in RPMI 1640 medium supplemented with 8% FCS. MTH cells were grown in RPMI 1640 medium supplemented with 8% FCS, 50 µM 2-mercaptoethanol, and 1.25 ng/ml IL-2 (Roche Molecular Biochemicals).

Preparation of Nuclear Extracts and Electophoretic Mobility Shift Assays (EMSAs)-- Cells were washed twice with ice-cold phosphate-buffered saline and once with hypotonic buffer (20 mM HEPES, pH 7.9, 1 mM EDTA, 0.1 mM EGTA, 2 mM MgCl2, 1 mM Na2VO4, 20 mM NaF, 1 mM dithiothreitol, 0.1 mM Pefabloc SC (Roche Molecular Biochemicals), 10 µg/ml leupeptin). Cells were suspended in buffer I (hypotonic buffer containing 0.2% Nonidet P-40) and incubated for 5 min on ice. Nuclei were pelleted by centrifugation at 15,000 × g for 20 min and re-suspended in buffer K (hypotonic buffer supplemented with 420 mM NaCl and 20% glycerol). After vigorous shaking for 30 min at 4 °C, the supernatants were collected and used as nuclear extract (19). Oligonucleotides corresponding to the -365 to -328 region of the lck proximal promoter (-365/-328, see section below) were subcloned into the KpnI site of pBluescript II (Stratagene). The fragment was cut out by Asp718I, labeled with [alpha -32P]dATP by Klenow large fragment (TaKaRa) and used as a probe. The nuclear extracts and the probe were incubated for 30 min at room temperature in the reaction buffer containing 10 mM HEPES, pH 7.9, 50 mM NaCl, 1 mM EDTA, 5% glycerol, 0.1% Nonidet P-40 (19). Each reaction contained 0.02 unit of poly[dI-dC] (Amersham Pharmacia Biotech). In some reactions, affinity-purified anti-mtbeta antibodies or normal rabbit IgG were added prior to incubation with the probe. Reactions were then subjected to 4% polyacrylamide gel electrophoresis in 0.25 × TBE buffer (Tris borate/EDTA buffer) and analyzed by autoradiography. Oligonucleotides with the following sequences (binding sequences for nuclear proteins are boldface) were used in EMSAs: -365/-328, 5'-TGTGGTTGAGTGGTGGGGGTAGGGGTGCTGGGGTAC-3' and 3'-CATGACACCAACTCACCACCCCCATCCCCACGACCC-5'; -365/328mut, 5'-GTACTGTGGTTGAGTGGTGCTGGTAGGGGTGCTGGG-3' and 3'-ACACCAACTCACCACGACCATCCCCACGACCCCATG-5'; delta A, 5'-AGAAGTTTCCATGACATCATGAATGGGGGTGGCAGA-3' and 3'-TTCAAAGGTACTGTAGTACTTACCCCCACCGTCTCT-5'; delta A-CRE, 5'-AGAAGTTTCCATAAGATGATGAATGGGGGTGGCAGA-3' and 3'-TTCAAAGGTATTCTACTACTTACCCCCACCGTCTCT-5'; delta A-G, 5'-AGAAGTTTCCATGACATCATGAATGGGGTGGCAGAG-3' and 3'-TCTTCAAAGGTACTGTAGTACTTACCCCACCGTCTCT-5'; IkarosBS, 5'-TCAGCTTTTGGGAATGTATTCCCTGTCA-3' and 3'-AGTCGAAAACCCTTACATAAGGGACAGT-5'.

Plasmid Construction-- The full-length mtbeta cDNA2 was subcloned into the EcoRI site of pcDNA3 (Stratagene), a eukaryotic expression vector driven by the human cytomegalovirus enhancer and promoter, resulting in pcDNA3-mtbeta . For the mtbeta antisense plasmid (pcDNA3-ASmtbeta ), the full-length mtbeta cDNA was subcloned in the opposite direction into the EcoRI site of pcDNA3. Various truncated fragments from the mouse lck proximal promoter were subcloned into the pGL2-Basic plasmid (Promega), which has a firefly luciferase gene without promoter or enhancer. For -3200/pGL2, the NotI-BamHI (positions -3200 to +37) fragment of the p1017 plasmid (20) containing the entire lck proximal promoter region was blunt-ended and ligated to the XhoI, HindIII-digested, blunt-ended pGL2-Basic plasmid. For -433GL2, two SmaI fragments (position -3200 to -1675 and -1675 to -433) were removed from -3200/pGL2 and self-ligated. For -240/pGL2, two KpnI fragments (position -3200 to -584 and -584 to -240) were removed from -3200/pGL2. For -584/pGL2, the KpnI fragment (position -584 to -240) from -3200/pGL2 was inserted into the KpnI site of -240/pGL2. To construct reporter plasmids carrying the B-factor binding site and TATA box, -365/-328 and -365/-328mut oligonucleotides (see section above) were inserted upstream of the TATA box of pLuc-S (gift from Drs. P. Doerfler and M. Busslinger (21)), resulting in pLuc-wild and pLuc-mut, respectively. Point mutations were introduced into the mtbeta binding sites of -3200/pGL2 and -433/pGL2 by PCR-based directed mutagenesis using -365/-328mut oligonucleotides to generate -3200-mut/pGL2 and -433-mut/pGL2, respectively.

RNA Isolation and Northern Blot Analysis-- Total RNA was extracted from various cell lines using the acid guanidine isothiocyanate-phenol-chloroform method. Fifteen micrograms of total RNA were fractionated through electrophoresis on 1% agarose gel in the presence of 0.66 M formaldehyde, transferred to nylon membranes (GeneScreen, DuPont). Mouse multiple tissue Northern blot was purchased from OriGene Technology (Rockville, MA). Membranes carrying RNA were hybridized with a 2.0-kilobase EcoRI fragment of mtbeta cDNA labeled with [alpha -32P]dCTP by the random priming method. After hybridization, membranes were analyzed using a BAS1000 Bio-Image Analyzer (Fuji Film, Tokyo, Japan). After removing the mtbeta probe, the membranes were re-hybridized with a human beta -actin probe to normalize the amount of RNA loaded per lane.

Generation of Polyclonal Antibodies-- A 1135-base pair EcoRV-ApaI fragment encoding Ser51 to Gly428 and a 1510-base pair ScaI-NotI fragment encoding Thr446 to Gly769 of mtbeta protein were subcloned into the bacterial expression vectors pGEX-4T-1 and pGEX-4T-2 (Amersham Pharmacia Biotech), respectively. The resulting plasmids were used to transform BL21(DE3)/pLysS (Novagen, Madison, WI). Recombinant glutathione S-transferase-mtbeta fusion proteins were induced with 1 mM isopropyl-beta -D-thiogalactopyranoside and affinity-purified by binding to glutathione-linked Sepharose beads (Amersham Pharmacia Biotech). The fusion proteins were further purified by gel filtration and used to immunize rabbits. Rabbit polyclonal anti-mtbeta antibodies were immunopurified on Sepharose-4B beads covalently coupled with the respective glutathione S-transferase-mtbeta fusion proteins used as immunogens.

Preparation of Cell Lysates and Western Blotting-- Cells were harvested and boiled in SDS-PAGE sample buffer (50 mM Tris, pH 6.8, 100 mM dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol) for 5 min. After centrifugation at 15,000 × g for 15 min, resulting clear cell lysates were subjected to SDS-8% PAGE and then electrophoretically transferred to a polyvinylidene difluoride membrane (Immobilon, Millipore, Bedford, MA) in transfer buffer (25 mM Tris, 200 mM glycine, 10% methanol). After blocking with 5% bovine serum albumin in TBS overnight at 4 °C, membranes were incubated with appropriately diluted primary antibodies. Membranes were washed with TBS containing 0.1% Tween 20 (TBS-T) and further incubated with horseradish peroxidase-conjugated antibodies against rabbit IgG (Cappel Organon Technica, Durham, NC). After washing, bound antibodies on membranes were detected using an ECL detection system (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) and x-ray film (Fuji Film). Splenic B cells were purified by negative sorting using anti-CD43 monoclonal antibody and a magnetic cell sorting system (Miltenyi Biotec, Bergisch Gladbach, Germany). Splenic T cells were purified by negative sorting using anti-B220 and anti-Mac1 monoclonal antibodies and magnetic cell sorting.

RT-PCR-- Total RNA or mRNA purified using Micro-FastTrack kit (Invitrogen) was subjected to cDNA synthesis using random hexamers (TaKaRa, Kyoto, Japan) and Superscript II reverse transcriptase (Life Technologies, Inc.). Serial dilution (3- or 2-fold) of the cDNA reaction mixtures was subjected to PCR amplification using the following primers: distal-sense, 5'-ATGTGAATAGGCCAGAAGAC-3'; proximal-sense, 5'-TCTGAGCTGACGATCTCGG-3'; lck antisense, 5'-GATCTTGTAATGTTTCACCAC-3'; beta -actin-sense, 5'-ACACTGTGCCCATCTACCAG-3'; beta -actin-antisense, 5'-CTAGAAGCACTTGCGGTGCA-3'; G3PDH (glyceraldehyde-3-phophate dehydrogenase)-sense, 5'-ACCACAGTCCATGCCATCAC-3'; G3PDH-antisense, 5'-TCCACCACCCTGTTGCTGTA-3'; HGPRT (hypoxantine guanine phophoribosyltransferase)-sense, 5'-CGTCGTGATTAGCGATGATGAACC-3'; HGPRT-antisense, 5'-ACTGCTTAACCAGGGAAAGCAAAG-3'. The conditions for PCR amplification were 30 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min for the lck or HGPRT transcripts and 27 cycles of the above for beta -actin or G3PDH transcripts using a PCR Thermal Cycler MP (TaKaRa). The resulting PCR products were separated by electrophoresis on 1% agarose gels and visualized by ethidium bromide staining.

Transfections and Luciferase Assays-- Cells (3 × 106) were suspended in 0.2 ml of Opti-MEM (Life Technologies, Inc.) and transferred to a 4-mm gap cuvette and mixed with 10 µg of reporter firefly luciferase plasmids, 1 µg of renilla luciferase plasmid pRL-TK (Promega). In some experiments, 10 µg of pcDNA3-mtbeta or a control pcDNA3 was added in addition to the reporter plasmids. Cells were transfected at 960 microfarads and 250 V using a Gene Pulser electroporation apparatus (Bio-Rad). Cells were harvested 12 h after transfection, and luciferase activity in cell lysates was measured using the Dual luciferase assay system (Promega) according to the manufacturer's recommendation. The firefly luciferase activity was normalized by the renilla luciferase activity to normalize for the transfection efficiency of each sample. In experiments using pcDNA3-ASmtbeta , EL4 cells were transfected with 15 µg of pcDNA3-ASmtbeta or control pcDNA3, together with 3 µg of reporter firefly luciferase plasmids and 1 µg of pRL-TK. Cells were harvested 30 h after transfection, and luciferase activity was measured. To analyze the amounts of mtbeta protein and the endogenous lck transcripts, cells were transfected with 10 µg of pcDNA3-ASmtbeta or pcDNA3, together with 1 µg of pEGFP-N1 (CLONTECH). Cells expressing GFP were sorted using a FACS Vantage cell sorter (Becton-Dickinson) at 30 or 36 h after transfection. Sorted cells were lysed and subjected to immunoblot analysis using anti-mtbeta or anti-tubulin antibodies or were subjected to RT-PCR analysis.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of a Nuclear Protein Binding to the lck Proximal Promoter-- A nuclear factor termed "B-factor" binds to a G-rich stretch located at -365 to -328 from the transcriptional initiation site of the lck proximal promoter, and its expression correlates well with the activity of the promoter (16). Because lck expression driven by the proximal promoter occurs early during lymphopoiesis (14, 15), and the level of p56lck greatly influences thymocyte maturation (6-8), we hypothesized that the B-factor would be one of the transcription factors playing critical roles in early lymphopoiesis. Ikaros regulates the early lymphopoiesis or the commitment for lymphocytes as demonstrated in the Ikaros-deficient mice (22). Ikaros has been shown to bind to a G-rich sequence of the CD3delta gene enhancer (23), although the high affinity binding sites for Ikaros are not G-rich (24). We examined whether the B-factor contains Ikaros or Ikaros-related proteins by EMSAs. Nuclear extract of a thymoma cell line, LSTRA, contains the B-factor binding to the -365 to -328 G-rich sequence (-365/-328) of the lck proximal promoter (Fig. 1A) as shown previously (16). The binding was specific, since it was competed by unlabeled -365/-328 oligonucleotides, but not by the -365/-328 oligonucleotides carrying mutations in the G-rich sequence (-365/-328mut). As shown in Fig. 1B, the binding of B-factor to the -365/-328 probe was competed by delta A sequences, a functional element of the CD3delta gene enhancer. The delta A consists of a CRE (cyclic AMP response element)-like region and a G-rich site similar to the B-factor binding site (25). The binding of B-factor to delta A was mediated by the G-rich site, because mutation at the G-rich site (delta A-G) but not at the CRE-like site (delta A-CRE) abrogated the competition with the -365/-328 probe. This binding characteristic of B-factor to delta A sequence was similar to that of Ikaros (23). However, the high affinity Ikaros binding oligonucleotides (IkarosBS) (24) did not show any competition with -365/-328. During the cloning of Ikaros, a cDNA clone encoding a zinc finger protein (mtbeta , see below) was simultaneously cloned by its ability to bind to the CD3delta enhancer.2 We examined whether antibodies against Ikaros or mtbeta could react with B-factor. Anti-Ikaros antibodies did not affect B-factor complex formation (Fig. 1C), confirming that Ikaros is not a component of B-factor. Interestingly, anti-mtbeta antibodies efficiently supershifted the B-factor complex (Fig. 1D). A similar result was obtained using nuclear extracts of EL4, a lymphoma cell line in which the lck proximal promoter is active (Fig. 1E). These results demonstrate that mtbeta is a component of the B-factor that binds to the -365 to -328 region of the mouse lck proximal promoter.


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Fig. 1.   Characterization of the nuclear protein, "B-factor," that binds to the sequence from -365 to -328 of the lck proximal promoter. A, the B-factor (arrow) in nuclear extracts from LSTRA. EMSA was performed using a fragment containing the sequence from -365 to -328 of the lck proximal promoter as a probe. The -365 to -328 (-365/-328) oligonucleotides or the oligonucleotides carrying mutations in the G-rich region (-365/-328mut) were used as competitors (10- and 50-fold molar excess over the labeled probe) to determine the binding specificity of the B-factor. B, the B-factor binds to delta A, the core enhancer sequences of the CD3delta gene enhancer. Unlabeled oligonucleotides with various G-rich sequences were used as competitors. delta A-CRE, delta A carrying the mutation in the CRE binding site; delta A-G, delta A carrying the mutation in the G-rich sequence; and Ikaros BS, a high affinity Ikaros binding sequence. C, Ikaros is not a component of the B-factor. Anti-Ikaros antiserum did not react with the B-factor. D, zinc finger protein; mtbeta that binds to delta A sequence is a component of the B-factor. The B-factor complex was supershifted by anti-mtbeta antibodies. E, the B-factor present in nuclear extracts from EL4.

mtbeta Is a Krüppel-type Zinc Finger Protein-- The deduced amino acid sequence of mtbeta contains an amino-terminal acidic domain, four tandem C2H2 Krüppel-type zinc finger motifs (26), and two basic domains, located upstream and downstream of the zinc finger cluster (data not shown). A data base search by BLAST identified a human homologue, htbeta , a 49-kDa protein that binds to the Vbeta 8.1 promoter and the Valpha silencer of the T cell receptor genes (17). The cDNA sequence encoding the NH2-terminal half of the mtbeta is 90% identical to htbeta , and their deduced amino acid sequences are 95% identical. The 3'-half of the mtbeta coding region has 91% identity with the 3'-untranslated region of the reported htbeta cDNA. These indicate that mtbeta is the murine homologue of htbeta and that the reported htbeta cDNA sequence has a one-base deletion that causes a frameshift and a premature stop codon. During this study, several cDNAs that have identical sequences with mtbeta have been reported: BFCOL1 that binds to the proximal promoters of the type I collagen genes (27) and BERF-1, a 89-kDa protein that binds to a muscle-specific enhancer of the beta -enolase gene (28). In addition, the rat and human homologue of the protein, ZBP-89, has been shown to bind to promoter regions of the gastrin gene (29) and the ornithine decarboxylase promoter (30). It has subsequently been reported that the same zinc finger protein also binds to the p21WAF1 gene (31), the matrix metalloproteinase-3 gene (32), the pTalpha gene (33), and the vimentin gene (34).

Recombinant mtbeta Binds to the lck Proximal Promoter-- We then asked whether recombinant mtbeta forms the B-factor complex. Recombinant mtbeta expressed in COS7 cells was detected as a band around 105 kDa in immunoblots (Fig. 2A). An endogenous simian homologue of mtbeta in COS7 cells was detected at the same position as the recombinant mtbeta when the blot was overexposed (data not shown). mtbeta protein appeared to migrate more slowly in SDS-PAGE than its estimated molecular size, as is consistent with the observation for BFCOL1 by Hasegawa et al. (27). In EMSA, a residual amount of the B-factor complex was detected in COS7 cells that derived from the endogenous simian mtbeta -homologue protein. Overexpression of mtbeta resulted in a significant increase of the amount of the B-factor complex (Fig. 2B). The entire complex was supershifted by the addition of anti-mtbeta antibodies. These results strongly indicate that mtB by itself, or in combination with proteins present in COS7 cells, forms the B-factor complex that binds to the -365 to -328 region of the lck promoter.


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Fig. 2.   The B-factor complex formation by recombinant mtbeta . A, Western blot analysis. COS7 cells were transfected with 10 µg of mtbeta expression vector or control plasmid. Cells were harvested at 48 h after transfection, and nuclear extracts were prepared. The resulting extracts (1 × 106 cells/lane) were separated, transferred to membrane, and were probed with anti-mtbeta antibodies. Molecular size markers are indicated on the left. B, EMSA. The amount of B-factor complex (arrow) was increased by expressing the recombinant mtbeta protein. Anti-mtbeta antibodies supershifted the B-factor complex.

Expression of Mtbeta in Various Cell Lines and Tissues-- In a previous study, the strong correlation between the lck proximal promoter activity and amounts of B-factor has been reported (16). We therefore examined the expression of mtbeta mRNA in various cell lines and tissues. As shown in Fig. 3A, two mRNA species, with estimated sizes of 9.0 and 4.2 kilobase, were detected. Mtbeta mRNA expression was observed in all cell lines tested and was independent of the lck proximal promoter activity. In mice, the mtbeta transcripts were ubiquitously expressed in various tissues. The mRNA was abundant in thymus where the proximal promoter is active; however, significant amounts of mRNA were also detected in all tissues, especially in the heart, kidney, and liver (Fig. 3B). We conclude that there is no correlation between lck proximal promoter activity and the expression levels of mtbeta mRNA.


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Fig. 3.   Northern blot analysis. Expression of mtbeta transcripts in various cell lines (A) and mouse tissues (B). RNA was prepared from LSTRA (thymoma), MTH (mature T), Ba/F3 (pro-B), WEHI231 (Immature B), BAL17 (mature B), and FDC-P1 (myeloid). Total RNA (15 µg) was separated and transferred onto a nylon membrane. A prepared multi-tissue membrane was purchased from OriGene Technologies. The blots were hybridized with mtbeta cDNA. The blots were stripped and subsequently hybridized with a beta -actin probe to normalize for the amount of loaded RNA.

The expression level of mtbeta protein might be controlled by post-transcriptional mechanisms. To test this possibility, we measured mtbeta protein levels by immunoblots of whole cell extracts isolated from various cell lines and primary mouse lymphoid cells. The mtbeta protein was detected in all tested cell lines, LSTRA (thymoma), BAL17 (mature B), Ba/F3 (proB), MTH (mature T), and EL4 (lymphoma) cell lines (Fig. 4A, left panel). Significant amounts of the mtbeta protein were also detected not only in thymocytes but also in splenic B cells and T cells (Fig. 4A, right panel). The expression of mtbeta protein was further confirmed by EMSAs. The B-factor was detected in the nuclear extract prepared from thymocytes as reported previously (16). We initially failed to detect either mtbeta protein or B-factor complex in extract from total splenocytes. However, the B-factor was present in the nuclear extract prepared from purified splenic B cells as well as mature T cells (Fig. 4B). High proteinase activity in total splenocytes may have caused degradation and prevented detection of mtbeta protein and the B-factor complex. Careful preparation of nuclear extracts revealed the presence of the B-factor, even in BAL17, Ba/F3, and MTH cell lines and mature splenic T cells (Fig. 4B).


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Fig. 4.   Expression of mtbeta protein and the B-factor is not restricted in T lineage cells. A, Western blot analysis of mtbeta in LSTRA (thymoma), BAL17 (mature B), Ba/F3 (pro-B), MTH (mature T), EL4 (lymphoma), thymocytes, purified splenic T cells (95% was CD3+), and purified splenic B cells (95% was B220+). Cells (2 × 105 cells) were boiled in 1 × SDS-PAGE sample buffer, and the insoluble materials were removed by centrifugation. Resulting cell lysates were separated, transferred to membrane, and were probed with anti-mtbeta antibodies. Molecular size markers are indicated on the left. B, EMSA. The B-factor (arrow) was detected in nuclear extracts prepared from thymocytes, purified splenic B cells (95% was B220+), BAL17, Ba/F3, MTH, and purified splenic T cells (95% was CD3+). Anti-mtbeta antibodies supershifted the B-factor complexes in all of tested extracts.

Lineage-specific Transactivation by mtbeta and Its Critical Function for the lck Proximal Promoter Activity-- To clarify the role of mtbeta in transactivation of the lck proximal promoter, various reporter plasmids were constructed and introduced into EL4 or mature BAL17. EL4 expressed mainly type I transcripts (9) transcribed from the proximal promoter, while BAL17 expressed only type II transcripts (9) from the distal promoter (Fig. 5A). First, we studied transactivation of a reporter construct that contains only the B-factor binding site of the proximal promoter and a TATA box (pLuc-wild) (Fig. 5B). The pLuc-wild construct showed significant promoter activity in parental EL4 cells, and the activity was augmented ~3-fold by overexpression of mtbeta . The promoter activity was not observed with a reporter (pLuc-mut) carrying mutations at the B-factor binding site on which mtbeta does not bind. Interestingly, pLuc-wild did not show significant promoter activity in BAL17, and the activity was not increased when mtbeta was overexpressed. We next studied the activity of reporter constructs with various deletions and mutations in the lck proximal promoter sequences (Fig. 5C). The fragment from -3200 to 0 of the promoter was active in EL4. Deletion of the fragment up to -584 did not affect promoter activity, whereas an additional deletion up to -433 resulted in a 3-fold increase of the activity. Further deletion of the fragment, including the B-factor binding site up to -240, did not impair the activity. However, the introduction of mutations into the B-factor binding site that abolishes mtbeta binding resulted in a significant reduction of the promoter activity of the -3200 and -433 fragments (compare -3200 versus -3200-mut and -433 versus -433-mut). All reporter constructs with the lck proximal promoter sequence did not show significant promoter activity in BAL17.


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Fig. 5.   Transcriptional activity of mtbeta on the lck proximal promoter. A, schematic representation of the lck gene promoter regions, and position of primers used for RT-PCR analysis are shown (left). Type I and II lck mRNAs are transcribed from the proximal and distal promoters, respectively. The relative ratio of type I and type II mRNAs in EL4 and BAL17 cells were measured by semiquantitative RT-PCR analysis. Serial dilutions (3-fold) of cDNA prepared from each cell line were subjected to PCR using sets of primers for type I (primers A and C) and type II (primers B and C) lck transcripts. beta -Actin cDNA was amplified (right lower panel) to calibrate the amounts of cDNA templates in each sample. The proximal lck promoter is mainly active in EL4, while the distal promoter is active in BAL17. B, mtbeta activates transcription from an artificial promoter consisting of the mtbeta binding site of the lck proximal promoter and a TATA-box (pLuc-wild) in EL4 but not in BAL17. Cells were transfected with 10 µg of reporter plasmid and 10 µg of mtbeta expression (+) or vector plasmid (-). Twelve hours after transfection, cells were harvested, lysed, and subjected to luciferase activity measurement. The luciferase activities are represented as percent activity of that produced by pGL2 control vector driven by the SV40 promoter. In pLuc-mut, mutations were introduced into the mtbeta binding site. The activity produced by a promoterless plasmid (0) is also shown. The results represent mean ± S.D. of multiple independent transfections. C, the binding of mtbeta is critical for the lck proximal promoter activity. Cells were transfected with 10 µg of luciferase reporter constructs carrying the various lengths of the lck proximal promoter region (-3200, -584, -433, -240, and 0) or the mutated promoter sequences (-3200-mut and -433-mut). The mtbeta binding site was destroyed by point mutations in the -3200-mut and -433-mut reporter constructs. The results are represented as percent luciferase activity observed with pGL2 control vector driven by SV40 promoter. The results represent mean ± S.D. of multiple independent transfections. In each experiment, the luciferase activities were normalized for transfection efficiency by measuring renilla luciferase activities encoded by a co-transfected pRL-TK plasmid. ND, not determined.

To confirm the role of mtbeta in transactivation of the lck proximal promoter, we reduced the protein expression level of mtbeta in EL4 by expressing mtbeta antisense mRNA. As shown in Fig. 6A, the amount of mtbeta protein was reduced to about 50% of control by transfection of the antisense plasmid. The activity of -3200 lck promoter fragment was significantly diminished, whereas that of the control SV40 promoter activity was not affected (Fig. 6B). Moreover, expression levels of the endogenous lck transcripts were also reduced (Fig. 6C). These results indicate that there is lineage-specific control for the mtbeta activity and that mtbeta plays a critical role in transactivation of the lck proximal promoter.


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Fig. 6.   Expression of mtbeta antisense reduces the lck proximal promoter activity. A, reduction of mtbeta protein level by expression of antisense mtbeta . EL4 cells were transfected with 10 µg of pcDNA3-ASmtbeta or pcDNA3, together with 1 µg of pEGFP-N1. Cells expressing GFP were sorted at 36 h after transfection. GFP-positive cells (5 × 104 cells) were lysed, and mtbeta protein levels were analyzed by immunoblot. Molecular size markers are indicated on the left. The blots were stripped and subsequently probed with anti-tubulin antibodies to normalize the amount of loaded proteins. The relative expression levels of mtbeta are indicated by mtbeta /tubulin ratio, which is set to 1 in cells transfected with vector control plasmid. B, relative luciferase activities of cells expressing mtbeta antisense plasmid. EL4 cells were transfected with 15 µg of pcDNA3-ASmtbeta or control pcDNA3, together with 3 µg of reporter plasmids (-3200/pGL2 or pGL2) and 1 µg of pRL-TK. Cells were harvested at 30 h after transfection, and luciferase activity in cell lysates was measured. The results represent mean ± S.D. of three independent transfections. C, reduced expression of the endogenous lck transcripts by expression of antisense mtbeta . EL4 cells were co-transfected with pcDNA3-ASmtbeta or control pcDNA3, together with pEGFP-N1 and GFP-positive cells were sorted at 30 h after transfection. cDNAs were synthesized, and serial dilutions (2-fold) of cDNA templates were subjected to PCR amplifications for lck, G3PDH, and HGPRT.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has been reported that a close correlation exists between transcriptional activities of the lck proximal promoter and the presence of the B-factor complex binding to the G-rich stretch of the promoter (16). In this work, we characterized and identified the B-factor, a potential transcriptional regulator of the lck proximal promoter. Our results indicated mtbeta , which has been previously cloned by its binding to the CD3delta enhancer in vitro, is a component of the B-factor. Anti-mtbeta antibodies supershifted the B-factor complex, and the recombinant mtbeta expressed in cell lines formed the B-factor complex.

Mtbeta is an 89-kDa zinc finger protein belonging to the Krüppel-type subfamily, whose members recognize the GC-rich or GT-rich sequences with their conserved DNA-binding zinc finger domains (26, 35). Mtbeta (identical to BFCOL1, BERF-1) and its human and rat homologues (htbeta , ZBP-89, ZFP148) are reported to bind regulatory regions of various genes, such as the Vbeta 8.1 promoter and the Valpha silencer of the T-cell receptor genes (17), the gastrin promoter (29), the type I collagen promoter (27), the beta -enolase enhancer (28), the ornithine decarboxylase promoter (30), the p21WAF1 promoter (31), the matrix metalloproteinase-3 promoter (32), the pTalpha enhancer (33), and the silencer element of the vimentin gene (34). Our data showing that mtbeta is ubiquitously expressed at both mRNA and protein levels are consistent with previously published reports and the fact that mtbeta functions in the various promoters and enhancers in various tissues. However, it should be noted that mtbeta regulates genes critical for maturation of T cells, such as lck, pTalpha gene, as well as TCR alpha  and beta  genes. Thus, mtbeta is one of the key transcriptional regulators controlling the T cell development.

In addition to its binding to a broad range of target genes, mtbeta appears to act as both transcriptional activator and repressor. As shown in this study, mtbeta is required for transactivating the proximal lck promoter. It also activates transcription from the Vbeta 8.1 promoter of the TCR gene and counteracts the silencing effect of the TCR alpha  gene silencer (17), and increased promoter activity of the p21WAF1 gene (31) and the matrix metalloproteinase-3 gene (32). Moreover, the binding site of mtbeta appears to be important for the pTalpha enhancer element (33). In contrast, mtbeta represses transcription from the gastrin gene (29), the beta -enolase gene (28), the ornithine decarboxylase gene (30), and the vimentin gene (34). It is currently unknown how mtbeta /BFCOL1/BERF-1 (htbeta /ZBP-89/ZFP148 in humans) manifests opposite activities on transcription of different genes. It has been shown that ZBP-89 competes with Sp1 for binding to the same element in the gastrin promoter (29) and inhibits the activation of the ornithine decarboxylase promoter by Sp1 (30). This might be one of the potential mechanisms by which mtbeta /BFCOL1/BERF-1 suppresses transcription from several promoter elements. A fascinating possibility is that mtbeta has different isoforms derived from alternative splicing, and each isoform has distinct transcriptional activities. To test this hypothesis, we performed RT-PCR to amplify various fragments of mtbeta cDNA using several combinations of primers. However, we could not detect any splicing variants of mtbeta cDNA either in thymocytes or splenocytes (data not shown). Another possibility is that interacting proteins exist that determine the DNA binding specificity and the transactivating activities of the mtbeta complex and whose expression is regulated in a tissue-specific manner. It is also possible that mtbeta receives post-transcriptional modifications in a tissue-specific manner. Endogenous as well as overexpressed mtbeta transactivated transcription from an artificial promoter consisting of B-factor binding sites and TATA-box in EL4 but not in BAL17. Moreover, mtbeta generally transactivates genes expressed in T cells such as lck and TCR alpha  and beta  genes, but represses gastrin, collagen, and beta -enolase genes expressed in non-T cells. These observations support the idea that there are tissue-specific mechanisms regulating activity of mtbeta . Basic Krüppel-like factor (BKLF), which is widely expressed in various tissues and binds to the CACCC motifs, is also a member of the Krüppel-like zinc finger protein subfamily (36). Although BKLF positively regulates the transcription from a promoter containing a single BKLF binding site, it represses activity of glucocorticoid receptor-mediated activation of a promoter containing three copies of CACCC motifs and glucocorticoid-responsive elements (36, 37). The NH2-terminal domain of BKLF is responsible for its repressive activity and interacts with a co-repressor protein, murine COOH-terminal-binding protein 2 (mCtBP2) (37). mCtBP2 interacts with BKLF and a number of mammalian transcription factors, such as Evi-1, AREB6, ZEB, and FOG, via the Pro-X-Asp-Leu-Ser (PXDLS) motif on the transcription factors (37). The mtbeta also carries several PXDLS-like motifs (PVDLQ (amino acids 112-116), PKDNS (amino acids 282-286)). mtbeta may associate with mCtBP2 or related molecules and exhibits suppressing activity in cells that fail to support proximal lck promoter activity. Our initial attempts, however, to detect associating molecules using glutathione S-transferase-mtbeta fusion proteins or modification of mtbeta such as phosphorylation have not been successful.

mtbeta is critical for the full activation of the lck proximal promoter activity, since the mutation of the mtbeta binding site of the promoter or the reduction of the mtbeta protein level significantly impaired the promoter activity. However, the overexpression of mtbeta in EL4 did not augment the lck proximal activity (data not shown). This may indicate that the coordinated interaction of mtbeta with T cell-specific transcription factors (whose expression level is limiting) is involved in the full activation of the lck proximal promoter in thymocytes. Binding sites for the T cell-specific factors TCF-1, LEF, and TCF-1alpha are present in a region highly homologous between the murine and human proximal promoters (16). TCF-1 expressed ectotopically in BAL17 cells, however, failed to drive the lck proximal promoter activity with endogenous mtbeta (data not shown), suggesting a complex cooperation of multiple transcription factors in transactivating the lck proximal promoter. A -240 lck promoter fragment lacking a mtbeta binding site is still active in EL4. It should be noted, however, that the -240 fragment (but not the -584 promoter fragment carrying a mtbeta binding site) failed to support thymocyte-specific transcription of the lacZ-hGH transgene construct in mice (16). The EL4 cell line might lack a negative regulator expressed in primary cells that suppresses the -240 promoter activity in the absence of mtbeta . Alternatively, EL4 might abundantly express positive transactivators binding to the -240 fragment whose activity is repressed by proteins bound to the -584 to -240 region. The mechanism that accounts for the discrepancy between the -240 promoter activity in EL4 and that in thymocytes remains unknown.

In addition to the positive regulators directing the lck proximal promoter activity in thymocytes, the silencers suppressing the promoter are likely to play roles in peripheral T cells. It has been reported that A2 complex binding to the sequence from -477 to -460 in the murine proximal promoter was detected in extracts from cells negative for the lck proximal promoter activity (16). It has also been reported that the -474 to -466 region in the human lck proximal promoter acts as a strong repressor in human tumor cell lines that do not express lck and binds proteins with molecular masses of 35 and 75 kDa (38). Deletion of the -584 to -433 region from our luciferase reporter constructs resulted in the enhancement of the promoter activity. These suppressive elements and binding factors are also critical in achieving tissue-specific expression of the lck proximal promoter in concert with the positive regulators, including mtbeta .

In summary, we identified a Krüppel-type zinc finger protein, mtbeta , as a transactivator of the lck proximal promoter. mtbeta is ubiquitously expressed and manifests a broad range of activities on various genes. However, mtbeta is presumably a critical transcription factor for the T cell development, since it positively regulates lck and pTalpha genes as well as TCR alpha  and beta  genes. Overexpressed mtbeta is active only in T-lineage cells, suggesting that there exists tissue-specific regulatory mechanisms to control mtbeta activity. Understanding the function of mtbeta should provide important insight into how T cell development and the thymocyte-specific expression of the lck proximal promoter are controlled and how one DNA-binding protein regulates different promoters positively and negatively.

    ACKNOWLEDGEMENT

We thank Drs. P. Doerfler and M. Busslinger for providing pLuc-S reporter constructs, Dr. J. Allen for providing oligonucleotides and probes, and all our colleagues for helpful discussions.

    FOOTNOTES

* This work was supported in part by Special Coordination Funds of the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

Supported in part by a Center of Excellence Fellowship from the Institute of Medical Science Program.

|| To whom correspondence should be addressed. Tel.: 81-3-5449-5264; Fax: 81-3-5449-5407; E-mail: takakis@ims.u-tokyo.ac.jp.

Dagger Dagger Supported by the Howard Hughes Medical Institute Predoctoral Fellowship in Biological Science.

Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M008387200

2 K. Georgopoulos, unpublished data.

    ABBREVIATIONS

The abbreviations used are: IL, interleukin; TCR, T cell receptor; EMSA, electrophoretic mobility shift assay; GFP, green fluorescent protein; FCS, fetal calf serum; RT-PCR, reverse transcriptase polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HGPRT, hypoxantine guanine phophoribosyltransferase; CRE, cyclic AMP response element; BKLF, basic Krüppel-like factor.

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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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