Spi-C, a Novel Ets Protein That Is Temporally Regulated during B Lymphocyte Development*

Mats BemarkDagger , Annica Mårtensson, David Liberg, and Tomas Leanderson§

From the Immunology Unit, Department of Cell and Molecular Biology, Lund University, P. O. Box 7031, S-220 07 Lund, Sweden

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A novel Ets protein was isolated by yeast one-hybrid screening of a cDNA library made from lipopolysaccharide-stimulated mouse splenic B cells, using the SP6 kappa  promoter kappa Y element as a bait. The novel Ets protein was most closely related to PU.1 and Spi-B within the DNA binding Ets domain and was therefore named Spi-C. However, Spi-C may represent a novel subgroup within the Ets protein family, as it differed significantly from Spi-B and PU.1 within helix 1 of the Ets domain. Spi-C was encoded by a single-copy gene that was mapped to chromosome 10, region C. Spi-C interacted with DNA similarly to PU.1 as judged by methylation interference, band-shift and site selection analysis, and activated transcription of a kappa Y element reporter gene upon co-transfection of HeLa cells. Spi-C RNA was expressed in mature B lymphocytes and at lower levels in macrophages. Furthermore, pre-B cell and plasma cell lines were Spi-C-negative, suggesting that Spi-C might be a regulatory molecule during a specific phase of B lymphoid development.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteins that are members of the Ets family of transcription factors are involved in a variety of developmental and cellular responses (1). At present around 20 distinct proteins within the family have been described, and all these bind to a similar, purine-rich DNA sequence element (1, 2). Given the similarity of their binding sites, and given that several of the proteins have overlapping pattern of expression, it has been difficult to define the individual function of each Ets protein. However, gene targeting experiments in mice have shown that Ets proteins are not functionally redundant (3-6), but that each Ets protein has a distinct function.

In the B lymphoid lineage, several of the Ets proteins are expressed; among these are Ets-1 (7, 8), elf-1 (9), PU.1 (Spi-1) (10), and Spi-B (11). However, none of these factors are restricted in their expression to B cells only. Ets-1, elf-1, and Spi-B are also expressed in T cells (3, 8, 9, 12), and PU.1 can be found in both lymphoid and myeloid cells (10, 13). Still, all four factors have been implicated in transcriptional regulation of B cell-specific genes. Hence, a binding site for Ets-1 has been found in the Ig heavy chain intron enhancer (14), while binding sites for elf-1 have been found in the Ig heavy chain 3' enhancer (15) and in a kappa  promoter (16). Binding sites for PU.1 have been found in the Ig heavy chain intron enhancer (14), the Ig kappa  3' enhancer (17), kappa  promoters (18), the lambda  2-4 enhancer (19), and the mb-1 (Igalpha ) and B-29 (Igbeta ) genes (20, 21), as well as in the Ig J-chain promoter (22). However, no target genes for PU.1 that can explain the phenotype observed in PU.1-/- animals have been identified.

Spi-B is closely related to PU.1, and no clear difference between the binding sequences of the two proteins has been defined (2). As the expression pattern between PU.1 and Spi-B is overlapping within the B cell lineage (3), it would not be surprising should the two proteins be functionally redundant. However, this is not the case, since, although PU.1-/- mice have a severe distortion of hematopoietic development (5, 6), Spi-B-/- mice form a normal myeloid and lymphoid compartment with the exception of a defective B cell receptor-mediated immune activation (23).

Here we describe the cloning and characterization of a novel Ets family protein, Spi-C, which was identified in an yeast one-hybrid screen due to its interaction with the SP6 kappa  promoter kappa Y element. The protein is mainly expressed in the peripheral B lymphoid compartment; it is not expressed in early B cell precursors or in pre-B cell lines and appears to be down-regulated as the B cell matures to the plasma cell stage.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Yeast Strains-- Four copies of a fragment containing the pentadecamer element and the kappa Y core element (16, 24) were cloned into the pHisi and pLacZi vectors (CLONTECH), between the XbaI and EcoRI sites, and SmaI and EcoRI sites, respectively. The vectors were linearized by XhoI or NcoI digestions and stably integrated into the YM 4271 yeast strain (CLONTECH). The integration was verified by Southern blotting of PCR1-amplified DNA from genomic yeast DNA using either Hisi- or LacZi-specific primers, using one copy of the inserted fragment as a probe (5'-TACTCTCAAACAGCTGTGTAATTTACTTTCC-3'). The EBF binding site yeast strain was constructed using the same protocol with four copies of the EBF binding CCCT element from the SP6 kappa  promoter (25). The obtained yeast strains did not grow on histidine-deficient plates at a 15 mM concentration of 3-amino-1,2,4-triazole (3-AT).

Yeast Library Construction and Screening-- BALB/c mouse spleens were minced and set up at a lymphocyte concentration of 1 × 106 lymphocytes/ml in Iscove's modified Dulbecco's medium with 5% fetal calf serum and 25 µg/ml LPS. After 72 h, the activated cells were washed once in phosphate-buffered saline and total RNA isolated using the acid phenol method. A total of approximately 1 mg of RNA was isolated, and poly(A)+ RNA was obtained by purification on an oligo(dT)-cellulose column (26). Double-stranded cDNA was prepared, inserted into predigested Hybri-ZAP lambda  arms and packaged as described for Stratagene's two-hybrid system. A total of 3.2 × 106 independent plaques were obtained with an average insert size of 1.1 kilobase pairs. The amplified stock was converted to its phagemid form as described by the manufacturer (Stratagene). 20 µg of phagemid was transfected into the yeast strain and spread on SD agar plates minus histidine and minus leucine, containing 15 mM 3-AT as described (CLONTECH). Growing clones were tested in a LacZi assay (CLONTECH), and positive clones were grown, plasmids isolated, transformed into bacteria, and finally retransformed into the screening yeast strain or the EBF control strain. cDNA clones that remained positive through these procedures were sequenced from the 5' end. A total of six E2A-containing, three E2-2-containing, and one Spi-C-containing clones were identified in the screen.

cDNA Library Screening and Sequencing-- The Hybri-ZAP lambda  library was spread on 90-mm plates at 22,000 plaque-forming units/plate and overlaid with sodium chloride magnesium to obtain 60 phage pools. These were screened via PCR using two Spi-C-specific primers (5'-GCAAACATTTCAAGACGCC-3' and 5'-CTGTACGGATTGGTGGAAGC-3'), the products separated by electrophoresis and blotted onto charged nylon membranes, which were probed with a polynucleotide kinase end-labeled oligonucleotide (5'-CAGACCTGTATTTGGAAGGA-3') as described by the manufacturer (Bio-Rad). Two positive pools were identified, and these were subsequently screened according to standard procedures using the original yeast Spi-C clone as probe and then excised as plasmids according to the manufacturer's instructions (Stratagene). The longest of the two clones was completely sequenced on each strand using the Sanger dideoxy method (26) with a combination of internal primers and restriction fragments cloned into pGEM 3Z (Promega). The original yeast clone was sequenced in parallel, and no differences between the two clones were detected in overlapping sequences.

In Vitro Translation, EMSA, and Methylation Interference Analysis-- The full-length Spi-C clone was cloned between the EcoRI and XhoI sites of the pcDNA3 vector (Invitrogen), while the partial Spi-C cDNA obtained from the yeast screening was cloned between the EcoRI and XhoI sites in a modified pcDNA3 vector in which an OCT2 ATG region (5'-AATTGCGGGCAGCATGGTTCATTCCAGCGAATT-3') had been inserted into the EcoRI site. The cDNAs were in vitro translated using Promega's coupled transcription/translation reticulocyte system from the T7 promoter. The EMSAs were performed as described (16), using between 1 and 0.1 µl of reticulocyte extract. Competitions were performed using 10, 30, 100, and 300 times, or 30, 100, 300, and 1000 times molar excess of competitor; the sequences of the competitors and probes can be found in Ref. 16. The methylation interference analyses were made as described (24) using protein translated from the yeast clone. After autoradiography of the wet gel for 4 h, the free and bound probes were cut out, eluted in 1× TE with 50 mM NaCl overnight, extracted with phenol, and ethanol-precipitated. The precipitated DNA was treated with piperidine as described (26) and subsequently separated on a 10% polyacrylamide gel electrophoresis, 1× TBE gel that was fixed, dried, and autoradiographed.

Recombinant Proteins, Antisera, and Site Selection-- The full-length or carboxyl-terminal part of Spi-C cDNA were amplified via PCR using Spi-C primers (full-length: 5'-ACGAGATCTATGACTTGTTGTATTGATC-3' against 5'-ACGGAATTCTCAGCTCTGGTAACTGG-3', carboxyl part: 5'-ACGA GATCTGAGGCTGTTCTCCAAAGA-3' against 5'-ACGGAATTCTCAGCTCTGG TAACTGG-3') and inserted between the BamHI and EcoRI sites of the pGEX-2 vector (Amersham Pharmacia Biotech) after restriction with BglII and EcoRI and verified by sequencing using the pGEX 5' primer (Amersham Pharmacia Biotech). The proteins were expressed and purified on glutathione-Sepharose according to the manufacturer's instructions (Amersham Pharmacia Biotech), and the carboxyl terminus-containing protein was further purified on a Q Column (Beckman) and was then used to raise a polyclonal sera in rabbits according to standard procedures. For the first round of site selection, an N oligonucleotide (5'-GCCACTGCGAATTCTCT(N)20AATGGGATCCCGTCGCA-3') was annealed to a [32P]polynucleotide kinase-labeled oligonucleotide, and fill-in was performed using Klenow enzyme. This oligonucleotide was then used in EMSA with the recombinant, full-length glutathione S-transferase-Spi-C protein. A piece of the gel that corresponded to the mobility of the Spi-C-DNA complex was cut out and eluted in 1× TBE overnight. The eluted DNA was amplified for 15 cycles in PCR using 5' and 3' primers (5' primer: 5'-GCCACTGCGAATTCTC-3', 3' primer: 5'-TGCGACGGGATCCCAT-3'), end-labeled, and purified by gel electrophoresis. The selection procedure was repeated twice more, and the final PCR products were cut with EcoRI and BamHI, inserted into the pGEM3Z vector (Promega), and sequenced.

RNA Extractions, Northern Blotting, and RT-PCR Assays-- Northern blotting was performed using standard protocols, and 2 µg of poly(A)+ RNA was loaded in each lane (26). Isolation of cells was done either by cell sorting (FACS Vantage, Beckton Dickinson) or by using magnetic MACS beads (Miltenyi Biotec). RNAs were prepared from tissue using a Pharmacia poly(A) RNA kit (the B cell lines), Qiaex spin columns (Qiagen; ES cells, lymph node and liver), or RNAzol B (Tel-test, Inc.; the rest). The RT reactions were performed using Superscript II (Life Technologies) according to the manufacturer's instructions, and the primers and probe used were the same as used in the library screen (see above). The amount of cDNA in each reaction was titrated using HPRT primers (27). Primers used to detect PU.1 expression in cell lines were described before (28). In some cases different exposures from the same blots are shown, but the HPRT exposure were always matched with the Ets probe exposure.

Transfection Constructs and Assay-- The 4× kappa Y reporter construct was made by inserting an oligonucleotide containing four copies of the kappa Y element and the TATA box from the SP6 kappa  promoter between the HindIII and XhoI sites of the pGL3 luciferase reporter vector (Promega), and the TATA box only containing was subsequently created by EcoRI digestion and re-ligation to remove the kappa Y elements. To obtain a PU.1 expression vector, an EcoRI/ApaI fragment containing the coding region of PU.1 was inserted into the pcDNA3 vector (Invitrogen). HeLa cells (2.5 × 106 cells/transfection) were transfected in duplicate using LipofectAMINE (Life Technologies, Inc.). Each transfection contained 0.2 µg of reporter plasmid and 0.3 µg of expression plasmid, as indicated. All co-transfections were normalized to contain 0.8 µg of DNA/transfection using the pcDNA3 plasmid. The cells were harvested and the luciferase activity determined after 40 h of incubation, and the mean value ± standard deviation from three experiments is shown.

Southern Blotting and Chromosome Mapping-- Southern blotting and the isolation of a genomic Spi-C clone was performed according to standard protocols (26). The chromosome mapping using FISH was performed by SeeDNA Biotech Inc., Toronto, Canada. Mouse chromosomes were prepared according to the published procedure. Briefly, lymphocytes were isolated from mouse spleen and cultured at 37 °C in RPMI 1640 medium supplemented with 15% fetal calf serum, 3 µg/ml concanavalin A, 10 µg/ml lipopolysaccharide, and 5 × 10-5 M mercaptoethanol. After 44 h, the cultures were treated with 0.18 mg/ml bromodeoxyuridine for an additional 14 h. The synchronized cells were washed and recultured at 37 °C for 4 h in alpha -minimal essential medium with thymidine (2.5 µg/ml). Chromosome slides were made by conventional method as used for human chromosome preparation (hypotonic treatment, fixation, and air-drying). As a probe, a 12.5-kilobase pair genomic fragment cloned from a I129 genomic library was used after biotinylation using a Life Technologies BioNick labeling kit at 15 °C for 1 h (29). FISH detection was performed as described (29, 30). Briefly, slides were baked at 55 °C for 1 h, and after treatment with RNase A the slides were denatured in 70% formamide in 2× SSC for 2 min at 70 °C, followed by dehydration with ethanol. Probes were denatured at 75 °C for 5 min in a hybridization mix consisting of 50% formamide, 10% dextran sulfate, and mouse cot I DNA, and prehybridized for 15 min at 37 °C before being loaded on the denatured slides. After overnight hybridization, slides were washed, detected, and amplified as described (29). FISH signals and DAPI banding pattern were recorded separately by photography, and the assignment of the FISH mapping data with chromosomal bands was achieved by superimposing FISH signals with DAPI-banded chromosomes (30).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Cloning of Spi-C, a kappa Y Interacting Protein-- Fig. 1A shows a schematic illustration of the mouse SP6 kappa  promoter. This immunoglobulin promoter contains several DNA elements that are poor activators of transcription per se but that will stimulate octamer-induced transcription (16, 24, 31). In this study we focused on the octamer upstream region that contains two transcriptional control elements, the bipartite pentadecamer element and the kappa Y element (16, 24, 32, 33). In an effort to identify the proteins that interact with these DNA elements, we used the sequence shown in Fig. 1A as bait in a yeast one-hybrid screen of a cDNA library made from LPS-stimulated mouse B cells (Fig. 1B). We isolated several E-box binding clones, all encoded by the E2A and E2-2 genes (data not shown), and in addition a clone containing a novel DNA sequence that we decided to call Spi-C. Also shown in Fig. 1B is a confirmation of the specificity of the screening procedure, using a control yeast strain that had an EBF binding site as bait. When using the pentadecamer/kappa Y yeast strain, Spi-C could rescue yeast growth on 3-AT-containing media while an EBF-containing plasmid could not. The opposite was true using the EBF control. Furthermore, in the absence of selection, the cDNA clones also activated the transcription of the LacZ genes as expected; Spi-C was positive only in the yeast strain used for screening, while EBF was positive only in the EBF bait strain.


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Fig. 1.   A, schematic drawing of the SP6 k promoter and the sequence used as bait in the one-hybrid screening. B, verification of specificity of the Spi-C expressing yeast clone and an EBF-expressing yeast clone as a control in combination with a yeast strain containing an EBF binding site as bait. The transformed yeast strains were then tested for their ability to grow in the presence and absence of 3-AT, or to activate the LacZ gene as indicated. C, the Spi-C cDNA from the original yeast clone was introduced into a vector containing the translational start region from Oct-2 in frame with the cDNA. After transcription and translation in vitro, Spi-C was tested for its ability to interact with 5' region of the SP6 kappa  promoter in EMSA. The Spi-C complex is indicated, and free probe is labeled F. The interaction between Spi-C and the probe was mapped by competition with the indicated competitors, and the result obtained is summarized in the lower part of the panel.

To investigate the binding specificity of the Spi-C gene product, the insert from the original yeast clone was transcribed and translated in vitro and the protein product analyzed by band-shift using the bait sequence as a probe. As shown in Fig. 1C, unlabeled probe competed efficiently for binding in this assay while a competitor that contained the SP6 kappa  promoter octamer and its 3'-flanking region did not. Further analysis of the region used as bait showed that a competitor that contained an intact kappa Y element competed efficiently for binding, while competitors with mutations of the kappa Y element or with a truncated kappa Y element did not. We concluded from these experiments that Spi-C interacted specifically with the kappa Y element in the SP6 kappa  promoter.

Spi-C Is a Member of the Ets Protein Family-- A full-length cDNA clone of Spi-C was isolated from a lambda  cDNA library made from LPS-stimulated B cells using the insert from the yeast clone as a probe. The full-length cDNA encompassed 1267 base pairs (accession no. AF098863), and contained an open reading frame starting with an ATG at position 144 and a stop codon at position 870. This would generate an open reading frame of 242 amino acids, with a predicted molecular mass of 28 kDa (Fig. 2A). Homology searches using the protein sequence showed that the protein had sequence similarity to the Ets proteins PU.1 and Spi-B in their DNA binding domain, while no significant homology was detected outside of this region. Hence, Spi-C appears to be a novel member of the Ets transcription factor family.


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Fig. 2.   A, the deduced amino acid sequence Spi-C with the Ets domain. B, an amino acid comparison between the Ets domain of the indicated Ets proteins. Boldface letters indicate amino acids conserved in all Ets proteins, and helices 1-3 of the Ets domain are indicated. C, the percentage of amino acid identity between Spi-B, PU.1, and Spi-C in the Ets domain or indicated subregions. The analysis was performed by hand. Identical amino acids in a given position were scored (X), and the percentage of identity calculated as X/N × 100, where N indicates the total number of amino acids in the analyzed region.

Fig. 2 (B and C) shows an analysis of the protein sequence homology in the Ets domain between Spi-C, PU.1, Spi-B, elf-1, and Ets-1. Outside the Ets domain, the amino acid sequence diverged quite extensively compared with the other Ets family proteins. With regard to the Ets domain, that of Spi-C matched those of PU.1 and Spi-B more closely than those of Ets-1 or elf-1, although all amino acids that are universally conserved in Ets proteins (34) were also conserved within the Ets domain of Spi-C (Fig. 2B). The amino acid identity within the Ets domains of Spi-B and PU.1 was 73%, while it was 59% or 63% when compared with Spi-C for PU.1 and Spi-B, respectively (Fig. 2C). When the alpha  helices within the Ets domain were analyzed separately, the degree of identity between PU.1/Spi-B and Spi-C was most pronounced in helix 3 (79%), while less marked in helix 2 (54-62%) and in helix 1 (31%). This is in contrast to the degree of identity between Spi-B and PU.1 that was very high (77%) also in helix 1 and 2. Since helix 2 and 3 entail the amino acids that contact DNA directly in PU.1 (35), the higher level of identity between Spi-B/PU.1 and Spi-C is not surprising, given that Spi-C binds a bona fide PU.1 binding site (18). Also, all amino acid positions shown by mutagenesis to be critical for PU.1 binding to DNA were conserved between PU.1 and Spi-C (36). We conclude from these results that Spi-C is more closely related to PU.1 and Spi-B than to other Ets proteins, but more diverged from these two proteins than those to each other. Given the low degree of identity in helix 1 of the Ets domain, Spi-C might be representing a novel subgroup within the Ets protein family.

Spi-C DNA Interactions Are Indistinguishable from Those of PU.1-- The interaction between Spi-C and the kappa Y element was further characterized and compared with that of PU.1 (18). First, the binding of the two proteins were studied via methylation interference analysis. Methylation of the two central G nucleotides within the Ets motif of the non-coding strand completely inhibited both Spi-C and PU.1 binding, while no specific interference could be detected by methylation of the coding strand (Fig. 3A). Upon longer exposures, detected partial interference by methylation of some of the A residues could also be detected, identically for both proteins (data not shown).


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Fig. 3.   A, methylation interference analyses was performed using both strands of the 5' region of the SP6 kappa  promoter with either in vitro translated PU.1 or Spi-C. Interfering positions are marked with arrowheads, and below the autoradiograph the sequence of the probe with interfering positions marked is shown. B, competition of Spi-C or PU.1 binding to the kappa Y element using competitors containing published PU.1 binding sites.

To further investigate the relationship between Spi-C and PU.1 DNA binding, we performed band-shift analysis using the kappa Y element as a probe, in vitro translated full-length Spi-C or PU.1 as ligands and various defined PU.1 sites as competitors. The in vitro translated, full-length Spi-C generated two complexes in the band-shift and also two bands in SDS-polyacrylamide gel electrophoresis (data not shown). The larger of these bands correlated in size with the predicted value for full-length Spi-C (28 kDa), and we therefore assume that the more abundant, faster migrating complex represents a partial break-down product with an intact DNA binding domain. That the DNA binding domain is not affected by the limited proteolysis is supported by the fact that in vitro translation of the original yeast clone generated only one product (Fig. 2C). As shown in Fig. 3B, Spi-C kappa Y binding could be competed by PU.1 binding sites from the SV40 enhancer (11), the kappa 3' enhancer (17), and the J-chain promoter (22). Furthermore, the amount of competitor needed to compete Spi-C binding was similar to that needed to compete PU.1 binding to the same probe. Finally, we performed a site-selection experiment using recombinant Spi-C to ask the question whether any novel Ets binding motifs could be revealed. As summarized in Table I, 90% of the selected templates contained established PU.1 binding sites. The most common was a GGA core motif (21/33 sequences), while 9 out of 33 sequences contained instead the variant AGA core found in the J-chain promoter (22) and in the macrophage colony-stimulating factor receptor (37). There was a clear preference for an A in the -4, -1, and +1 positions, while a preference for A or T could be seen in the -3 and +3 positions. We conclude from these data that Spi-C and PU.1 bind to overlapping DNA elements with similar selectivity.

                              
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Table I
Binding site preference for Spi-C
Site selection was performed as described under "Experimental Procedures," and plasmids containing inserts were randomly selected and sequenced. Thirty out of 33 plasmids contained previously described PU. 1 binding sites and were used for the compilation above. In the compiled sequence, a higher degree of base preference is indicated with uppercase letters (present in over 65% of the sequences) while lower degree of preference is in lowercase letters.

Spi-C Is Preferentially Expressed in Mature B Cells-- To characterize the expression pattern of Spi-C, we performed semi-quantitative RT-PCRs from a panel of mouse tissues and purified cell populations (Fig. 4A). High expression was found in spleen and significant expression was also detected in lymph node and bone marrow. Low expression could also be detected in thymus, Peyer's patches, and liver, while no expression was seen in heart, striated muscle, or kidney. We subsequently analyzed the Spi-C expression in purified cell populations. No Spi-C expression was detected in embryonic stem cells, double-negative or double-positive thymocytes, or purified peripheral CD4+ or CD8+ T cells. On the other hand, purified macrophages expressed Spi-C RNA at low but detectable levels, and B220+ peripheral B lymphocytes at higher levels. Thus, Spi-C seems to be preferentially expressed in the macrophage and B lymphoid lineage.


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Fig. 4.   A, semiquantitative RT-PCR analysis for Spi-C and HPRT RNA expression in the indicated organs and purified cell types. BM, bone marrow; DP, thymic double-positive (CD4+ CD8+) T cells; ES, embryonic stem cells. B, semiquantitative RT-PCR analysis for Spi-C and PU.1 RNA expression in the indicated organs and cell lines. C, Northern blot analysis of 2 µg of poly(A)+ RNA from the indicated cell lines probed with either a Spi-C or a GADPH probe.

We next investigated the expression of Spi-C RNA in various cell lines, as a complement to the tissue and polyclonal cell analysis discussed above. For comparison, the expression of PU.1 RNA was also investigated in these preparations while all samples had first been normalized using HPRT expression (data not shown). Fig. 4B shows that the macrophage cell line J774 showed low but significant Spi-C expression, concordant with the observed expression in freshly isolated macrophages. More interestingly, when cell lines representing various stages of B lymphoid differentiation were analyzed for Spi-C expression, only cell lines having a phenotype of mature B cells were positive. The two plasmacytoma cell lines J558 and S194 were negative, as were the pre-B cell lines 230-238, 18-81, and 70Z/3. The 70Z/3 cell line differentiates to a mature B cells upon addition of LPS (38), but such induction did not induce Spi-C expression. Hence, it appears as if Spi-C expression is temporally regulated during B lymphoid differentiation and is preferentially expressed in mature, non-secretory B cells.

We finally investigated the expression of Spi-C by Northern blotting using poly(A)+ RNA from two plasmacytomas and from the B cell lymphoma K46R (Fig. 4C). The Spi-C probe hybridized with a single band migrating below the 18 S RNA marker in the K46R lane, while no hybridization was detected in the lanes with plasmacytoma RNA. All three lanes gave a similar hybridization signal with the GADPH control probe. The size of the Spi-C transcript correlates favorably with the isolated cDNA clone and the low expression level with the RT-PCR data above.

Spi-C Can Transactivate a kappa Y Reporter Gene Construct in HeLa Cells-- We subsequently wanted to determine whether Spi-C was a transcriptional activator as a first characterization of its function. To this end, 0.2 µg of a reporter construct, containing four copies of the kappa Y element from the SP6 kappa  promoter in front of the luciferase gene, was co-transfected into HeLa cells together with 0.3 µg of an empty expression vector or a PU.1- or a Spi-C-containing expression vector (Fig. 5). All transfections were adjusted to include 0.8 µg of total DNA using the empty expression vector, except the control transfection with the kappa Y reporter only. Co-transfection of the Spi-C expression vector induced an almost 10-fold increase in luciferase activity compared with that seen after co-transfection of the empty expression vector. Co-transfection of the PU.1 expression vector induced a slightly higher luciferase activity than that observed with Spi-C at the same concentration, while the addition of PU.1 and Spi-C expression vectors together resulted in luciferase activity at the same level as seen with PU.1 alone. Transfection of the expression vectors alone or together with a luciferase vector containing a TATA box only did not induce any significant luciferase activity (data not shown). We conclude from these experiments that Spi-C is a transcriptional activator of similar efficacy as PU.1.


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Fig. 5.   Co-transfection in HeLa cells of a kappa Y luciferase reporter gene with expression vectors containing Spi-C or PU.1 as indicated. The mean value and standard deviation from three experiments is shown.

Spi-C Is Encoded by a Single-copy Gene Mapping to Chromosome 10, Region C-- We finally wanted to determine the genomic representation of Spi-C and therefore performed a genomic Southern blot. As shown in Fig. 6A, digestion of genomic DNA with four different enzymes gave a single strongly hybridizing band when probed with a Spi-C cDNA probe. With the BamHI and XbaI digests, a weaker hybridizing band of higher mobility was also observed. For the XbaI digest, this can be explained by the presence of an XbaI site in the Spi-C coding region while a BamHI site is present within the Spi-C locus.2


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Fig. 6.   A, Southern blot of E14 embryonic stem cell DNA digested with the indicated restriction enzymes and probed with a Spi-C probe. B, FISH mapping of the Spi-C locus in mouse. The left panel shows the FISH signals on the chromosome, while the right panel shows the same mitotic figure stained with DAPI to identify mouse chromosome 10. C, diagram of FISH mapping results for probe Spi-C. Each dot represents the double FISH signals detected on mouse chromosome 10.

We subsequently cloned the Spi-C gene and used a 12-kilobase pair fragment containing the complete Spi-C locus for chromosomal mapping using FISH (Fig. 6B). Under the conditions used, the FISH detection efficiency was 91% (among 100 checked mitotic figures, 91 of them showed signals on one pair of chromosomes). DAPI banding was used to identify the specific chromosome, and an assignment between signals from the probe and mouse chromosome 10 was obtained. The detailed position was further determined to region C based on the summary from 10 photos (Fig. 6C). Hence, Spi-C is encoded by a single-copy gene mapping to chromosome 10, region C in the mouse genome.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

The expression of several transcription factors has been shown to be essential for B cell development (39, 40). Some of these are obligate for the formation of several hematopoietic lineages, while others seem to be selectively involved in B cell development. Examples of transcription factors of both these types can be found within the Ets family of transcription factors. For example, the phenotypes of mice lacking either PU.1 or the closely related Spi-B, which have a partially overlapping expression patterns within the B cell compartment, differ significantly. In mice homozygous for an inactivated PU.1 gene defects in the development of T and B lymphocytes, granulocytes and monocytes were observed (5, 6). In mice devoid of Spi-B, the phenotype was milder and only antigen receptor signaling in B cells during immune responses seemed to be perturbed, while the remaining hematopoietic system appeared to be normal (23).

Here we describe a novel Ets family protein, Spi-C, that seems to be preferentially expressed within the B cell compartment and has an expression pattern distinct from that of Spi-B and PU.1. A low level of Spi-C expression was detected in the macrophage cell line J774 and in purified macrophages, but whether this is representative for the whole macrophage lineage remains to be investigated. Furthermore, the Spi-C RNA expression detected in liver and thymus could be due to the fact that these tissues contained a low number of B cells that expressed Spi-C. Upon very long exposures, a low level of Spi-C expression could also be detected in heart and kidney RNA (data not shown). The conclusion that Spi-C expression in some of these organs was derived from contaminating B cells is strengthened by the finding that purified, double-negative thymocytes as well as peripheral CD4+ or CD8+ T cells did not express Spi-C, while purified B220+ cells did. Lymphoid organs that contain mature B cells like spleen, lymph node, and bone marrow did express Spi-C RNA. As a comparison, PU.1 is expressed in cells of the myeloid lineage (13) and both PU.1 and Spi-B are expressed in T lymphocytes (3). Furthermore, Spi-C appears to be expressed during a window of B cell differentiation representing peripheral, mature B cells but neither early B cells nor plasma cells, a finding that also distinguishes Spi-C expression from that of PU.1 and Spi-B. We did not detect any Spi-C expression in embryonic stem cells using RT-PCR while several expressed sequence tags can be found in the NCBI data base that appears to be derived from Spi-C. Some of these have been cloned from a cDNA library prepared from blastocysts, which might indicate an additional role for Spi-C during early development. In conclusion, we show evidence that Spi-C has an expression pattern distinct from that of other Ets proteins and we would like to propose that it has a unique function during B lymphoid differentiation. The elucidation of that putative function must await the targeted inactivation of the Spi-C gene in the mouse germline.

Spi-C is related to Spi-B and PU.1 in the DNA binding Ets domain based on amino acid sequence homology, while it showed no homology to other Ets proteins outside this domain. Furthermore, PU.1 and Spi-B are more closely related to each other than to Spi-C. Spi-C may therefore be considered to represent a novel subgroup of Ets proteins, especially considering the lower degree of amino acid sequence identity compared with Spi-B and PU.1 within helix 1 of the Ets domain (1, 41). The DNA binding characteristics of Spi-C were very similar to those of PU.1. In methylation interference assays using the SP6 kappa  promoter kappa Y element, the same protection patterns were observed using either PU.1 or Spi-C. Furthermore, several PU.1 binding sites competed as efficiently for Spi-C binding as for PU.1 binding, and a site selection experiment selected defined PU.1 target sequences in >90% of the templates. The only significant difference in binding site preference between the two that could be noted was a clear preference for an A in position -2 for Spi-C, while PU.1 selects a G in this position (2). Thus, it appears as if these two Ets proteins are alternative ligands to the same recognition sequences in mature B cells, although a minor divergence could be detected. This could be expected, given the conservation of all residues in helix 2 and 3 of the Ets domain that has been shown to interact with DNA either by structural analysis or mutagenesis (34, 35). On the other hand, given the sequence divergence in helix 1 of the Ets domain, some difference in sequence recognition of motifs outside the Ets core could be envisioned. For example, amino acids in the NH2 terminus of PU.1 helix 1 form water-mediated or phosphate backbone interactions with DNA (34). It should also be considered that Ets proteins have been described to mainly exert their function as members of higher order protein complexes (19, 42-44), which might qualitatively influence the protein/DNA interaction. For example, the DNA interaction of GABPalpha is of lower affinity than that observed for the GABPalpha /GABPbeta heterodimer, although only the former contains an Ets domain (45). It has also been shown that the part of the Ets domain of GABPalpha that interacts with GABPbeta is in helix 1 (44). It is thus tempting to speculate that Spi-C might have a distinct preference from that of PU.1 and Spi-B with regard to protein/protein interactions and, as a consequence, a distinct function with regard to gene regulation both qualitatively and quantitatively. The identification of a selective ligand for Spi-C is obviously an interesting topic for future studies, as is the definition of its target genes.

Spi-C was cloned using the SP6 kappa  promoter kappa Y element as bait. This element is only found in some kappa  promoters, and the role of Spi-C in the regulation of kappa  transcription is difficult to envision, especially since Spi-C seems to be turned off in plasma cells. Rather, the Ets motifs that have been shown to be critical for the control of immunoglobulin expression should be interacting with PU.1 or other Ets proteins at this late stage of B cell differentiation (14, 17-19, 22). One obvious possibility is that the relevant target gene(s) for Spi-C is not immunoglobulin but that our cloning approach rendered its isolation fortuitously. On the other hand, we could show by co-transfection that Spi-C contains a transcriptional activation domain of similar efficiency as PU.1. Whether the potential of Spi-C as a transcriptional activator, or its target sequence specificity, is modified if proper adapter molecules (19) are present remains to be determined.

Finally, the Spi-C locus was mapped to chromosome 10, region C in the mouse. No homogeneous syntenic region with regard to the human genome can be found, but several markers from this region map to human chromosome 12q22-24 (46). Interestingly, several chromosomal translocations in non-Hodgkin's lymphomas have been described in this region (50). It should also be pointed out that, upon a data base search, a sequence corresponding a human homolog of Spi-C was identified on human chromosome 4p16.3 in the Huntington's disease region (GenBank accession no. Z68163). However, this locus most likely corresponds to a pseudogene, since it is a near perfect match of the Spi-C cDNA with the exception of a deleted region that potentially corresponds to a splice-intermediate. Furthermore, no synteny is evident between mouse chromosome 10 and 4p16 (46). However, the sequence of the human pseudogene would indicate that the Spi-C gene has about a 70% homology between the mouse and the human genome. In conclusion, our initial characterization indicates that Spi-C is an important regulator of B lymphocyte development, but the elucidation of its biological function must thus await further experimentation in vitro and in vivo.

    ACKNOWLEDGEMENTS

We thank Dr. M. Sigvardsson for critical reading of the manuscript and members of the Immunology Unit in Lund for reagents.

    FOOTNOTES

* This work was supported by grants from the Swedish Cancer Society, Swedish Medical Research Council, Kock's Foundation, Crafoord Foundation, and Österlunds Foundation.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF098863.

Dagger Current address: Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom.

§ To whom correspondence should be addressed. Fax: 46-46-2224218; E-mail: Tomas.Leandersson{at}immuno.lu.se.

2 D. Liberg, unpublished results.

    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; LPS, lipoppolysaccharide; RT, reverse transcription; FISH, fluorescence in situ hybridization; 3-AT, 3-amino-1,2,4-triazole; EMSA, electrophoretic mobility shift assay; DAPI, 4',6-diamidino-2-phenyl-indole; GABP, GA-binding protein; EBF, early B cell factor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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