Nucleic Acids Research, Vol. 18, No. 18 5495

Astrocytes and glioblastoma cells express novel octamerDNA binding proteins distinct from the ubiquitous Oct-1 and B cell type Oct-2 proteins Edgar Schreiber*, Keith Harshman, Iris Kemler, Ursula Malipiero1, Walter Schaffner and Adriano Fontana1

Institut fur Molekularbiologie 11 der Universitat ZOrich, Honggerberg, 8093 ZOrich and 1Abteilung fur Klinische Immunologie, Departement fur Innere Medizin, Universitatsspital, Haldeliweg 4, 8044 ZOrich, Switzerland Received May 25, 1990; Revised and Accepted August 15, 1990

ABSTRACT The 'octamer' sequence, ATGCAAAT or its complement ATTTGCAT, is a key element for the transcriptional regulation of immunoglobulin genes in B-lymphocytes as well as a number of housekeeping genes in all cell types. In lymphocytes, the octamerbinding protein Oct-2A and variants thereof are thought to contribute to the B-cell specific gene expression, while the ubiquitous protein Oct-1 seems to control general octamer site-dependent transcription. Various other genes, for example interleukin-1 and MHC class 11 genes, contain an octamer sequence in the promoter and are expressed in cells of both the immune and nervous systems. This prompted us to analyze the octamer-binding proteins in the latter cells. Using the electrophoretic mobility shift assay, at least six novel octamer binding proteins were detected in nuclear extracts of cultured mouse astrocytes. These proteins are differentially expressed in human glioblastoma and neuroblastoma cell lines. The nervous system-derived (N-Oct) proteins bound to the octamer DNA sequence in a manner which is indistinguishable from the Oct-1 and Oct-2A proteins. The relationship of the N-Oct proteins to Oct-i and Oct-2A was analyzed by proteolytic clipping bandshift assays and by their reactivity towards antisera raised against recombinant Oct-I and Oct-2A proteins. On the basis of these assays, all N-Oct-factors were found to be distinct from the ubiquitous Oct-I and the lymphoid-specific Oct-2A proteins. In melanoma cells that contain the N-Oct-3 factor, a transfected lymphocyte-specific promoter was neither activated nor was it repressed upon cotransfection with an Oct-2A expression vector. We therefore speculate that N-Oct-3 and other N-Oct factors have a specific role in gene expression in cells of the nervous system.

*

To whom

correspondence should be addressed

INTRODUCTION The selectivity of tissue-specific gene expression depends primarily on the interplay of transcription factors present in a given cell type with their cognate recognition sequences in the promoter and enhancer regions of the gene to be transcribed (for review see Maniatis et al. 1987, Muller et al. 1988a). The particular arrangement of the DNA modules forming these control elements allows distinct regulatory factors to function coordinately in potentiating messenger RNA synthesis (for review see Ptashne 1988; Johnson and McKnight, 1989; Mitchell and Tjian, 1989). The octamer sequence motif, ATGCAAAT (or its complement ATTTGCAT), is a paradigm of a regulatory element employed in transcriptional activation of both ubiquitously expressed genes and cell-type specific genes (reviewed in Schreiber et al. 1989). The octamer motif, first identified in the promoters of the histone H2B gene (Harvey et al. 1982) and in the light chain and heavy chain immunoglobulin genes (Falkner and Zachau, 1984; Parslow et al., 1984), is now recognized, with a certain degree of sequence variation, to be an important element in the promoters and enhancers of a number of tissue-specific eukaryotic genes. Interestingly, included in this octamer-responsive group are a number of genes, such as the human T cell receptor (3-chain enhancer (Krimpenfort et al. 1988), and the major histocompatibility antigen complex (MHC class II) HLA DQf32 and DRax (Miwa and Strominger 1987, Sherman et al. 1989) the expression of which is cell-type specific but not restricted to Bcells. In addition, the octamer sequence is also present in the control regions of many housekeeping genes such as the U2 small nuclear RNA genes as well as some viruses, e.g. SV40 virus, herpes simplex virus and Adenovirus (reviewed in Schreiber et al. 1989). The apparent selective use of the octamer sequence element in promoting ubiquitous and cell type-specific gene expression may be accomplished by the selective binding of a ubiquitously present octamer binding protein (Oct-i) or cell typespecific octamer binding proteins (Oct-2A and Oct-2B), respectively (for Ref. see Miller et al. 1988a and Schreiber et

5496 Nucleic Acids Research, Vol. 18, No. 18 al. 1989). Alternatively, the presence and position of other factor binding sites might influence the exhibited affinity for the octamer binding site. Of course, relative intracellular/ (nuclear) concentration of all factors could influence octamer-site occupancy in both models. The cDNAs encoding the ubiquitously expressed Oct-i and the lymphoid-specific Oct-2A proteins have been cloned (Sturm et al. 1988, Muller et al. 1988b; Staudt et al. 1988, Clerc et al. 1988, Scheidereit et al. 1988). Ectopic overexpression of the Oct-2A cDNA in a non-lymphoid cell led to transcription activation of a lymphoid-specific promoter construction consisting of an octamer site with a TATA-box (Muller et al. 1988b). This result suggests that in different cell types the transcriptional selectivity of octamer-containing genes depends on the presence of different cell type-specific octamer binding proteins. Indeed, the list of Oct-binding proteins with novel tissue distributions is increasingly long: Barberis et al. (1987) reported on a testisspecific Oct- protein in sea urchin and Cox et al. (1988) noted a novel Oct-protein in extracts from a melanoma cell line; a developmentally regulated octamer-binding acivity in F9 embryocarcinoma cells was described (Lenardo et al., 1989; Scholer et al. 1989); a broad overview of novel octamer binding proteins present in various organs, including brain and embryonic tissues from mouse and rat was described by Scholer et al. (1989); three Oct-related transcripts were isolated from rat brain mRNA by 'PCR-cloning' and shown to hybridize selectively to different regions in the brain (He et al. 1989). An indication of expression of Oct-2 mRNA in certain glioma cell lines was given by Staudt et al. (1988). All of these data point to the existence of a large but related family of octamer-binding proteins having a wide and varied tissue distribution. In the present study we have investigated in more detail the presence of octamer binding proteins in cultured mouse astrocytes as well as a wide variety of transformed cells of the nervous system and have thoroughly analyzed their relationship to known Oct-proteins. We demonstrate that the astrocytes express novel octamer binding proteins which we have termed N-Oct-2cz and ,B, N-Oct-3, N-Oct-4 and N-Oct-5a/5b. Data are presented which describe the differential expression of these proteins in nervous system derived tumor cells, and characterize the biochemical features of these proteins as well as the role of N-Oct-3 in transcription activation.

MATERIALS AND METHODS Cell lines and preparation of nuclear extracts BJA-B cells were cultured in RPMI-1640 medium (Sigma) containing 10% fetal calf serum (FCS), 100 U/ml penicillin and 100 jg streptomycin. The nervous system derived tumor cell lines tested consisted of four well characterized human glioblastoma cell lines (line LN-18, 309, 382, 215), three neuroblastoma cell lines (line LAN-1, SK-N-BE and SK-N-LE), and one melanoma line (Bowes) and were kindly provided by Dr. N. de Tribolet, Neurosurgical Service, Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland. Rat C6 glioma cells were obtained from Flow Laboratories. The cell lines used have been characterized in studies of Biedler et al., 1987; Bissell er al., 1974; Schnegg et al., 1981; Seeger et al., 1977; Sherman et al., 1989. The cells were grown in culture flasks (Falcon, 175 cm2 growth area) in DMEM supplemented with 10% FCS and Lglutamin (0.3 mg/ml). Astrocytes were prepared from brain of newborn

1987.

ICR +/ + mice and cultured

as

described

by

Frei et al.,

Nuclear extracts were prepared as described by Schreiber et al. (1989) with minor modifications: buffers A and C contained Leupeptin (Boehringer Mannheim) as an additional protease inhibitor at 4 itg/ ml buffer while Buffer C contained 20% Glycerol. Methylation interference assay This assay (Siebenlist and Gilbert, 1980) was performed as described by Kemler et al. (1989). Preparative SDS-PAGE and renaturation of DNA-binding activity These procedures were performed as described by Schreiber et al. (1988) with minor modifications: a 12% SDS-PAGE gel was used and the guanidiniumhydrochloride denatured nuclear proteins were renatured by adding 50 y1 buffer X50 without glycerol and microdialyzed against 200 ml buffer X50 with 20% glycerol for 12 hrs at 4 'C. Preparation of Oct-1 and Oct-2a Antisera Recombinant Oct-I and Oct-2a were used as antigens for the production of polyclonal antisera. The Oct-2a cDNA (Muller et al., 1988) and a 3' truncation of the Oct-I cDNA (Sturm et al., 1988) were expressed in E. coli under the control of an IPTGinducible T7 RNA polymerase gene (Studier and Moffat, 1986). Following induction, the insoluble recombinant protein was isolated in the inclusion body fraction and injected (150 yg) without further purification into New Zealand white rabbits. The rabbits were boosted (100 ,ug) 3 weeks following the first injection and tested for an immune response by Western blotting after an additional 10 days. Both sera were determined to have sufficiently high titers and were used without purification in the described experiments. Probes for bandshift analysis The DdeI-Hinfl fragment (nucleotides position 518-566) from the IgH enhancer was subcloned into the SalI-site of pUC 18. A 51 bp SalI-fragment was prepared from this clone, dephosphorylated with CIAP (Boehringer Mannheim), and 5'-end labelled with 'y-32p-ATP and T4 polynucleotide kinase. A specific activity of 1 x 106/pmol 5'-end was usually achieved. For competition experiments, an unlabelled fragment containing 4 copies of the above mentioned sequence was used (octa+); the (octa -) fragment contained 4 copies of a sequence in which the octanucleotide motif was eliminated by mutation from ATGCAAAT to CTGAACAT. Bandshift assay and proteolytic clipping The procedures were performed exactly as described by Schreiber et al. (1988) Transfections and RNase protection mapping. Plasmid A and B used in this study are identical to constructs '5' and '4' respectively, used by Muiller et al. (1988b). Construct C in this study is identical to plasmid pbG (Banerji et al. 1981) containing the SV40 enhancer 3' of the rabbit fl-globin gene. COS-7 and Bowes melanoma cells were split the day before transfection; at the time of transfection a confluency of 50% was usually desired. 10 Ag of reporter plasmid was mixed with 3 itg of OVEC-REF (Westin et al. 1987), the internal standard, and 5 itg of sonicated herring sperm carrier DNA and applied as CaPO4 coprecipitates to the cells for 12 hrs; 3 ml of a 25 % solution DMSO was given to the TBS-washed cells for 3 min

Nucleic Acids Research, Vol. 18, No. 18 5497 and after its removal the cells were incubated in 10 ml medium for 30 more hrs. Cytoplasmic RNA was extracted according to Gough (1988) and the RNase protection assay with a ,3-globin SP6 riboprobe was performed as described by Muller et al.

(1988b). RESULTS Nervous system-derived tumor cell lines contain novel octamer binding proteins Nuclear extracts were prepared from seven neuroblast cell lines: four human glioblastoma cell lines (line LN-18, 308, 382 and

215) and three human neuroblastoma cell lines (line LAN-1, SKN-BE and SK-N-LE). Furthermore, one human melanoma cell line (BOWES) was included as a control in the study, as melanocytes, like neuroblasts, originate from the neuroepithelial plate. Nuclear extracts containing the thoroughly characterized tissue-specific octamer binding proteins Oct-2A and Oct-2B were prepared from BJA-B lymphocytes. Nuclear extracts from COS-7 monkey kidney epithelial-like cells and from mouse L-cells (fibroblasts), prepared in the same manner and with the same buffers as the other samples, were included as (non tissue-specific) controls containing only Oct-I protein.

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Figure 1A: Autoradiograph of a bandshift experiment showing octamer DNA binding nuclear proteins in various cell lines: BJA-B, a human Burkitt lymphoma line; human glioblastoma cell lines LN-18, 308, 382, 215; human neuroblastoma cell lines LAN-1, SK-N-BE, SK-N-LE and human Bowes melanoma cells, monkey kidney fibroblasts COS-7, and mouse fibroblasts (L-cells). The origin of the cell lines is described in Materials and Methods. The schematic drawing on the right half illustrates the bandshift pattern and nomenclature of Oct-proteins in B-lymphocytes and nervous system derived tumor cell lines. The radiolabelled probe was the 51 bp DdeI-Hinfl fragment from the mouse IgH enhancer that contains the octamer sequence, ATTTGCAT. Figure 1B: Expression of octamer DNA binding proteins in the brain. Extracts prepared from cultured murine astrocytes (lane 3), rat C6 glioma cells (lane 4), or from homogenized brain of either newborn (lane 2) or 14 day old mice (lane 1) were tested in the bandshift assay and compared with the retardation pattern of BJA-B lymphocytes (lane 5). Figure 1C: Specificity of the octamer binding activities of nuclear extracts prepared from cells of the nervous system. The specificity of the retarded complexes formed by Oct-proteins of the various cells indicated is demonstrated by addition of excess unlabelled competitor DNA containing either the intact octamer motif (Ddel-Hinfl IgH enhancer fragment described in Material and Methods) or a mutated octamer motif differing in three out of eight nucleotides (see Material and Methods).

5498 Nucleic Acids Research Vol. 18, No. 18 A radiolabelled 51 bp DdeI-Hinfl octamer-containing fragment from the murine immunoglobulin heavy chain enhancer was incubated with these extracts to test their octamer binding activity in the electrophoretic mobility shift (bandshift) assay. As shown in Figure lA, extracts from BJA-B lymphocytes give rise to three prominent protein DNA complexes in a long-run bandshift experiment Oane 1). The slowest migrating complex is due to binding of Oct-i, the ubiquitously expressed protein. The second most retarded complex is due to binding of the lymphocyte-specific protein Oct-2B followed by the most prominent Oct-2A protein complex (Schreiber et al. 1988; MUller et al. 1988b). In addition to these previously described Oct-protein complexes, we noted a faint and faster migrating band at the lower end of the Oct-2A complex. We tentatively designated this binding activity Oct-2C. By analyzing tumor cell lines derived from the nervous system, several octamer-DNA protein complexes were detected (lanes 2-9): 1) a typical Oct-I complex indistinguishable from the Oct-i complex in BJA-B nuclear extracts, 2) a. doublet band designated N-Oct-2 (N-for nervous system), which migrates close to, but slightly slower than the Oct-2A complex from BJA-B cells, and 3) a faster migrating DNA-protein complex termed N-Oct-3 which co-migrates with the Oct-2C complex in BJA-B extracts. Unlike lymphocyte nuclear extracts, none of the extracts from the different human nervous system cell lines displayed the Oct-2B complex. It was not possible to distinguish the cell lines according to their origin as neuroblastoma, glioblastoma or melanoma based on the composition of Oct-proteins in the corresponding nuclear extracts. The Oct-I protein was present in nuclear cell extracts of all cell lines tested; the combination of Oct-I with N-Oct-3 or Oct-i together with N-Oct-2 and N-Oct-3 was observed in some glioblastoma and neuroblastoma cell lines. None of the cell lines expressed N-Oct-2 in the absence of N-Oct-3. In COS-7 cells, L-cells (lanes 10 and 11), and HeLa cells (data not shown) only the Oct-I protein was detected. To show that the novel complexes detected are not due to proteolytic degradation occuring during extract preparation with these particular cell lines, we examined the integrity of high molecular weight proteins in these extracts by comparative SDSpolyacrylamide electrophoresis (data not shown). Furthermore, we performed a comparative bandshift experiment with these extracts, a BJA-B extract, and an SpI-transcription factor binding site. All extracts produced bandshifts characteristic of the presence of un-proteolyzed Spl (data not shown).

",_~A

human SK-N-BE neuroblastoma cells (Fig. lA, lane 7). The same data were obtained when nuclear extracts were taken from astrocyte cultures established from newborn rats as described by Massa et al. (1987) (data not shown). We conclude that astrocytes, but not the various glioblastoma or neuroblastoma tumor cell lines, contain the full spectrum of Oct-proteins in the nervous system: N-Oct-2 , N-Oct-3, N-Oct-4, N-Oct-5a/b and the ubiquitous Oct-i protein. Identical DNA binding specificity of B-cell derived Oct-2 and N-Oct-2 and N-Oct-3 The specificity of the octamer DNA-protein interactions was analyzed by competition binding experiments. A 500-fold molar excess of an unlabelled oligonucleotide containing either the wildtype octamer sequence (ATGCAAAT) or a mutated octamer (CTGAACAT) was used to compete for binding of nuclear proteins to the radiolabelled bandshift probe. As shown in Figure 1C, the binding of Oct-i, N-Oct-2 , N-Oct-3 and N-Oct-Sa/b was efficiently competed by an unlabelled DNA containing the genuine octamer binding site (lanes 7-12). In contrast, the mutated octamer sequence did not compete for binding (lanes 13-18). These data indicate that the novel octamer binding proteins interact specifically with the octamer DNA sequence and not with other sequences on the fragment used for the bandshift experiments. To identify the contact points of protein binding to the octamer element we performed a methylation interference analysis (Siebenlist and Gilbert, 1980). An end-labelled octamer bandshift probe (oligo oct+/hep-; Kemler et al. 1989) was partially methylated and used for a preparative bandshift assay with extracts from SK-N-BE and from BJA-B cells. As seen in Figure 2, formation of the N-Oct-2 complex (ane 1) as well as the NOct-3 complex (lane 2) was prevented by methylation over the same nucleotides of the octamer region as for the Oct-2A protein from BJA-B cells (lane 3). We conclude therefore that the mode of DNA recognition is the same for all three octamer binding proteins tested.

3

;

Presence of additional N-Oct proteins in extracts from cultured astrocytes To rule out the formal possibility that the novel octamer factors were only detectable in long-term cultured human tumor cell lines, we tested for their presence in extracts from cultured astrocytes derived from brains of newborn mice and from extracts prepared from total brain of adult and newborn mice. We included in this experiment a rat glioma cell line (C6) to ask whether the novel Oct-complexes are conserved inter-species. As shown in Figure iB, (lane 4), the extract from the C6 cells also produced the Oct-i and the N-Oct-3 complex. In addition to these, three more complexes were detected with the astrocytes (lane 3) and brain extracts (lane 1 and 2): the N-Oct-2 complex, a faint complex, termed N-Oct-4, running below N-Oct-3, and two fast, comigrating complexes, referred to as N-Oct-5a and N-Oct-Sb. The N-Oct-5a/b complexes were less abundant in

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Figure 2: Autoradiograph of a methylation interference analysis with the radiolabelled octamer bandshift probe and Oct-2A from B-cells, N-Oct-2 and NOct-3 proteins from SK-N-BE cells reveals identical contact sites over the octamer element. The nucleotides the metiylation of which completely or partally interferes with protein binding are indicated by solid or open triangles, respectively.

_

Nucleic Acids Research, Vol. 18, No. 18 5499

Isolation of octamer binding proteins from neuroblastoma cells Preparative SDS-PAGE of nuclear extracts and subsequent recovery and renaturation of size-separated proteins is a simple and straightforward method to isolate and compare different octamer binding proteins (Schreiber et al. 1988). Nuclear extract from SK-N-BE neuroblastoma cells was loaded onto a 12% SDSprotein gel. The Oct-I binding activity eluted from a region of the SDS-PAGE gel corresponding to a MW of 90-100 kDa. This value is in accordance with published data (Fletcher et al. 1987). The N-Oct-2 binding activity eluted from a region around 60 kDa (+/- 5 kDa) and the N-Oct-3 binding activity eluted from a region around 50 kDa (+/- 5 kDa). In spite of extensive efforts, we did not succeed in achieving renaturation of the NOct-4 and the N-Oct-5 complexes. Figure 3A (lanes 1-4) shows an autoradiograph of a bandshift experiment with renatured Octproteins isolated from SK-N-BE neuroblastoma cells. The specificity of the Oct-proteins: DNA interactions was demonstrated by binding competition analysis with a 100-fold molar excess of an unlabelled wildtype octamer binding site (lanes 5-7) and an otherwise identical DNA fragment which contained a mutated octamer binding site (lanes 8-10). Note that the N-Oct-2 complex appeared to consist of two independent binding activities which migrated closely together when tested under native conditions (see Figure 1) but were clearly separable after physical separation on a denaturing gel and subsequent renaturation. It is conceivable that a low MW component was removed from the complex during the denaturation/separation process. We have tentatively designated the proteins responsible for the slower migrating component and faster migrating complexes as N-Oct-2or and N-Oct-23, respectively. This latter protein was also present in the gel eluate containing the N-Oct-3 protein. Combination of both gel slice eluates yielded an additive increase in N-Oct-2f3 binding activity; the same result was seen when both (still unrenatured) protein eluates were co-renatured (data not shown). We conclude, therefore, that the N-Oct-2 complex seen in the extracts of astrocytes and some neuroblastoma cell lines consists of two

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separable entities with MW of ca. 60 kDa each, namely N-Oct-2a and N-Oct-2,3. N-Oct-2 and N-Oct-3 also bind to the heptamer element of the IgH promoter A characteristic feature of known Oct-proteins is their remarkable flexibility in binding site recognition (Sturm et al. 1988; Thali et al. 1988). The Oct-1 and Oct-2A/B proteins do not only bind to the octamer motif (ATGCAAAT), but also to the unrelated heptamer motif CTCATGA present in the IgH gene promoter, though with lower affinity (Poellinger and Roeder, 1989; Kemler et al., 1989). We asked whether the novel Oct-proteins present in the nervous system cells are also able to bind to this heptamer sequence. To this end, we incubated a radiolabelled oligonucleotide containing the heptamer element and a mutated octamer sequence (hep+ oct; Kemler et al., 1989) with renatured fractions from size-separated octamer binding proteins derived from SK-N-BE cells. As shown in Figure 3B, the neuroblastoma derived Oct-1, N-Oct-2 and N-Oct-3 proteins can specifically bind to the heptamer motif (lanes 1 and 2), and thus display the same feature of heterologous sequence recognition as the Oct-proteins from B-lymphocytes.The N-Oct-protein: heptamer-DNA interaction was efficiently competed with excess of unlabelled DNA with an octamer- binding site (lanes 3 and 4) but not with a DNA fragment containing a mutated octamer sequence (lanes 5 and 6).

Oct-2A from B-cells is different from the N-Oct-2 complex By using the proteolytic clipping bandshift assay technique (Marzouki et al. 1986) it was possible to distinguish the three major forms of octamer DNA binding proteins detected in B lymphocytes (Schreiber et al. 1988). Since the Oct-2A protein from B-cells comigrates with the N-Oct-2 complex in a bandshift gel we asked whether the Oct-2A protein is related to the a or :-form of this complex. To this end, the Oct-2A protein from BJA-B cells and the N-Oct-2 complex from SK-N-BE cells were isolated following SDS-PAGE, renatured, and, after a preincubation in a bandshift binding reaction, exposed to defined

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Figure 3A: Autoradiograph of a bandshift experiment after separation of N-Oct-2a/2fl and N-Oct-3 by preparative SDS-PAGE and renaturation. Nuclear extracts prepared from SK-N-BE neuroblastoma cells were separated on a denaturing protein gel; after cutting the gel into slices, the proteins were eluted and subjected to a denaturation/renaturation protocol as described in Materials and Methods The three octamer DNA binding complexes detected, which correspond to Oct-i, N-Oct-2a/2(3 and N-Oct-3, were used for a competition experiment as described in legend to Figure IC. Figure 3B: N-Oct-proteins bind also to the heptamer element of the IgH promoter. Isolated N-Oct-2a/2(3 and N-Oct-3 protein (see Figure 3A) were tested by bandshift assay using a radiolabelled heptamer motif (see Material and Methods) and either intact or mutated octamer competitor DNAs as described in legend to Figure IC.

5500 Nucleic Acids Research, Vol. 18, No. 18

amounts of trypsin which cleaves proteins after arginine or lysine residues. As seen in Figure 4A, increasing amounts of trypsin clipped the Oct-2A protein of BJA-B cells into five polypeptides. Even though the degradation pattern of the neuroblastoma NOct-2 complex differed somewhat from that of Oct-2A, note that three small truncated polypeptides (most probably derived from N-Oct-2a) comigrate with peptides derived from the Oct-2A protein. Although the N-ct-2(3 protein seems to be more resistant to trypsin than N-Oct-2a its contribution to the lower molecular weight fragments is not clear. We are therefore unable at this point to assess its contribution to the clipping pattern seen in Figure 4A and its relationship to N-Oct-2a. We conclude that both N-Oct 2 proteins are distinct from the Oct-2A protein of B-cells. We next used the ArgC protease, which cleaves proteins specifically after arginine residues, to analyze the relationship between Oct-2A from B-cells and the N-Oct-3 protein from Bowes melanocytes. Figure 4B shows that the clipping pattern exhibited by N-Oct-3 was seemingly similar to that of Oct-2A. The change in the molecular weights of the two different polypeptides due to the action of ArgC is the same, even though the starting molecular weights are dissimilar. This observation can be interpreted to mean that both proteins contain a homologous domain that is removed upon ArgC digestion. However, the Oct-2A protein appeared to be more sensitive towards ArgC proteolysis than the N-Oct-3 protein. A similar lower sensitivity to ArgC proteolysis was observed for the NOct-3 protein in SK-N-BE extracts (data not shown). Therefore,the extent of the relation between these proteins remains an open question. The Oct-I complex from extracts of Bowes (Figure 4B) and SK-N-BE cells (data not shown) is considered to be identical with the ubiquitous Oct-I protein since it displayed the same degradation pattern as that described for the Oct-i protein of HeLa and BJA-B cells (Schreiber et al. 1988). Antibodies directed against Oct-i or Oct-2A do not recognize N-Oct proteins To further investigate the relationship of N-Oct-factors to Oct-I and Oct-2, we used antisera raised against recombinant Oct-I or Oct-2 proteins. Nuclear proteins from BJA-B. lymphocytes, Bowes melanoma cells, and from astrocytes were preincubated

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with dilutions of either antiserum and then tested for octamerDNA sequence binding. From Figure 5A it is evident that the anti-Oct-l-antibodies specifically affected DNA-binding of the Oct-l protein. Neither the B-cell type factors Oct-2A and Oct-2B nor the N-Oct factors were neutralized. The preimmune serum did not affect any of the Oct-proteins (data not shown). Next, we used the anti-Oct-2-antibodies: Figure 5B shows that all NOct factors and Oct-I remained unaffected in DNA-binding and migration even at high antiserum concentrations, whereas virtually all DNA binding activity of Oct-2A and (less pronounced) of Oct-2B proteins were affected. The interactions of the antibodies with Oct-2 gave rise to high molecular weight complexes and at higher concentrations of the antiserum to elimination of DNA binding. We conclude that all N-Oct proteins do not contain epitopes which can be recognized by the two polyclonal antisera directed against the Oct-l and the Oct-2 proteins respectively. Therefore, the N-Oct proteins are most likely structurally different from Oct-I and Oct-2.

Transcription from a B cell-specific promoter is not stimulated in N-Oct-3 containing Bowes melanocytes The octamer sequence in conjunction with a TATA box constitutes a B-cell specific promoter (Dreyfus et al. 1987, Wirth et al. 1987, Muller et al. 1988). A simple model proposes that the transcription activation potential of the lymphoid-specific Oct-2A protein is sufficient to activate this promoter, whereas the ubiquitous Oct-I protein is only able to activate transcription in conjunction with an additional, closely bound transcription factor (reviewed in Schaffner,1989). We asked whether the N-Oct-3 protein in Bowes melanocytes is able to 'transactivate' such a minimal, B-cell specific promoter. Bowes melanocytes (containing Oct-I and N-Oct-3) and Cos7 kidney fibroblasts (containing only Oct-i) were transfected with the target plasmids outlined in Figure 6. Construct A contains the octamer sequence separated from the TATA box of the (3globin reporter gene by 5 bp. Construct B, which contains a mutated octamer sequence, and construct C, which contains the wild-type 3-globin promoter, were used as negative and positive controls, respectively. After transient expression of these constructs, the cytoplasmic RNA was isolated and reporter

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Figure 4A: Autoradiograph of a proteolytic clipping bandshift analysis. The SDS-PAGE purified Oct-2A protein from B-cells and N-Oct-2ca and (3 from SK-N-BE cells were pre-incubated with the radiolabelled bandshift probe and subjected to limited proteolysis by addition of the indicated amount of trypsin. After 10 min. the 'bandshift'/protease mixture was loaded on a bandshift gel. The clipping pattern is illustrated schematically on the right hand side. The faster migrating band seen with N-Oct-2 in the absence of trypsin is a degradation product of N-Oct-2. Figure 4B: Proteolytic clipping bandshift analysis of Oct-proteins with increasing amounts of ArgC protease and nuclear extracts from HeLa cells (lane 1), HeLa cells transfected with the Oct-2A cDNA (lane 2) (Muller et al. 1989) and Bowes melanoma cells (lane 3). The schematic drawing illustrates the clipping pattern of the Oct-2A and the N-Oct-3 protein.

Nucleic Acids Research, Vol. 18, No. 18 5501 transcripts monitored by RNase protection mapping. Figure 6 shows that the B cell-specific octamer-TATA promoter (construct A) remained silent in Bowes melanocytes as well as in Cos7 cells. To exclude a possible repressor function of N-Oct 3 perhaps analogous to the one proposed for NF-A 11 (Lenardo et al. 1989), we co-transfected construct A and the Oct-2A cDNA in an expression vector (Muller et al. 1988b). This resulted in an activation of this promoter comparable to the strong activation seen with the (3-globin promoter (construct C; data not shown). This result indicates that the N-Oct-3 protein, in the amounts present in Bowes melanoma cells, is neither competent to 'transactivate' a simple octamer-TATA promoter nor to prevent promoter activation by the Oct-2A protein.

the bandshift assay; in their nomenclature the Oct-2 band corresponds to our N-Oct-2 complex, Oct-3 to N-Oct-3, Oct-7 to N-Oct4 and Oct9/10 to N-Oct 5a/b. In this report we describe the differential distribution of these proteins in both astrocytes and tumor cell lines of the nervous system. Further, we have studied the DNA binding characteristics of the N-Oct-2 and NOct-3 proteins and analyzed their relationship to the Oct-proteins from the immune system. Finally, we tested the role of N-Oct-3 as a putative transcription factor.

B-cell and nervous system derived Oct-proteins have indistinguishable features of DNA-binding The nuclear proteins from nervous system cells share the same properties of specific binding to the octamer motif as the ubiquitous Oct-I protein and the lymphoid-prominent Oct-2A and Oct-2B proteins by several criteria. The binding of the N-Oct proteins to the octamer motif was competed efficiently by an excess of unlabelled octamer binding site, but was not affected when the binding site was mutated. The DNA binding domains of Oct-I and Oct-2A share a 60 amino acid region referred to as the homeodomain, first described in factors regulating early development in Drosophila, and by the adjacent region, designated the POU-specific domain. The POU-domain is highly conserved among the transcriptional activators Pit-1, a factor essential for prolactin and growth

DISCUSSION The present work shows that the nervous system expresses a set of Oct-proteins which is distinct from that expressed by the immune system. The nervous system derived octamer binding proteins have been designated as Oct-I (the ubiquitous species), N-Oct-2ca and 2(3, N-Oct-3, N-Oct4 and N-Oct-5a/5b on the basis of their mobility in the bandshift assay. While this work was in progress, Scholer et al. (1989) published an overview of octamer binding proteins from extracts of various tissues including brain of mouse and rat. A similar picture was seen with A

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Figure 5A: Autoradiograph of a bandshift analysis showing that anti-Oct-I-antibodies eliminated DNA-binding of Oct-I but not of N-Oct proteins. Nuclear extracts (2y1) from BJA-B lymphocytes, Bowes melanoma cells, and from mouse astoytes were preincubated in the presence (+) or absence (-) of 1 IA of anti-Oct-i-antibodies prior to addition of DNA. Figure 5B: Antibodies against the Oct-2A protein do not recognize N-Oct proteins. Autoradiograph of a bandshift analysis showing a titration of anti-Oct-2-antibodies with nuclear extracts from BJA-B lymphocytes, Bowes melanoma cells and from mouse astrocytes. The amount of antiserum is indicated (in !d) at the bottom of each lane.

A B C A B C _ t

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Figure 6: Autoradiograph of an RNase protection mapping of transfected ,-globin test and reference genes in Bowes melanoma and COS-7 cells. The test plasmids upper arrow points to the protected signal of the test gene and the lower

are schematically outlined on the right hand side and are further described in the text. The arrows to the signal of the cotransfected reference gene.

5502 Nucleic Acids Research, Vol. 18, No. 18 hormone gene expression in pituitary cells (Ingraham et al. 1988), Oct-i and Oct-2 and the Caenorhabditis gene unc-86 (reviewed in Herr et al. 1988 and Garcia-Blanco et al. 1989). The methylation interference pattern generated on the octamer sequence is identical for N-Oct-2 proteins, N-Oct-3, and Oct-2A. Furthermore, like the known Oct-1, Oct-2A, and Oct-2B proteins (Kemler et al. 1989), the N-Oct-3 protein and the N-Oct-2 proteins recognize also the heptamer element CTCATGA of the IgH promoter. We therefore suspect the DNA binding domains of N-Oct-2a and and N-Oct-3 contain homologies with the POU-homeo DNA binding domain of known Oct-proteins. In order to isolate additional members of the POU-domain containing family of transcription factors, oligonucleotide primers that comprised the conserved nucleotide residues of the four POU domains were used to amplify cDNAs from rat brain mRNA by the polymerase chain reaction (He et al. 1989). Three amplification products, referred to as (brain) Brn-1, Brn-2, and Brn-3, were shown to be highly homologous, but not identical, to known POU-domains. Furthermore, the amplification products were shown to differentially hybridize in situ to certain segments and regions of the brain (He et al. 1989). It is possible that at least some of these amplified transcripts correspond to the novel octamer binding proteins described in this study. Recently, Monuki et al. (1989) and He et al. (1989) isolated cDNAs encoding a POU-domain containing factor, termed SCIP (or tst-1 respectively), that is present in myelin-forming glia cells but absent in astrocytes. The expression of this factor is cAMP inducible and thought to be involved as a primary step in the cascade of progressive determination of myelination. Although all sequenced genes of genuine octamer DNA-binding proteins contain POU-domains, the reverse may not be true; the actual DNA binding sites for the brn 1-3 factors and the SCIP (tst-1) protein remain to be determined. Since the SCIP factor is absent in astrocytes, we think that it was not detected in our experiments.

Differential distribution of N-Oct proteins in glial and neuronal cells Substantial differences were observed in the expression pattern of the N-Oct proteins in different human glioblastoma and neuroblastoma cell lines. Both cell types displayed the N-Oct-2 complex together with the N-Oct-3 protein or the N-Oct-3 protein alone. Therefore, these data did not make it possible to distinguish the cell lines according to their origin as glial or neuronal. Remarkably, LN-18 and 308 glioblastoma cells contained only the Oct-1 protein. Whether this result represents a cell line pecularity remains unclear. We have now analyzed 20 more neuroblastoma and glioblastoma cell lines derived from human tumors; all of these displayed either N-Oct-3 alone or N-Oct-3 together with the N-Oct-2 complex (our unpublished results). We have yet to find a cell line displaying the N-Oct-2 complex in the absence of the N-Oct-3 protein. Astrocytes can be classified as epitheloid type 1 and stellar type-2. These two populations belong to two different cell lineages; bipotential glial precursors differentiate into type-2 astrocytes and oligodendrocytes while type 1 astrocytes belong to a different lineage (Raff et al. 1983). Under the culture conditions used in the present experiments, more than 90% of the astrocytes express the Ran-2 antigen, a marker for type-I astrocytes (Massa et al. 1987). It is striking that fully differentiated type-I astrocytes in culture express all of the N-Oct proteins detectable under our assay conditions.

The relationship of N-Oct proteins with Oct-2 from B-cells The N-Oct-2 complex, the N-Oct-3 protein, and the Oct-2A protein from BJA-B cells were compared by proteolytic clipping bandshift analysis in order to assess structural similarities and differences. In the case of N-Oct-2, advanced proteolytic cleavage results in the production of polypeptides which comigrate with polypeptides produced by similar cleavage of Oct-2A. This might indicate a conserved homology in the DNA binding region between these proteins. A similar phenomenon was observed when the clipping patterns of Oct-2A, -2B and Oct-I were compared (Schreiber et al. 1988), namely the use of high protease concentrations resulted in the appearance of similarly migrating polypeptides. These results were interpreted to reflect the high degree of homology in the DNA binding domain in these proteins. This prediction was subsequently confirmed by the cloning of the cDNAs encoding Oct-I and Oct-2 (Herr et al. 1988). The clipping patterns of N-Oct-2a and a using trypsin digestion were found to be different, suggesting that N-Oct-2a and NOct-2,B are distinct proteins. These data in addition to the immunological data lead us to conclude that the N-Oct-2 a and ( proteins are not identical to the Oct-2A protein from B-cells. When the protease ArgC was used to analyze the relationship between Oct-2A and N-Oct 3, we saw a similar change in molecular weight upon proteolytic cleavage which could reflect a structural relationship in certain domains between Oct-2A and N-Oct-3. However, the N-Oct-3 protein, the N-Oct-2 cx/3 complex, and all other N-Oct factors were not at all recognized by specific antisera directed against recombinant Oct-I or Oct-2A protein. Therefore, we consider it most likely that the N-Oct factor genes are different from the oct-I or oct-2 genes. This finding contrasts with a report indicating Oct-2 to be present also in nervous system cells (Scholer et al., 1989). Additionally, Staudt et al. (1988) showed that a human glioblastoma cell lines expressed the Oct-2 mRNA when tested by Northern blot using an Oct-2 cDNA probe. Furthermore, in situ hybridization with an RNA probe containing the entire POUdomain revealed expression of Oct-2 mRNA in rat brain tissue (He et al. 1989). Since we have clearly shown that the Oct-2A protein from B-cells is absent from astrocytes and from cell lines established from tumors of the nervous system, we believe that the RNA hybridization results which imply oct-2 expression in the brain can be explained by extensive sequence homologies between the Oct-2 gene and N-Oct-factor gene(s). Sequence homologies are most likely to occur in the POU-homeo DNAbinding region. Alternatively, if the oct-2 gene is expressed in the brain in the absence of detectable quantities of Oct-2 protein, one must consider a mechanism that blocks translation of oct-2 transcripts or prevents DNA-binding of the protein e.g. by an inhibiting factor. A lymphoid-specific promoter is neither stimulated nor repressed in N-Oct-3 containing cells The octamer motif present in the immunoglobulin heavy and light chain promoters and in the heavy chain enhancer plays an important role as a determinant of B cell specificity (reviewed in Schreiber et al. 1989). However, the octamer motif is not only restricted to immunoglobulin promoters; in addition to some housekeeping genes, it is also present in promoters of specifically expressed genes such as the ones coding for MHC class II protein (Sherman et al. 1989), the interleukin 1, and the granulocyte

Nucleic Acids Research Vol. 18, No. 18 5503 colony stimulating factor (J. Blaszczynski, personal communication). The transcription of these genes is inducible in astrocytes (Fontana et al. 1982 and 1984; Malipiero et al. 1989) and thus N-Oct factors may be involved in their regulation. It is also conceivable that target promoters/enhancers of N-Oct factors contain the heptamer site CTCATGA, since we have shown that N-Oct proteins can specifically bind to this sequence. Another candidate for activation by N-Oct factors is the human calcitonin/a-CGRP gene. This gene is expressed in neural and thyroid tissues and contains an octamer site at position -160 (Broad et al., 1989). It is also conceivable that N-Oct factors serve to downregulate certain genes. This hypothesis gained support by a recent report of Kemp et al. (1990). These authors could show that the octamer motif mediates transcriptional repression of Herpes simplex virus immediate early genes in C 1300 neuroblastoma cells. To investigate the specificity of NOct-3 factor, we have assayed for activation and repression of a reporter gene that contains an octamer-TATA promoter, which is readily activated in HeLa cells by ectopic overexpression of Oct-2A protein but not by the endogenous Oct-I protein (Miller et al. 1988b). This promoter construct was not activated after transfection into Bowes melanoma cells, which express both Oct-I and N-Oct-3 protein. A trivial explanation for this observation could be a low N-Oct-3 factor concentration; ectopic overexpression of the N-Oct-3 cDNA, which is yet to be cloned, may clarify this issue. Co-transfection of the Oct-2A cDNA, however, gave strong activation of the reporter gene in Bowes cells, similar to the earlier findings with HeLa cells (Miller et al., 1988b). At least under our assay conditions, N-Oct-3 protein did not interfere with the activation of the lymphoid specific promoter upon co-expression with Oct-2A protein. We consider it likely that N-Oct-3 is a transcriptional activator for genes specifically expressed in cells of the nervous system and related cell types.

ACKNOWLEDGMENTS We thank Fritz Ochsenbein for his expert graphical work and Dr. Michael M. Miiller-Immergliick for providing us with the plasmids used in this study. We are obliged to Dr. Sandro Rusconi for critical reading of the manuscript and Jacek Blaszczynski for helpful discussions. This work was supported by grants of the Swiss National Science Foundation, by the Kanton of Zurich, and by the Swiss National Multiple Sclerosis Society.

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