The EMBO Journal vol.9 no. pp.
F25-1634, 1990
Transcription factor Oct-2A contains functionally redundant activating domains and works selectively from a promoter but not from a remote enhancer position in non-lymphoid (HeLa) cells Michael M.MUller-ImmerglUck, Walter Schaffner and Patrick Matthias Institut fur Molekularbiologie II der Universitiit Zurich, CH-8093 Zurich, Switzerland
Communicated by W.Schaffner
In non-lymphoid cells such as HeLa cells, ectopic expression of the lymphocyte-specific transcription factor Oct-2A can activate reporter genes whose promoters consist of a single octamer sequence (ATTTGCAT) upstream of a TATA box. While the factor is strongly active in a promoter position, it fails as an enhancer factor: an enhancer consisting of multiple copies of the octamer sequence placed downstream of the reporter gene is not active in HeLa cells, even at high concentration of Oct-2A. In B lymphoid cells, however, the same enhancer is highly active. This could mean that an additional factor is required for enhancer activation in B cells. Furthermore, we have tested the transcriptional activation potential of Oct-2A with a series of N-terminal and C-terminal deletions. We show that a glutamine-rich domain near the N-terminus is required for full activity. Otherwise, large segments of the N-terminal half or the entire C-terminal region are dispensable in our assay, as long as the deletions do not impinge on the conserved POU domain which is sufficient for DNA binding. Vhile N-terminal and C-terminal regions can functionally compensate for each other, a combined deletion that only retains the POU domain is a strong down mutation. We also fmd that activity depends on the promoter structure of the reporter gene: the POU domain by itself shows some activity with a promoter where the octamer sequence is located very close to the TATA box, but no activity with another promoter construction where the octamer sequence is located further upstream. The two promoters also respond differently to the deletion of the glutamine-rich stretch important for transcriptional activation. From these experiments we consider it likely that the natural octamer factor variants can selectively activate the different naturally occurring octamercontaining promoters. Key words: enhancer/Oct-2A(OTF-2)/POU/promoter/tissuespecific transcription/transactivation
Introduction The octamer site ATGCAAAT (or its complement ATTTGCAT) is present in all immunoglobulin (Ig) promoters (Falkner and Zachau, 1984; Parslow et al., 1984) and in the Ig heavy chain (IgH) enhancers (Falkner and Zachau, 1984; Maeda et al., 1987). The same octamer
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sequence is also found in promoters and enhancers of other cellular and viral genes which display no cell type specificity. For example, it is present in the promoters of histone H2B genes, in the promoters of U2 and U6 small nuclear RNA genes and the simian virus SV40 enhancer (Falkner et al., 1986; Harvey et al., 1982; Barberis et al., 1987; LaBella et al., 1988; Mattaj et al., 1985; Zenke et al., 1986; Rosales et al., 1987; for a recent compilation of octamer sequencecontaining genes, see Schreiber et al., 1989a). Three major octamer binding activities have been identified and thoroughly characterized: Oct-I (also called NF-A1, OTFI, NFIII, OBP100, oct-B3) has an apparent relative molecular mass (Mr) of 90 000-100 000 (90-100K) and has been found in all cell types investigated (Pruijn et al., 1986; Singh et al., 1986; Staudt et al., 1986; Fletcher et al., 1987; Rosales et al., 1987; O'Neill and Kelly, 1988; Sturm et al., 1988). Oct-2A (also called NF-A2, OTF2, oct-B2) has an apparent Mr Of 60K and has been found in a few cell types only, notably in B lymphocytes where its presence also correlates with transcription of Ig genes (Staudt et al., 1986; Gerster et al., 1987; Rosales et al., 1987; Scheidereit et al., 1987). Oct-2B is another lymphoid-specific protein of - 75K and is related to Oct-2A (Schreiber et al., 1988). A number of other octamer binding proteins have been identified in various tissues and cell lines: several octamer factors in neural tissues Oct4 -Oct0 (Scholer et al., 1989a,b), an embryonal carcinoma (EC) cell line-specific factor NF-A3 (Lenardo et al., 1989), another protein in malignant melanoma (independently also called NF-A3; Cox et al., 1988), a testis-specific factor Oct-T (Barberis et al., 1987, 1989) and two high mobility group proteins which preferentially bind both AT rich sequences and the octamer sequence (Eckner and Birnstiel, 1989). We and others have isolated cDNAs encoding the lymphocyte-specific transcription factor Oct-2A. Like the protein it codes for, the Oct-2A RNA is expressea almost exclusively in lymphoid cells (Clerc et al., 1988; MullerImmergluck et al., 1988; Scheidereit et al., 1988; Staudt et al., 1988; see also He et al., 1989). Consistent with the finding that the octamer sequence is important for B cellspecific transcription (Mason et al., 1985; Dreyfus et al., 1987; Gerster et al., 1987; Wirth et al., 1987), the presence of Oct-2A in non-lymphoid cells is sufficient to activate, via the octamer sequence motif, a lymphocyte-specific promoter (Muller-Immergluck et al., 1988). Furthermore Oct-2A can also activate promoters containing octamer-related sites derived from homeotic genes (Thali et al., 1988). The deduced protein sequence of Oct-2A (Clerc et al., 1988; Ko et al., 1988; Muller-Immergluck et al., 1988; Scheidereit et al., 1988) shows a region of high homology to Oct-i (Sturm et al., 1988), as well as to two other tissue-specific factors, Pit-l (Bodner et al., 1988; Ingraham et al., 1988) and Unc-86 (Finney et al., 1988). The region of homology -
1625
M.M.Muller-immergluck, W.Schaffner and P.Matthias
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Fig. 1. Activity of the OCTA(l) promoter driven by various enhancers at the same downstream position determined by RNase protection analysis. Lanes 1, no enhancer; lanes 2, SV40 enhancer; lanes 3, IgH enhancer; lanes 4, one octamer sequence; lanes 5, six octamer sequences in tandem. (A) Promoter activity in BJAB cells. (B) in HeLa cells. (C) in HeLa cells expressing cotransfected Oct-2A protein. (D) structure of the reporter gene drawn to scale except for the enlarged promoter region. The location where the various enhancers are inserted is indicated by an arrow. For sequence of the OCTA(l) promoter, see Figure 4D.
among these proteins has been called the POU domain and contains two conserved subregions: a POU-specific box and a POU-homeobox (Herr et al., 1988). The POU-homeobox is 30% homologous to homeoboxes found in many genes regulating development (Gehring, 1987). Mutagenesis experiments have indicated that the entire POU domain is likely to represent the DNA binding region of this class of proteins (Herr et al., 1988; Sturm et al., 1988). The recent detection of other POU domain proteins in mammalian neural tissues (He et al., 1989) and Caenorhabditis elegans (Biirglin et al., 1989) could be indicative of a complex regulatory network with overlapping specificities. In this paper we present evidence that the cDNA encoded Oct-2A acts preferentially as a promoter factor: in a heterologous system, it fails to activate a remote synthetic enhancer consisting of multiple octamer sites even though the same enhancer is highly active in B cells. This suggests that either another Oct-2 related factor is responsible for enhancer activation or that perhaps a second factor is required together with Oct-2A in order to activate the enhancer. By testing deleted versions of Oct-2A for their transactivation potential, we identified a glutamine-rich region in the N terminus important for transactivation of two different test promoters. Two weaker activating domains in the C terminus and within the POU domain can only be monitored with one or the other test promoter. This suggests that a different promoter architecture can influence the response to the same transactivator. -
1626
Results In HeLa cells Oct-2A acts preferentially as a promoter transcription factor Previous experiments have shown that multimerized octamer sites from various origins behave as a lymphoid-specific enhancer, capable of activating the fl-globin promoter from an upstream (Tanaka et al., 1988) or downstream position (Gerster et al., 1987; Tanaka et al., 1988). By cotransfection experiments we have recently demonstrated that cloned Oct-2A can specifically stimulate reporter plasmids containing an octamer site in the promoter (MullerImmergluck et al., 1988). In order to see whether Oct-2A can also account for the enhancer activity observed with multiple octamer sites in a downstream position, we constructed a series of plasmids which all contain the OCTA(1) promoter. This minimal promoter consists of the ,3-globin TATA-box and an octamer binding site (Figure 4D) and responds well to transactivation by Oct-2A (MullerImmergluck et al., 1988 and Figures 4B and 5C). In addition, the plasmids contain either no enhancer or one of the following enhancers inserted downstream of the fl-globin gene (Figure ID): the SV40 enhancer, the IgH enhancer, a single octamer, or six octamer motifs in tandem (1 xOCTA or 6 x OCTA) (Gerster et al., 1987). We first tested the activity of these plasmids in B cells (BJA-B). The plasmids containing the SV40 enhancer, the IgH enhancer, or the 6 x OCTA enhancer were all highly
Oct-2A transcription factor function
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F25-1634, 1990
Transcription factor Oct-2A contains functionally redundant activating domains and works selectively from a promoter but not from a remote enhancer position in non-lymphoid (HeLa) cells Michael M.MUller-ImmerglUck, Walter Schaffner and Patrick Matthias Institut fur Molekularbiologie II der Universitiit Zurich, CH-8093 Zurich, Switzerland
Communicated by W.Schaffner
In non-lymphoid cells such as HeLa cells, ectopic expression of the lymphocyte-specific transcription factor Oct-2A can activate reporter genes whose promoters consist of a single octamer sequence (ATTTGCAT) upstream of a TATA box. While the factor is strongly active in a promoter position, it fails as an enhancer factor: an enhancer consisting of multiple copies of the octamer sequence placed downstream of the reporter gene is not active in HeLa cells, even at high concentration of Oct-2A. In B lymphoid cells, however, the same enhancer is highly active. This could mean that an additional factor is required for enhancer activation in B cells. Furthermore, we have tested the transcriptional activation potential of Oct-2A with a series of N-terminal and C-terminal deletions. We show that a glutamine-rich domain near the N-terminus is required for full activity. Otherwise, large segments of the N-terminal half or the entire C-terminal region are dispensable in our assay, as long as the deletions do not impinge on the conserved POU domain which is sufficient for DNA binding. Vhile N-terminal and C-terminal regions can functionally compensate for each other, a combined deletion that only retains the POU domain is a strong down mutation. We also fmd that activity depends on the promoter structure of the reporter gene: the POU domain by itself shows some activity with a promoter where the octamer sequence is located very close to the TATA box, but no activity with another promoter construction where the octamer sequence is located further upstream. The two promoters also respond differently to the deletion of the glutamine-rich stretch important for transcriptional activation. From these experiments we consider it likely that the natural octamer factor variants can selectively activate the different naturally occurring octamercontaining promoters. Key words: enhancer/Oct-2A(OTF-2)/POU/promoter/tissuespecific transcription/transactivation
Introduction The octamer site ATGCAAAT (or its complement ATTTGCAT) is present in all immunoglobulin (Ig) promoters (Falkner and Zachau, 1984; Parslow et al., 1984) and in the Ig heavy chain (IgH) enhancers (Falkner and Zachau, 1984; Maeda et al., 1987). The same octamer
Oxford University Press
sequence is also found in promoters and enhancers of other cellular and viral genes which display no cell type specificity. For example, it is present in the promoters of histone H2B genes, in the promoters of U2 and U6 small nuclear RNA genes and the simian virus SV40 enhancer (Falkner et al., 1986; Harvey et al., 1982; Barberis et al., 1987; LaBella et al., 1988; Mattaj et al., 1985; Zenke et al., 1986; Rosales et al., 1987; for a recent compilation of octamer sequencecontaining genes, see Schreiber et al., 1989a). Three major octamer binding activities have been identified and thoroughly characterized: Oct-I (also called NF-A1, OTFI, NFIII, OBP100, oct-B3) has an apparent relative molecular mass (Mr) of 90 000-100 000 (90-100K) and has been found in all cell types investigated (Pruijn et al., 1986; Singh et al., 1986; Staudt et al., 1986; Fletcher et al., 1987; Rosales et al., 1987; O'Neill and Kelly, 1988; Sturm et al., 1988). Oct-2A (also called NF-A2, OTF2, oct-B2) has an apparent Mr Of 60K and has been found in a few cell types only, notably in B lymphocytes where its presence also correlates with transcription of Ig genes (Staudt et al., 1986; Gerster et al., 1987; Rosales et al., 1987; Scheidereit et al., 1987). Oct-2B is another lymphoid-specific protein of - 75K and is related to Oct-2A (Schreiber et al., 1988). A number of other octamer binding proteins have been identified in various tissues and cell lines: several octamer factors in neural tissues Oct4 -Oct0 (Scholer et al., 1989a,b), an embryonal carcinoma (EC) cell line-specific factor NF-A3 (Lenardo et al., 1989), another protein in malignant melanoma (independently also called NF-A3; Cox et al., 1988), a testis-specific factor Oct-T (Barberis et al., 1987, 1989) and two high mobility group proteins which preferentially bind both AT rich sequences and the octamer sequence (Eckner and Birnstiel, 1989). We and others have isolated cDNAs encoding the lymphocyte-specific transcription factor Oct-2A. Like the protein it codes for, the Oct-2A RNA is expressea almost exclusively in lymphoid cells (Clerc et al., 1988; MullerImmergluck et al., 1988; Scheidereit et al., 1988; Staudt et al., 1988; see also He et al., 1989). Consistent with the finding that the octamer sequence is important for B cellspecific transcription (Mason et al., 1985; Dreyfus et al., 1987; Gerster et al., 1987; Wirth et al., 1987), the presence of Oct-2A in non-lymphoid cells is sufficient to activate, via the octamer sequence motif, a lymphocyte-specific promoter (Muller-Immergluck et al., 1988). Furthermore Oct-2A can also activate promoters containing octamer-related sites derived from homeotic genes (Thali et al., 1988). The deduced protein sequence of Oct-2A (Clerc et al., 1988; Ko et al., 1988; Muller-Immergluck et al., 1988; Scheidereit et al., 1988) shows a region of high homology to Oct-i (Sturm et al., 1988), as well as to two other tissue-specific factors, Pit-l (Bodner et al., 1988; Ingraham et al., 1988) and Unc-86 (Finney et al., 1988). The region of homology -
1625
M.M.Muller-immergluck, W.Schaffner and P.Matthias
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Fig. 1. Activity of the OCTA(l) promoter driven by various enhancers at the same downstream position determined by RNase protection analysis. Lanes 1, no enhancer; lanes 2, SV40 enhancer; lanes 3, IgH enhancer; lanes 4, one octamer sequence; lanes 5, six octamer sequences in tandem. (A) Promoter activity in BJAB cells. (B) in HeLa cells. (C) in HeLa cells expressing cotransfected Oct-2A protein. (D) structure of the reporter gene drawn to scale except for the enlarged promoter region. The location where the various enhancers are inserted is indicated by an arrow. For sequence of the OCTA(l) promoter, see Figure 4D.
among these proteins has been called the POU domain and contains two conserved subregions: a POU-specific box and a POU-homeobox (Herr et al., 1988). The POU-homeobox is 30% homologous to homeoboxes found in many genes regulating development (Gehring, 1987). Mutagenesis experiments have indicated that the entire POU domain is likely to represent the DNA binding region of this class of proteins (Herr et al., 1988; Sturm et al., 1988). The recent detection of other POU domain proteins in mammalian neural tissues (He et al., 1989) and Caenorhabditis elegans (Biirglin et al., 1989) could be indicative of a complex regulatory network with overlapping specificities. In this paper we present evidence that the cDNA encoded Oct-2A acts preferentially as a promoter factor: in a heterologous system, it fails to activate a remote synthetic enhancer consisting of multiple octamer sites even though the same enhancer is highly active in B cells. This suggests that either another Oct-2 related factor is responsible for enhancer activation or that perhaps a second factor is required together with Oct-2A in order to activate the enhancer. By testing deleted versions of Oct-2A for their transactivation potential, we identified a glutamine-rich region in the N terminus important for transactivation of two different test promoters. Two weaker activating domains in the C terminus and within the POU domain can only be monitored with one or the other test promoter. This suggests that a different promoter architecture can influence the response to the same transactivator. -
1626
Results In HeLa cells Oct-2A acts preferentially as a promoter transcription factor Previous experiments have shown that multimerized octamer sites from various origins behave as a lymphoid-specific enhancer, capable of activating the fl-globin promoter from an upstream (Tanaka et al., 1988) or downstream position (Gerster et al., 1987; Tanaka et al., 1988). By cotransfection experiments we have recently demonstrated that cloned Oct-2A can specifically stimulate reporter plasmids containing an octamer site in the promoter (MullerImmergluck et al., 1988). In order to see whether Oct-2A can also account for the enhancer activity observed with multiple octamer sites in a downstream position, we constructed a series of plasmids which all contain the OCTA(1) promoter. This minimal promoter consists of the ,3-globin TATA-box and an octamer binding site (Figure 4D) and responds well to transactivation by Oct-2A (MullerImmergluck et al., 1988 and Figures 4B and 5C). In addition, the plasmids contain either no enhancer or one of the following enhancers inserted downstream of the fl-globin gene (Figure ID): the SV40 enhancer, the IgH enhancer, a single octamer, or six octamer motifs in tandem (1 xOCTA or 6 x OCTA) (Gerster et al., 1987). We first tested the activity of these plasmids in B cells (BJA-B). The plasmids containing the SV40 enhancer, the IgH enhancer, or the 6 x OCTA enhancer were all highly
Oct-2A transcription factor function
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