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Lymphoid-specific transcription mediated by the conserved octamer site: Who is doing what?
seminars in I M M U N OL OG Y, Vol 10, 1998: pp. 155]163
Lymphoid-specific transcription mediated by the conserved octamer site: Who is doing what? Patrick MatthiasU m constant ŽC. region.5,6 This motif is also present in several of the enhancers found at the 39 end of the IgH locus,7 ] 11 as well in the promoters of various B cell-specific genes, for example B29 12 or CD20.13 Interestingly, the octamer motif is found in different relative orientations in the Ig heavy and light chain gene promoters, indicating that the proteins recognizing this element have a large degree of functional flexibility. Transfection experiments using variously truncated Ig VH or VL promoter constructs first identified the functional importance of the octamer site for Ig promoter function and B cell-specific activity.4,14,15 These data were corroborated by in vitro transcription experiments testing the activity of Ig promoter constructs in transcriptional extracts derived from B or non-B Že.g. HeLa. cells.16 In addition, it was shown that insertion of an octamer site into an unrelated core promoter is sufficient to render the resulting chimeric promoter B cell-specific in transfection or in vitro transcription assays.17 ] 19 Likewise, multimerisation of a small fragment derived from the IgH J-C intron enhancer and containing the octamer site is sufficient to create a potent B cell-specific enhancer.20 Most importantly, transgenic mice were generated containing a rearranged IgH gene or mutant versions of it in which different regulatory sites had been eliminated.21 These studies showed that mutation of the IgH promoter octamer site reduced activity over twentyfold in lymphoid cells and identified this element as the most important one for VH promoter function. By contrast, the octamer site of the IgH J-C intron enhancer did not appear to play a major role in these assays.21 Additional support for a crucial role of the octamer site for Ig gene transcription came from somatic cell fusion experiments in which reporter constructs under the control of Ig gene regulatory elements Že.g. a kappa light chain gene promoter. were first stably integrated into B cell lines. The resulting reporter cell lines were then fused either to fibroblasts or to a T cell line ŽBW5147. and expression of
The conserved octamer site found in the promoters of Immunoglobulin (Ig) genes has been identified early on as a key element for B cell-specific Ig promoter activity and has also been associated with expression of other B cell-specific genes. Several transcription factors } ubiquitous or cellrestricted } interact specifically with the octamer motif in B cells: Oct-1 and Oct-2 are POU domain transcription factors that bind directly to the octamer site and thereby can recruit the coactivator OBF-1 to the promoters of various genes. Yet, in spite of intense work, the precise role played by each of these factors for transcription activation through the octamer motif is unclear. In particular, genetic inactivation in mice of the lymphoid cell-specific transcription factor Oct-2 or of the B cell-specific coactivator OBF-1 led to surprising findings that are being discussed. Key words: octamer site r Oct-1 r Oct-2 r OBF-1 r immunoglobulin transcription Q1998 Academic Press Ltd
The octamer site and lymphoid-specific transcription FOR MORE THAN 10 years the genes coding for Immunoglobulins ŽIg. have served as a model system for the study of B cell-specific transcription. Numerous regulatory elements and cognate trans-acting factors that are important for the transcription of Ig genes have been identified Žreviewed in Refs 1,2.. In particular, the so-called octamer site -ATGCAAAT- has drawn a lot of attention. This site was first identified as a motif conserved in virtually all of the Ig heavy ŽH. and light ŽL. chain variable ŽV. region promoters,3,4 as well as in the enhancer that is present in the intron found between the joining ŽJ. and U
From the Friedrich Miescher Institute, Maulbeerstr. 66, PO Box 2543, 4058 Basel, Switzerland Q1998 Academic Press Ltd 1044-5323r 98r 020155q 09 $25.00r 0r si980117
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the reporter gene was subsequently measured in the hybrids.22 ] 24 In each case, expression of the reporter gene was rapidly extinguished, paralleling the downregulation of the endogenous Ig gene. For this phenomenon, the Ig octamer site was found to be essential;23,24 replacement of the kappa promoter octamer site by an Sp1 or NF1 binding site rendered the promoter resistant to extinction.23 In addition, it was shown that the expression of several of the transcription factors binding to the octamer site Žsee below. was also downregulated in the fusion process.23,25,26 Thus, it appeared that the octamer site, either as a natural element of Ig promoters, or as an artificial component of chimeric promoters or synthetic enhancers, is able to mediate B cell-specific transcription activation. Furthermore, the octamer site is also a conserved and functionally important element of the promoters of ubiquitous genes, such as the histone H2B 27 or the small nuclear RNA U2 or U6 genes.28,29 The dual role played by the octamer motif for lymphoidspecific or ubiquitous transcription regulation must therefore be explained by the existence of cellspecific and ubiquitous transcription factors and also by promoter-specific features such as, for example, distance between the octamer motif and the core promoter or presence of nearby binding sites for additional transcription factors.
monstrated that the B cell-specific Oct-2 protein had the capacity to trans-activate cotransfected reporter p lasm id s in an o ctam e r site - d e p e n d e n t manner.32,39 ] 41 These results led to the early notion that the B cell-specific activity associated with the octamer site was primarily mediated by the Oct-2 protein. RNA analysis studies showed that Oct-1 expression is virtually ubiquitous, as predicted from the protein]DNA assays. In contrast Oct-2, which comprises several isoforms generated by alternative splicing,42 was found to be expressed at low levels in B cells of early differentiation stages, such as pro- or pre-B cells and at higher levels in more mature B cells.43,44 In pre-B cells Oct-2 expression can be further enhanced by treatment of the cells with, e.g. bacterial lipopolysaccharide ŽLPS., a polyclonal B cell mitogen. Furthermore, Oct-2 expression can also be induced in T cell lines or primary T cells by the phorbol ester 12-myristate 13-acetate ŽPMA. and ionomycin, or by specific antigen.45 Finally, Oct-2 expression has also been detected in the nervous system, in the kidney and in testis.
OBF-1 (aka OCA-B, Bob1), a lymphoid-specific transcriptional coactivator Recently, several groups cloned the gene coding for a novel protein that specifically interacts with Oct-1 and Oct-2. This protein has been variously dubbed OBF-1 ŽOct Binding Factor 146 ., OCA-B 47 or Bob-1 48 and its existence had been postulated on the basis of either biochemical experiments suggesting the presence in B cells of coactivatorŽs. for Oct factors,49,50 or as a means of explaining the phenotype of Oct-2-de ficient mice Žsee below.. OBF-1 is a novel 256-amino acid proline-rich protein that has very little homology to other proteins in the databases. Expression of OBF-1, at the protein or RNA level, is highly B cell-specific and is not observed in most other cell types examined.46 ] 48 Within the B cell lineage, OBF-1 expression is found in cells of all stages of differentiation Žpro-, pre- and mature B cells. at constant levels.51 Through its interaction with either Oct-1 or Oct-2, OBF-1 can be recruited to a subset of octamer sites and thereby co-activate gene transcription, as was demonstrated with octamer sitecontaining reporter plasmids either in transient transfections or in vitro transcription assays.46 ] 48 The mechanism of transcription coactivation has yet to be examined in greater detail, but can be at least partly explained by the presence of a strong transcription
Oct factors: Oct-1 and Oct-2 In parallel to the above-mentioned studies defining the functional role of the octamer site, protein]DNA interaction assays identified nuclear factors binding specifically to this motif. Oct-1 was identified as a ubiquitous protein and Oct-2 as a mostly lymphoid cell-restricted protein. The simultaneous cloning of these factors by several laboratories30 ] 33 led to the identification of a novel family of transcription factors, the POU proteins.34 These factors are characterized by the presence of an approximately 150 amino acid long bipartite DNA binding domain, the POU domain, which comprises a novel ‘POU-specific’ domain linked to a variant homeodomain.34 Both of these regions contain helix-turn-helix motifs required for high affinity binding to DNA. By now a large number of POU proteins have been identified in many different organisms and these proteins have been implicated in developmental regulation in several systems Žfor reviews, see Refs 35]38.. Transfection studies performed in non-B cells de156
Octamer site and b cell-specific transcription
by gene targeting either in a B cell line ŽWEHI 231. 58 or in embryonic stem cells, from which mice were subsequently generated.59 The data obtained from these experiments can be summarized as follows. In the oct-2yry WEHI cells transcription of endogenous Ig genes ŽIgL or IgH. was surprisingly not affected. Octamer site-containing reporter plasmids introduced transiently into these Oct-2-deficient B cells could be put in two categories. Ž1. Reporter plasmids containing an octamer site in their promoter were unaffected by the lack of Oct-2 and were still highly expressed in the mutant B cells. Such reporters, however, were only weakly active in non-B cells Žcontaining only Oct-1 ., suggesting the presence in B cells of at least one additional factor that would be important for full activity. This factor is thought to be OBF-1. Ž2. By contrast, reporters containing a synthetic octamer site enhancer inserted at the 39 of the gene were only weakly active in the Oct-2-deficient B cells, indicating that Oct-2 is required for this ‘activation at a distance’ through octamer sites.58 However, because such reporter plasmids could not be activated by Oct-2 alone in non-B cells, the existence of another B cell-specific cofactor has also been postulated here.40,60 To organize these results in a framework, a model has been put forward suggesting that in B cells there are two distinct pathways of gene activation through the octamer motif ŽFigure 1.. The first pathway, promoter]proximal, does not require Oct-2 and can be mediated by Oct-1 in association with a Bcell-specific component, most likely OBF-1 ŽFigure 1A.. The second pathway, reflecting activation from a distance, can not operate via Oct-1 and requires Oct-2, also in conjunction with a B-cell-specific activity ŽFigure 1B.. Additional genetic experiments with Gal4-Oct-2 fusion proteins identified the C terminus of Oct-2 as being necessary and sufficient to mediate this effect in B cells.61,62 Furthermore, expression of both OBF-1 and Oct-2 together in non-B cells was not sufficient to mediate activation at a distance.51 Thus, an additional activity, as yet undefined but specific for Oct-2, has been postulated to be present in B cells. This activity could be another transcriptional coactivator like OBF-1, but could also be an enzyme such as, e.g. a kinase that would modify Oct-2 specifically in B cells and thus endow it with a novel functional property. The Oct-2-deficient mice also showed a surprising phenotype. These mutant mice develop normally but always die within hours of birth for unknown reasons, perhaps related to the neurogenic expression of Oct-
activation domain in the C-terminal moiety of OBF1.52 The N-terminal portion of OBF-1 contains the domain that mediates interaction with Oct factors.46,53 Recruitment of OBF-1 in a ternary complex is influenced both by the POU domain and by the DNA site. While OBF-1 interacts efficiently in vitro with the POU domains of Oct-1 and Oct-2 Žboth of which are present in B lymphocytes., it fails to interact with other POU proteins, such as Oct-4, Oct-6 or Pit-1. 46 This selectivity of interaction has been recently mapped to a few key residues located in the POU domain at the beginning of the POU-specific first helix and at the end of the POU-homeodomain third helix Žsee Ref 54; Sauter and Matthias, submitted.. At the DNA level, it has been shown that positionq 5 of the octamer site is critical and has to be an A as in the consensus octamer site Ž-ATGCAAAT- . in order to allow efficient ternary complex formation.53,55 This observation was further extended by the finding that, at high concentration, the N-terminal portion of OBF-1 Žamino acids 1]118. can bind to octamer site DNA in the absence of POU domain55 and also by our recent studies with phosphothioate-modified oligonucleotides showing that several positions on the DNA backbone influence the efficiency of ternary complex formation ŽSauter and Matthias, submitted.. Thus, the picture that currently emerges is that OBF-1 interacts with multiple residues in the POU domain and also with several positions on the DNA and thereby forms an intimate molecular ‘clamp’ between the two halves of the POU domain and the cognate DNA site. Recently it was found that OBF-1 is also transiently expressed in various T cell lines and in primary T cells following activation in vitro by PMA and ionomycin or by anti-CD3 antibody treatment.56,57 The kinetics of induction is rapid, suggesting that OBF-1 may participate in regulating the program of gene activation that is characteristic of T cell activation. The induction of OBF-1 in T cells was dissected into two discrete steps: Ž1. OBF-1 gene induction and concomitant protein production; and Ž2. phosphorylation of OBF-1 on Ser 184 leading to formation of a transcriptionally active form of the protein.57 Because OBF-1 appears to be phosphorylated on the same residue in B cells, it has been postulated that in these cells the OBF-1 kinase is constitutively active.57
Genetic inactivation of Oct-2: unexpected results The B cell-specific factor Oct-2 has been inactivated 157
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Figure 1. Model describing the different modes of transcription activation through octamer sites in lymphoid cells. Oct factors ŽOct-1 or Oct-2 . are schematically depicted bound to the DNA. The two ovals represent the POU specific and the POU homeodomain and the N-terminal and C-terminal activation domains are indicated ŽN-ter, C-ter .. ŽA. Promoter pathway of gene activation. Octamer sites in promoters can be bound by Oct-1 or Oct-2 together with the coactivator OBF-1 Žif the site is permissive., leading to transcription activation. ŽB. Enhancer pathway of gene activation. Multimerized enhancer octamer sites Žbut also octamer sites found in natural enhancers?. are bound by Oct-2. In B cells an activity distinct form OBF-1 and requiring the C terminal domain of Oct-2 mediates transcription activation from such multimerized octamer sites.
2. B cell precursors are found in normal numbers in these mice and IgM positive cells are found, but in somewhat reduced numbers.59 Upon in vitro mitogenic stimulation with bacterial LPS, oct-2yry B cells failed to proliferate and secrete antibody normally. In Abelson virus-transformed B cells derived from these mice transcription of several putative target genes, such as IgH, IgL, or B29 ŽIg b ., was found to be unaffected.59 Because oct-2yry mice die at birth, in vivo analysis
of B cell development or Ig levels had to rely on reconstitution of SCID or RAG1yry mice with oct2yry fetal liver cells.63 In such chimeras the B and T cell compartments developed essentially normally, indicating that B cell development and Ig transcription could proceed in the absence of Oct-2. In these mice serum Ig levels were reduced, in particular IgM, IgG1, IgG3 and IgG2b; surprisingly, IgA levels were normal. In a proliferation assay, splenic B cells from such chimeric mice failed to respond to stimulation 158
Octamer site and b cell-specific transcription
by LPS or anti-m , two polyclonal T cell-independent ŽTI. B cell activators. This defect is manifested by an arrest in the G1 phase of the cell cycle.63 Thus, the lack of Oct-2 appears to affect B cells at a late stage of their development. Recently, it was shown that RAG1yry mice reconstituted with oct-2yry B cells fail to develop a robust immune response to either TI and also, surprisingly, to T cell-dependent ŽTD. antigens.64 Thus, although initial Ig gene transcription as well as B cell development proceed essentially normally in the absence of Oct-2, late steps of the immune response are dependent on this protein. The seeming lack of deleterious effect of the Oct-2 ablation on Ig gene transcription was originally explained by invoking a functional redundancy between Oct-2 and the coactivator OBF-1. According to that model, in Oct-2-deficient mice OBF-1 } in concert with Oct-1 } ensures that Ig genes are efficiently transcribed and thus permits a normal B cell development. Several experiments had suggested that OBF-1 has a unique function for Ig gene transcription. In particular, it was shown that Oct-2, when expressed at physiological Žlow. levels in fibroblasts by stable transfection, was unable to activate octamer-dependent reporter plasmids.65 Likewise, in in vitro transcription assays with purified proteins, neither Oct-1 nor Oct-2 activated Ig promoter reporter plasmids on their own,66,67 but when partially purified OBF-1 protein was added to the reaction efficient transcription was obtained.50 These experiments therefore suggested that the coactivator OBF-1 is the obligatory factor for Ig promoter activation, in conjunction with Oct-1 or Oct-2.
indirectly. When stimulated in vitro with, e.g. LPS or LPS and IL-4, splenocytes from OBF-1yry mice respond fairly normally, as measured either at the level of Ig production Žby Northern blotting or ELISA., or at the level of cellular proliferation.68 In addition, Ig class switching also takes place essentially normally in vitro and relatively normal numbers of B cells are found with switched IgH isotypes on their surface.69 Surprisingly, in splenocytes from these mice transcription of Ig m or Ig k was found to be unaffected, but this was in good agreement with the normal IgM serum levels observed.68,70 In one case it was shown that transcription of isotype-switched Ig genes ŽIgG. was somewhat reduced in the absence of OBF-1, but this effect appeared to be highly dependent on the stimulus used to induce switching.69 Thus, evaluation of the potential role of OBF-1 for transcription of already switched Ig genes will require further study. One could imagine that OBF-1 is indeed required for transcription from V region promoters, but only for a subset of them. In that scenario, OBF-1-defi cient mice would express only a limited number of V genes Žthose that are not dependent on OBF-1 for their activation., but this would go unrecognized in an analysis done by Northern blot. To examine this, hybridomas were generated with LPS-stimulated splenic cells obtained from normal or OBF-1-defi cient mice; subsequently, IgM producing clones were identified, their V genes cloned and their corresponding DNA sequence determined. However, this analysis did not reveal a significant difference in V gene usage between wild-type and OBF-1yry mice, which show a normal Ig repertoire ŽSchubart et al, submitted.. Thus, in spite of the in vitro evidence to the contrary, the link between OBF-1 and Ig gene transcription has not yet been clearly made in vivo. The most striking feature of the lack of OBF-1 is that deficient mice have a dramatically impaired capacity to mount an immune response following immunization with TI and TD antigens. This impairment correlates with a complete lack of germinal center formation in the spleen of immunized mice.68 In addition, the phenotype can be completely transferred by injection of OBF-1yry bone marrow into irradiated wt recipient mice, both at the level of B cell development as measured by FACS, as well as at the level of the defective immune response. In addition, through in vivo and in vitro assays, we showed that in OBF-1yry mice T cell function appears to be normal.68 Furthermore, we also found that induction of the IL-2 and IL-4 genes in OBF-1yry T cells is
Genetic inactivation of OBF-1: more unexpected results Recently several groups including ours generated OBF-1-deficient mice by gene targeting.68 ] 70 These mice are born normally and appear healthy. Early B cell development in the bone marrow, identified by the expression of markers such as c-kit or IL-2 receptor a chain in conjunction with the pan B cell marker B220, appeared normal. However, mature recirculating B cells ŽB220 highrIgM low and coexpressing IgD. were present at significantly reduced levels. In addition, the spleen of these animals contained approximately two to fourfold less B cells. While IgM serum levels were normal, the levels of all IgGs as well as IgA were strongly reduced, indicating that OBF-1 has an impact on Ig production, directly or 159
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normal.56 Therefore the primary defect in OBF-1-deficient mice appears to lie within the B cells.
the double mutant mice obtained showed that B cell development in the bone marrow was indeed essentially normal and that peripheral lymphoid organs such as spleen or lymph nodes were, remarkably, empty of B cells and filled up mostly with T cells ŽSchubart et al, submitted.. These data thus indicate that OBF-1, in addition to its paramount role for in vivo immune response, is also necessary for the development of B cells at the time when they exit the bone marrow and enter the periphery ŽFigure 2..
OBF-1 has an important role for the bone marrow-derived B cell developmental pathway In the course of characterizing B cell development in OBF-1yry mice by FACS, we identified an antibody showing a dramatically different labeling pattern between wt and knockout mice. This antibody, called mAb493, labels an as yet unidentified protein of approximately 135 kDa found on B cells representing early stages of B cell development. It distinguishes pro-preBI, preBII and immature B cells, which are all mAb493q, from mature B cells which are mAb493y ŽRolink et al, submitted.. Therefore, practically all B cells in the bone marrow of a normal mouse are mAb493q and only the long-lived recirculating IgM low IgD high CD23q mature B cells are mAb493y. By contrast, in the spleen only approximately 20% of all B cells are mAb493q and these represent immature B cells that have just entered the peripheral B cell pool ŽRolink et al, submitted.. Precisely these mAb493q splenic B cells were found to be lacking in OBF-1yry mice, while mAb493q cells were found in OBF-1yry bone marrow at normal levels ŽSchubart et al, submitted.. These and other data indicated that OBF-1yry B cells are impaired at the transition from immature to mature B cells, which corresponds to the exit from the bone marrow to the periphery. In that context, the finding that OBF-1 deficient mice have B cells in their peripheral organs, albeit less than normal mice, represented a seeming paradox. If OBF-1yry B cells are impaired at the bone marrowrperiphery transition, then where are the splenic B cells coming from? A possibility is that splenic B cells in OBF-1yry mice might be derived from the early wave of B cell generation Žreviewed in Ref 71., that takes place primarily in the fetal liver and is dependent on the cytoplasmic tyrosine kinase Btk 72 and on the paired domain transcription factor Pax-5.73 To test this hypothesis at the genetic level Schubart et al crossed OBF-1-deficient mice with mice of the CBArN strain, that are deficient in Btk, owing to a natural null mutation in the gene ŽXid.. Based on the model described above, the prediction was that in such double mutant mice early B cell development in the bone marrow should be largely normal and that in contrast development of B cells in the periphery should be strongly affected. Analysis of
Redundancy versus specificity: who is doing what? From the results mentioned above it is clear that the study of Oct factors and of the coactivator OBF-1 has revealed many very interesting and unexpected aspects of the transcriptional control in lymphocytes. However, the critical target genes whose expression is altered in the absence of Oct-2 or OBF-1 and which might explain the phenotype observed in the corresponding knockout mice, remain to be identified. In the case of Oct-2, a few target genes have been described that are not expressed anymore in cells derived from oct-2yry mice, such as CD36, which encodes a membrane glycoprotein implicated in signal transduction74 and CRISP-3, a cysteine-rich secreted protein whose function is unknown.75 Yet, although these genes clearly require Oct-2 to be expressed in mouse B cells, lack of their expression is unlikely to explain the phenotype of the oct-2yry mice and therefore other target genes still have to be found. In the case of OBF-1, no specific target gene has been identified yet and this is one of the pressing issues for the future. These different transcription factors have been first identified and were subsequently cloned through the study of Ig gene regulation; yet their precise role in the regulation of Ig gene transcription remains to be elucidated. In spite of a number of experiments suggesting the contrary, it is still possible that in vivo Oct-2 and OBF-1 are to a certain extent redundant, at least with respect to Ig promoter activation. To elucidate this, mice lacking both Oct-2 and OBF-1 will certainly be instrumental and they are currently being generated in our laboratory. Also the role of the ubiquitous factor Oct-1 will have to be evaluated in vivo. And what is the function, if any, of Oct-2 and OBF-1 in T cells? No doubt, the study of Oct factors and of their coactivators will provide more surprises in the future. 160
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Figure 2. Schematic model of the antigen independent phase of B lymphoid development and its requirement for Btk, Pax-5 and OBF-1 Žafter Schubart et al, submitted.. ŽA. The early developmental wave of B cells that initiates in various organs of the fetusrneonate is largely dependent on Pax-5 w73x and Btk w72x. The point at which Btk blocks this developmental pathway has not been defined precisely yet. In adult mice these B cells ŽB1. are found predominantly in the peritoneum but also to some extent in other peripheral organs. ŽB. In the bone marrow, progression through the pre-BII stage fails in the absence of Pax-5 and a mutated Btk gene causes selective disadvantages at the transition from pre-BII to immature B cells w76x; however, neither here nor in the spleen Žsee below. is a developmental block observed in the Btk mut background. ŽC. In a normal mouse immature B cells migrate from the bone marrow to the spleen where they develop into more mature B cells and loose expression of the antigen recognized by mAb493. The transition of immature B cells from the bone marrow to the periphery is greatly impaired in OBF-1-deficient mice and peripheral lymphoid organs that are usually populated with bone marrow derived B cells, are filled with B cells that are Btk dependent and therefore most likely originated from the fetalrneonatal phase of B lymphopoeisis. In Btk mut mice the maturation to mature B cells is affected moderately. For simplicity the scheme does not cover the developmental aspects taking place after B cells have entered the spleen or the peritoneum and does not indicate the impaired immune response in the absence of OBF-1. Abbreviations: GL, Ig genes in germline configuration; DJ-H, D-J rearrangement in the Ig heavy chain gene; VDJ-H, V-DJ heavy chain Ig gene rearrangement; VJ-L, V-J Ig light chain gene rearrangement.
Acknowledgements I wish to thank all the members of my laboratory for discussions and in particular Patrick Sauter, Steffen Massa, Daniel Schubart and Steffen Junker ŽAarhus University. for help with the figures and critical reading of the manuscript.
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defines a new class of RNA polymerase II enhancer. Genes Dev 2:1764]1778 Sturm RA, Das G, Herr W Ž1988. The ubiquitous octamerbinding protein Oct-1 contains a POU domain with a homeo box subdomain. Genes Dev 2:1582]1599 Scheidereit C, Cromlish JA, Gerster T, Kawakami K, Balmaceda CG, Currie RA, Roeder RG Ž1988. A human lymphoid-specific transcription factor that activates immunoglobulin genes is a homeobox protein. Nature 336:551]557 Muller MM, Ruppert S, Schaffner W, Matthias P Ž1988. A ¨ cloned octamer transcription factor stimulates transcription from lymphoid-specific promoters in non-B cells. Nature 336:544]551 Clerc RG, Corcoran LM, LeBowitz JH, Baltimore D, Sharp PA Ž1988. The B-cell-specific Oct-2 protein contains POU boxand homeo box- type domains. Genes Dev 2:1570]1581 Herr W, Sturm RA, Clerc RG, Corcoran LM, Baltimore D, Sharp PA, Ingraham HA, Rosenfeld MG, Finney M, Ruvkun G, Horvitz HR Ž1988. The POU domain: a large conserved region in the mammalian pit- 1, oct- 1, oct- 2 and Caenorhabditis elegans unc-86 gene products. Genes Dev 2:1513]1516 Rosenfeld MG Ž1991. POU-domain transcription factors: pou-er-ful developmental regulators. Genes Dev 5:897]907 Scholer ¨ HR Ž1991. Octamania: the POU factors in murine development. Trends Genet 7:323]329 Wegner M, Drolet DW, Rosenfeld MG Ž1993. POU-domain proteins: structure and function of developmental regulators. Curr Opin Cell Biol 5:488]498 Ryan AK, Rosenfeld MG Ž1997. POU domain family values: flexibility, partnerships, and developmental codes. Genes Dev 11:1207]1225 Tanaka M, Herr W Ž1990. Differential transcriptional activation by Oct-1 and Oct-2: interdependent activation domains induce Oct-2 phosphorylation. Cell 60:375]386 Muller-Immergluck MM, Schaffner W, Matthias P Ž1990. ¨ 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 ŽHeLa. cells. Embo J 9:1625]1634 Gerster T, Balmaceda CG, Roeder RG Ž1990. The cell typespecific octamer transcription factor OTF-2 has two domains required for the activation of transcription. Embo J 9:1635]1643 Wirth T, Priess A, Annweiler A, Zwilling S, Oeler B Ž1991. Multiple Oct2 isoforms are generated by alternative splicing. Nucleic Acids Res 19:43]51 Staudt LM, Clerc RG, Singh H, LeBowitz JH, Sharp PA, Baltimore D Ž1988. Cloning of a lymphoid-specific cDNA encoding a protein binding the regulatory octamer DNA motif. Science 241:577]580 Miller CL, Feldhaus AL, Rooney JW, Rhodes LD, Sibley CH, Singh H Ž1991. Regulation and a possible stage-specific function of Oct-2 during pre-B-cell differentiation. Mol Cell Biol 11:4885]4894 Kang SM, Tsang W, Doll S, Scherle P, Ko HS, Tran AC, Lenardo MJ, Staudt LM Ž1992. Induction of the POU domain transcription factor Oct-2 during T-cell activation by cognate antigen. Mol Cell Biol 12:3149]3154 Strubin M, Newell JW, Matthias P Ž1995. OBF-1, a novel B cell-specific coactivator that stimulates immunoglobulin promoter activity through association with octamer-binding proteins. Cell 80:497]506 Luo Y, Roeder RG Ž1995. Cloning, functional characterization, and mechanism of action of the B-cell-specific transcriptional coactivator OCA-B. Mol Cell Biol 15:4115]4124 Gstaiger M, Knoepfel L, Georgiev O, Schaffner W, Hovens CM Ž1995. A B-cell coactivator of octamer-binding transcription factors. Nature 373:360]362
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49. Pierani A, Heguy A, Fujii H, Roeder RG Ž1990. Activation of octamer-containing promoters by either octamer-binding transcription factor 1 ŽOTF-1 . or OTF-2 and requirement of an additional B-cell-specific component for optimal transcription of immunoglobulin promoters. Mol Cell Biol 10:6204]6215 50. Luo Y, Fujii H, Gerster T, Roeder RG Ž1992. A novel B cell-derived coactivator potentiates the activation of immunoglobulin promoters by octamer-binding transcription factors. Cell 71:231]241 51. Schubart DB, Sauter P, Massa S, Friedl EM, Schwarzenbach H, Matthias P Ž1996. Gene structure and characterization of the murine homologue of the B cell-specific transcriptional coactivator OBF-1. Nucleic Acids Res 24:1913]1920 52. Pfisterer P, Zwilling S, Hess J, Wirth T Ž1995. Functional characterization of the murine homolog of the B cell- specific coactivator BOB.1rOBF.1. J Biol Chem 270:29870]29880 53. Gstaiger M, Georgiev O, van Leeuwen H, van der Vliet P, Schaffner W Ž1996. The B cell coactivator Bob1 shows DNA sequence-dependent complex formation with Oct-1rOct-2 factors, leading to differential promoter activation. Embo J 15:2781]2790 54. Babb R, Cleary MA, Herr W Ž1997. OCA-B is a functional analog of VP16 but targets a separate surface of the Oct-1 POU domain. Mol Cell Biol 17:7295]7305 55. Cepek KL, Chasman DI, Sharp PA Ž1996. Sequence-specific DNA binding of the B-cell-specific coactivator OCA-B. Genes Dev 10:2079]2088 56. Sauter P, Matthias P Ž1997. The B cell-specific coactivator OBF-1 ŽOCA-B, Bob-1 . is inducible in T cells and its expression is dispensable for IL-2 gene induction. Immunobiology 197:293]302 57. Zwilling S, Dieckmann A, Pfisterer P, Angel P, Wirth T Ž1997. Inducible expression and phosphorylation of coactivator BOB.1rOBF.1 in T cells wsee commentsx. Science 277:221]225 58. Feldhaus AL, Klug CA, Arvin KL, Singh H Ž1993. Targeted disruption of the Oct-2 locus in a B cell provides genetic evidence for two distinct cell type-specific pathways of octamer element- mediated gene activation. Embo J 12:2763]2772 59. Corcoran L, Karvelas M, Nossal GJ, Ye ZS, Jacks T, Baltimore D Ž1993. Oct-2, although not required for early B-cell development, is critical for later B-cell maturation and for postnatal survival. Genes Dev 7:570]582 60. Annweiler A, Muller-Immergluck M, Wirth T Ž1992. Oct2 transactivation from a remote enhancer position requires a B- cell-restricted activity. Mol Cell Biol 12:3107]3116 61. Annweiler A, Zwilling S, Wirth T Ž1994. Functional differences between the Oct2 transactivation domains determine the transactivation potential of individual Oct2 isoforms. Nucleic Acids Res 22:4250]4258 62. Friedl EM, Matthias P Ž1995. Transcriptional activation and repression, two properties of the lymphoid-specific transcription factor Oct-2a. Eur J Biochem 234:308]316
63. Corcoran LM, Karvelas M Ž1994. Oct-2 is required early in T cell-independent B cell activation for G1 progression and for proliferation. Immunity 1:635]645 64. Humbert PO, Corcoran LM Ž1997. oct-2 gene disruption eliminates the peritoneal B-1 lymphocyte lineage and attenuates B 2 cell maturation. J Immunol 159:5273]5284 65. Pfisterer P, Annweiler A, Ullmer C, Corcoran LM, Wirth T Ž1994. Differential transactivation potential of Oct1 and Oct2 is determined by additional B cell-specific activities. Embo J 13:1655]1663 66. LeBowitz JH, Kobayashi T, Staudt LM, Baltimore D, Sharp PA Ž1988. Octamer-binding proteins from B or heLa cells stimulate transcription of the immunoglobulin eavy chain promoter in vitro. Genes Dev 2:1227]1237 67. Johnson DG, Carayannopoulos L, Capra JD, Tucker PW, Hanke JH Ž1990. The ubiquitous octamer-binding proteinŽs. is sufficient for transcription of immunoglobulin genes. Mol Cell Biol 10:982]990 68. Schubart DB, Rolink A, Kosco-Vilbois MH, Botteri F, Matthias P Ž1996. B-cell-specific coactivator OBF-1rOCA-BrBob1 required for immune response and germinal centre formation. Nature 383:538]542 69. Kim U, Qin XF, Gong S, Stevens S, Luo Y, Nussenzweig M, Roeder RG Ž1996. The B-cell-specific transcription coactivator OCA-BrOBF-1rBob-1 is essential for normal production of immunoglobulin isotypes. Nature 383:542]547 70. Nielsen PJ, Georgiev O, Lorenz B, Schaffner W Ž1996. B lymphocytes are impaired in mice lacking the transcriptional co- activator Bob1rOCA- BrOBF1. Eur J Immunol 26:3214]3218 71. Hardy RR, Carmack CE, Li YS, Hayakawa K Ž1994. Distinctive developmental origins and specificities of murine CD5 q B cells. Immunol Rev 137:91]118 72. Hayakawa K, Hardy RR, Herzenberg LA Ž1986. Peritoneal Ly-1 B cells: genetic control, autoantibody production, increased lambda light chain expression. Eur J Immunol 16:450]456 73. Nutt SL, Urbanek P, Rolink A, Busslinger M Ž1997. Essential functions of Pax5 ŽBSAP. in pro-B cell development: difference between fetal and adult B lymphopoiesis and reduced V-to-DJ recombination at the IgH locus. Genes Dev 11:476]491 74. Konig H, Pfisterer P, Corcoran LM, Wirth T Ž1995. Identification of CD36 as the first gene dependent on the B-cell differentiation factor Oct-2. Genes Dev 9:1598]1607 75. Pfisterer P, Konig H, Hess J, Lipowsky G, Haendler B, Schleuning WD, Wirth T Ž1996. CRISP-3, a protein with homology to plant defense proteins, is expressed in mouse B cells under the control of Oct2. Mol Cell Biol 16:6160]6168 76. Hendriks RW, de BM, Maas A, Dingjan GM, Karis A, Grosveld F Ž1996. Inactivation of Btk by insertion of lacZ reveals defects in B cell development only past the pre-B cell stage. Embo J 15:4862]4872
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Lymphoid-specific transcription mediated by the conserved octamer site: Who is doing what? Patrick MatthiasU m constant ŽC. region.5,6 This motif is also present in several of the enhancers found at the 39 end of the IgH locus,7 ] 11 as well in the promoters of various B cell-specific genes, for example B29 12 or CD20.13 Interestingly, the octamer motif is found in different relative orientations in the Ig heavy and light chain gene promoters, indicating that the proteins recognizing this element have a large degree of functional flexibility. Transfection experiments using variously truncated Ig VH or VL promoter constructs first identified the functional importance of the octamer site for Ig promoter function and B cell-specific activity.4,14,15 These data were corroborated by in vitro transcription experiments testing the activity of Ig promoter constructs in transcriptional extracts derived from B or non-B Že.g. HeLa. cells.16 In addition, it was shown that insertion of an octamer site into an unrelated core promoter is sufficient to render the resulting chimeric promoter B cell-specific in transfection or in vitro transcription assays.17 ] 19 Likewise, multimerisation of a small fragment derived from the IgH J-C intron enhancer and containing the octamer site is sufficient to create a potent B cell-specific enhancer.20 Most importantly, transgenic mice were generated containing a rearranged IgH gene or mutant versions of it in which different regulatory sites had been eliminated.21 These studies showed that mutation of the IgH promoter octamer site reduced activity over twentyfold in lymphoid cells and identified this element as the most important one for VH promoter function. By contrast, the octamer site of the IgH J-C intron enhancer did not appear to play a major role in these assays.21 Additional support for a crucial role of the octamer site for Ig gene transcription came from somatic cell fusion experiments in which reporter constructs under the control of Ig gene regulatory elements Že.g. a kappa light chain gene promoter. were first stably integrated into B cell lines. The resulting reporter cell lines were then fused either to fibroblasts or to a T cell line ŽBW5147. and expression of
The conserved octamer site found in the promoters of Immunoglobulin (Ig) genes has been identified early on as a key element for B cell-specific Ig promoter activity and has also been associated with expression of other B cell-specific genes. Several transcription factors } ubiquitous or cellrestricted } interact specifically with the octamer motif in B cells: Oct-1 and Oct-2 are POU domain transcription factors that bind directly to the octamer site and thereby can recruit the coactivator OBF-1 to the promoters of various genes. Yet, in spite of intense work, the precise role played by each of these factors for transcription activation through the octamer motif is unclear. In particular, genetic inactivation in mice of the lymphoid cell-specific transcription factor Oct-2 or of the B cell-specific coactivator OBF-1 led to surprising findings that are being discussed. Key words: octamer site r Oct-1 r Oct-2 r OBF-1 r immunoglobulin transcription Q1998 Academic Press Ltd
The octamer site and lymphoid-specific transcription FOR MORE THAN 10 years the genes coding for Immunoglobulins ŽIg. have served as a model system for the study of B cell-specific transcription. Numerous regulatory elements and cognate trans-acting factors that are important for the transcription of Ig genes have been identified Žreviewed in Refs 1,2.. In particular, the so-called octamer site -ATGCAAAT- has drawn a lot of attention. This site was first identified as a motif conserved in virtually all of the Ig heavy ŽH. and light ŽL. chain variable ŽV. region promoters,3,4 as well as in the enhancer that is present in the intron found between the joining ŽJ. and U
From the Friedrich Miescher Institute, Maulbeerstr. 66, PO Box 2543, 4058 Basel, Switzerland Q1998 Academic Press Ltd 1044-5323r 98r 020155q 09 $25.00r 0r si980117
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the reporter gene was subsequently measured in the hybrids.22 ] 24 In each case, expression of the reporter gene was rapidly extinguished, paralleling the downregulation of the endogenous Ig gene. For this phenomenon, the Ig octamer site was found to be essential;23,24 replacement of the kappa promoter octamer site by an Sp1 or NF1 binding site rendered the promoter resistant to extinction.23 In addition, it was shown that the expression of several of the transcription factors binding to the octamer site Žsee below. was also downregulated in the fusion process.23,25,26 Thus, it appeared that the octamer site, either as a natural element of Ig promoters, or as an artificial component of chimeric promoters or synthetic enhancers, is able to mediate B cell-specific transcription activation. Furthermore, the octamer site is also a conserved and functionally important element of the promoters of ubiquitous genes, such as the histone H2B 27 or the small nuclear RNA U2 or U6 genes.28,29 The dual role played by the octamer motif for lymphoidspecific or ubiquitous transcription regulation must therefore be explained by the existence of cellspecific and ubiquitous transcription factors and also by promoter-specific features such as, for example, distance between the octamer motif and the core promoter or presence of nearby binding sites for additional transcription factors.
monstrated that the B cell-specific Oct-2 protein had the capacity to trans-activate cotransfected reporter p lasm id s in an o ctam e r site - d e p e n d e n t manner.32,39 ] 41 These results led to the early notion that the B cell-specific activity associated with the octamer site was primarily mediated by the Oct-2 protein. RNA analysis studies showed that Oct-1 expression is virtually ubiquitous, as predicted from the protein]DNA assays. In contrast Oct-2, which comprises several isoforms generated by alternative splicing,42 was found to be expressed at low levels in B cells of early differentiation stages, such as pro- or pre-B cells and at higher levels in more mature B cells.43,44 In pre-B cells Oct-2 expression can be further enhanced by treatment of the cells with, e.g. bacterial lipopolysaccharide ŽLPS., a polyclonal B cell mitogen. Furthermore, Oct-2 expression can also be induced in T cell lines or primary T cells by the phorbol ester 12-myristate 13-acetate ŽPMA. and ionomycin, or by specific antigen.45 Finally, Oct-2 expression has also been detected in the nervous system, in the kidney and in testis.
OBF-1 (aka OCA-B, Bob1), a lymphoid-specific transcriptional coactivator Recently, several groups cloned the gene coding for a novel protein that specifically interacts with Oct-1 and Oct-2. This protein has been variously dubbed OBF-1 ŽOct Binding Factor 146 ., OCA-B 47 or Bob-1 48 and its existence had been postulated on the basis of either biochemical experiments suggesting the presence in B cells of coactivatorŽs. for Oct factors,49,50 or as a means of explaining the phenotype of Oct-2-de ficient mice Žsee below.. OBF-1 is a novel 256-amino acid proline-rich protein that has very little homology to other proteins in the databases. Expression of OBF-1, at the protein or RNA level, is highly B cell-specific and is not observed in most other cell types examined.46 ] 48 Within the B cell lineage, OBF-1 expression is found in cells of all stages of differentiation Žpro-, pre- and mature B cells. at constant levels.51 Through its interaction with either Oct-1 or Oct-2, OBF-1 can be recruited to a subset of octamer sites and thereby co-activate gene transcription, as was demonstrated with octamer sitecontaining reporter plasmids either in transient transfections or in vitro transcription assays.46 ] 48 The mechanism of transcription coactivation has yet to be examined in greater detail, but can be at least partly explained by the presence of a strong transcription
Oct factors: Oct-1 and Oct-2 In parallel to the above-mentioned studies defining the functional role of the octamer site, protein]DNA interaction assays identified nuclear factors binding specifically to this motif. Oct-1 was identified as a ubiquitous protein and Oct-2 as a mostly lymphoid cell-restricted protein. The simultaneous cloning of these factors by several laboratories30 ] 33 led to the identification of a novel family of transcription factors, the POU proteins.34 These factors are characterized by the presence of an approximately 150 amino acid long bipartite DNA binding domain, the POU domain, which comprises a novel ‘POU-specific’ domain linked to a variant homeodomain.34 Both of these regions contain helix-turn-helix motifs required for high affinity binding to DNA. By now a large number of POU proteins have been identified in many different organisms and these proteins have been implicated in developmental regulation in several systems Žfor reviews, see Refs 35]38.. Transfection studies performed in non-B cells de156
Octamer site and b cell-specific transcription
by gene targeting either in a B cell line ŽWEHI 231. 58 or in embryonic stem cells, from which mice were subsequently generated.59 The data obtained from these experiments can be summarized as follows. In the oct-2yry WEHI cells transcription of endogenous Ig genes ŽIgL or IgH. was surprisingly not affected. Octamer site-containing reporter plasmids introduced transiently into these Oct-2-deficient B cells could be put in two categories. Ž1. Reporter plasmids containing an octamer site in their promoter were unaffected by the lack of Oct-2 and were still highly expressed in the mutant B cells. Such reporters, however, were only weakly active in non-B cells Žcontaining only Oct-1 ., suggesting the presence in B cells of at least one additional factor that would be important for full activity. This factor is thought to be OBF-1. Ž2. By contrast, reporters containing a synthetic octamer site enhancer inserted at the 39 of the gene were only weakly active in the Oct-2-deficient B cells, indicating that Oct-2 is required for this ‘activation at a distance’ through octamer sites.58 However, because such reporter plasmids could not be activated by Oct-2 alone in non-B cells, the existence of another B cell-specific cofactor has also been postulated here.40,60 To organize these results in a framework, a model has been put forward suggesting that in B cells there are two distinct pathways of gene activation through the octamer motif ŽFigure 1.. The first pathway, promoter]proximal, does not require Oct-2 and can be mediated by Oct-1 in association with a Bcell-specific component, most likely OBF-1 ŽFigure 1A.. The second pathway, reflecting activation from a distance, can not operate via Oct-1 and requires Oct-2, also in conjunction with a B-cell-specific activity ŽFigure 1B.. Additional genetic experiments with Gal4-Oct-2 fusion proteins identified the C terminus of Oct-2 as being necessary and sufficient to mediate this effect in B cells.61,62 Furthermore, expression of both OBF-1 and Oct-2 together in non-B cells was not sufficient to mediate activation at a distance.51 Thus, an additional activity, as yet undefined but specific for Oct-2, has been postulated to be present in B cells. This activity could be another transcriptional coactivator like OBF-1, but could also be an enzyme such as, e.g. a kinase that would modify Oct-2 specifically in B cells and thus endow it with a novel functional property. The Oct-2-deficient mice also showed a surprising phenotype. These mutant mice develop normally but always die within hours of birth for unknown reasons, perhaps related to the neurogenic expression of Oct-
activation domain in the C-terminal moiety of OBF1.52 The N-terminal portion of OBF-1 contains the domain that mediates interaction with Oct factors.46,53 Recruitment of OBF-1 in a ternary complex is influenced both by the POU domain and by the DNA site. While OBF-1 interacts efficiently in vitro with the POU domains of Oct-1 and Oct-2 Žboth of which are present in B lymphocytes., it fails to interact with other POU proteins, such as Oct-4, Oct-6 or Pit-1. 46 This selectivity of interaction has been recently mapped to a few key residues located in the POU domain at the beginning of the POU-specific first helix and at the end of the POU-homeodomain third helix Žsee Ref 54; Sauter and Matthias, submitted.. At the DNA level, it has been shown that positionq 5 of the octamer site is critical and has to be an A as in the consensus octamer site Ž-ATGCAAAT- . in order to allow efficient ternary complex formation.53,55 This observation was further extended by the finding that, at high concentration, the N-terminal portion of OBF-1 Žamino acids 1]118. can bind to octamer site DNA in the absence of POU domain55 and also by our recent studies with phosphothioate-modified oligonucleotides showing that several positions on the DNA backbone influence the efficiency of ternary complex formation ŽSauter and Matthias, submitted.. Thus, the picture that currently emerges is that OBF-1 interacts with multiple residues in the POU domain and also with several positions on the DNA and thereby forms an intimate molecular ‘clamp’ between the two halves of the POU domain and the cognate DNA site. Recently it was found that OBF-1 is also transiently expressed in various T cell lines and in primary T cells following activation in vitro by PMA and ionomycin or by anti-CD3 antibody treatment.56,57 The kinetics of induction is rapid, suggesting that OBF-1 may participate in regulating the program of gene activation that is characteristic of T cell activation. The induction of OBF-1 in T cells was dissected into two discrete steps: Ž1. OBF-1 gene induction and concomitant protein production; and Ž2. phosphorylation of OBF-1 on Ser 184 leading to formation of a transcriptionally active form of the protein.57 Because OBF-1 appears to be phosphorylated on the same residue in B cells, it has been postulated that in these cells the OBF-1 kinase is constitutively active.57
Genetic inactivation of Oct-2: unexpected results The B cell-specific factor Oct-2 has been inactivated 157
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Figure 1. Model describing the different modes of transcription activation through octamer sites in lymphoid cells. Oct factors ŽOct-1 or Oct-2 . are schematically depicted bound to the DNA. The two ovals represent the POU specific and the POU homeodomain and the N-terminal and C-terminal activation domains are indicated ŽN-ter, C-ter .. ŽA. Promoter pathway of gene activation. Octamer sites in promoters can be bound by Oct-1 or Oct-2 together with the coactivator OBF-1 Žif the site is permissive., leading to transcription activation. ŽB. Enhancer pathway of gene activation. Multimerized enhancer octamer sites Žbut also octamer sites found in natural enhancers?. are bound by Oct-2. In B cells an activity distinct form OBF-1 and requiring the C terminal domain of Oct-2 mediates transcription activation from such multimerized octamer sites.
2. B cell precursors are found in normal numbers in these mice and IgM positive cells are found, but in somewhat reduced numbers.59 Upon in vitro mitogenic stimulation with bacterial LPS, oct-2yry B cells failed to proliferate and secrete antibody normally. In Abelson virus-transformed B cells derived from these mice transcription of several putative target genes, such as IgH, IgL, or B29 ŽIg b ., was found to be unaffected.59 Because oct-2yry mice die at birth, in vivo analysis
of B cell development or Ig levels had to rely on reconstitution of SCID or RAG1yry mice with oct2yry fetal liver cells.63 In such chimeras the B and T cell compartments developed essentially normally, indicating that B cell development and Ig transcription could proceed in the absence of Oct-2. In these mice serum Ig levels were reduced, in particular IgM, IgG1, IgG3 and IgG2b; surprisingly, IgA levels were normal. In a proliferation assay, splenic B cells from such chimeric mice failed to respond to stimulation 158
Octamer site and b cell-specific transcription
by LPS or anti-m , two polyclonal T cell-independent ŽTI. B cell activators. This defect is manifested by an arrest in the G1 phase of the cell cycle.63 Thus, the lack of Oct-2 appears to affect B cells at a late stage of their development. Recently, it was shown that RAG1yry mice reconstituted with oct-2yry B cells fail to develop a robust immune response to either TI and also, surprisingly, to T cell-dependent ŽTD. antigens.64 Thus, although initial Ig gene transcription as well as B cell development proceed essentially normally in the absence of Oct-2, late steps of the immune response are dependent on this protein. The seeming lack of deleterious effect of the Oct-2 ablation on Ig gene transcription was originally explained by invoking a functional redundancy between Oct-2 and the coactivator OBF-1. According to that model, in Oct-2-deficient mice OBF-1 } in concert with Oct-1 } ensures that Ig genes are efficiently transcribed and thus permits a normal B cell development. Several experiments had suggested that OBF-1 has a unique function for Ig gene transcription. In particular, it was shown that Oct-2, when expressed at physiological Žlow. levels in fibroblasts by stable transfection, was unable to activate octamer-dependent reporter plasmids.65 Likewise, in in vitro transcription assays with purified proteins, neither Oct-1 nor Oct-2 activated Ig promoter reporter plasmids on their own,66,67 but when partially purified OBF-1 protein was added to the reaction efficient transcription was obtained.50 These experiments therefore suggested that the coactivator OBF-1 is the obligatory factor for Ig promoter activation, in conjunction with Oct-1 or Oct-2.
indirectly. When stimulated in vitro with, e.g. LPS or LPS and IL-4, splenocytes from OBF-1yry mice respond fairly normally, as measured either at the level of Ig production Žby Northern blotting or ELISA., or at the level of cellular proliferation.68 In addition, Ig class switching also takes place essentially normally in vitro and relatively normal numbers of B cells are found with switched IgH isotypes on their surface.69 Surprisingly, in splenocytes from these mice transcription of Ig m or Ig k was found to be unaffected, but this was in good agreement with the normal IgM serum levels observed.68,70 In one case it was shown that transcription of isotype-switched Ig genes ŽIgG. was somewhat reduced in the absence of OBF-1, but this effect appeared to be highly dependent on the stimulus used to induce switching.69 Thus, evaluation of the potential role of OBF-1 for transcription of already switched Ig genes will require further study. One could imagine that OBF-1 is indeed required for transcription from V region promoters, but only for a subset of them. In that scenario, OBF-1-defi cient mice would express only a limited number of V genes Žthose that are not dependent on OBF-1 for their activation., but this would go unrecognized in an analysis done by Northern blot. To examine this, hybridomas were generated with LPS-stimulated splenic cells obtained from normal or OBF-1-defi cient mice; subsequently, IgM producing clones were identified, their V genes cloned and their corresponding DNA sequence determined. However, this analysis did not reveal a significant difference in V gene usage between wild-type and OBF-1yry mice, which show a normal Ig repertoire ŽSchubart et al, submitted.. Thus, in spite of the in vitro evidence to the contrary, the link between OBF-1 and Ig gene transcription has not yet been clearly made in vivo. The most striking feature of the lack of OBF-1 is that deficient mice have a dramatically impaired capacity to mount an immune response following immunization with TI and TD antigens. This impairment correlates with a complete lack of germinal center formation in the spleen of immunized mice.68 In addition, the phenotype can be completely transferred by injection of OBF-1yry bone marrow into irradiated wt recipient mice, both at the level of B cell development as measured by FACS, as well as at the level of the defective immune response. In addition, through in vivo and in vitro assays, we showed that in OBF-1yry mice T cell function appears to be normal.68 Furthermore, we also found that induction of the IL-2 and IL-4 genes in OBF-1yry T cells is
Genetic inactivation of OBF-1: more unexpected results Recently several groups including ours generated OBF-1-deficient mice by gene targeting.68 ] 70 These mice are born normally and appear healthy. Early B cell development in the bone marrow, identified by the expression of markers such as c-kit or IL-2 receptor a chain in conjunction with the pan B cell marker B220, appeared normal. However, mature recirculating B cells ŽB220 highrIgM low and coexpressing IgD. were present at significantly reduced levels. In addition, the spleen of these animals contained approximately two to fourfold less B cells. While IgM serum levels were normal, the levels of all IgGs as well as IgA were strongly reduced, indicating that OBF-1 has an impact on Ig production, directly or 159
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normal.56 Therefore the primary defect in OBF-1-deficient mice appears to lie within the B cells.
the double mutant mice obtained showed that B cell development in the bone marrow was indeed essentially normal and that peripheral lymphoid organs such as spleen or lymph nodes were, remarkably, empty of B cells and filled up mostly with T cells ŽSchubart et al, submitted.. These data thus indicate that OBF-1, in addition to its paramount role for in vivo immune response, is also necessary for the development of B cells at the time when they exit the bone marrow and enter the periphery ŽFigure 2..
OBF-1 has an important role for the bone marrow-derived B cell developmental pathway In the course of characterizing B cell development in OBF-1yry mice by FACS, we identified an antibody showing a dramatically different labeling pattern between wt and knockout mice. This antibody, called mAb493, labels an as yet unidentified protein of approximately 135 kDa found on B cells representing early stages of B cell development. It distinguishes pro-preBI, preBII and immature B cells, which are all mAb493q, from mature B cells which are mAb493y ŽRolink et al, submitted.. Therefore, practically all B cells in the bone marrow of a normal mouse are mAb493q and only the long-lived recirculating IgM low IgD high CD23q mature B cells are mAb493y. By contrast, in the spleen only approximately 20% of all B cells are mAb493q and these represent immature B cells that have just entered the peripheral B cell pool ŽRolink et al, submitted.. Precisely these mAb493q splenic B cells were found to be lacking in OBF-1yry mice, while mAb493q cells were found in OBF-1yry bone marrow at normal levels ŽSchubart et al, submitted.. These and other data indicated that OBF-1yry B cells are impaired at the transition from immature to mature B cells, which corresponds to the exit from the bone marrow to the periphery. In that context, the finding that OBF-1 deficient mice have B cells in their peripheral organs, albeit less than normal mice, represented a seeming paradox. If OBF-1yry B cells are impaired at the bone marrowrperiphery transition, then where are the splenic B cells coming from? A possibility is that splenic B cells in OBF-1yry mice might be derived from the early wave of B cell generation Žreviewed in Ref 71., that takes place primarily in the fetal liver and is dependent on the cytoplasmic tyrosine kinase Btk 72 and on the paired domain transcription factor Pax-5.73 To test this hypothesis at the genetic level Schubart et al crossed OBF-1-deficient mice with mice of the CBArN strain, that are deficient in Btk, owing to a natural null mutation in the gene ŽXid.. Based on the model described above, the prediction was that in such double mutant mice early B cell development in the bone marrow should be largely normal and that in contrast development of B cells in the periphery should be strongly affected. Analysis of
Redundancy versus specificity: who is doing what? From the results mentioned above it is clear that the study of Oct factors and of the coactivator OBF-1 has revealed many very interesting and unexpected aspects of the transcriptional control in lymphocytes. However, the critical target genes whose expression is altered in the absence of Oct-2 or OBF-1 and which might explain the phenotype observed in the corresponding knockout mice, remain to be identified. In the case of Oct-2, a few target genes have been described that are not expressed anymore in cells derived from oct-2yry mice, such as CD36, which encodes a membrane glycoprotein implicated in signal transduction74 and CRISP-3, a cysteine-rich secreted protein whose function is unknown.75 Yet, although these genes clearly require Oct-2 to be expressed in mouse B cells, lack of their expression is unlikely to explain the phenotype of the oct-2yry mice and therefore other target genes still have to be found. In the case of OBF-1, no specific target gene has been identified yet and this is one of the pressing issues for the future. These different transcription factors have been first identified and were subsequently cloned through the study of Ig gene regulation; yet their precise role in the regulation of Ig gene transcription remains to be elucidated. In spite of a number of experiments suggesting the contrary, it is still possible that in vivo Oct-2 and OBF-1 are to a certain extent redundant, at least with respect to Ig promoter activation. To elucidate this, mice lacking both Oct-2 and OBF-1 will certainly be instrumental and they are currently being generated in our laboratory. Also the role of the ubiquitous factor Oct-1 will have to be evaluated in vivo. And what is the function, if any, of Oct-2 and OBF-1 in T cells? No doubt, the study of Oct factors and of their coactivators will provide more surprises in the future. 160
Octamer site and b cell-specific transcription
Figure 2. Schematic model of the antigen independent phase of B lymphoid development and its requirement for Btk, Pax-5 and OBF-1 Žafter Schubart et al, submitted.. ŽA. The early developmental wave of B cells that initiates in various organs of the fetusrneonate is largely dependent on Pax-5 w73x and Btk w72x. The point at which Btk blocks this developmental pathway has not been defined precisely yet. In adult mice these B cells ŽB1. are found predominantly in the peritoneum but also to some extent in other peripheral organs. ŽB. In the bone marrow, progression through the pre-BII stage fails in the absence of Pax-5 and a mutated Btk gene causes selective disadvantages at the transition from pre-BII to immature B cells w76x; however, neither here nor in the spleen Žsee below. is a developmental block observed in the Btk mut background. ŽC. In a normal mouse immature B cells migrate from the bone marrow to the spleen where they develop into more mature B cells and loose expression of the antigen recognized by mAb493. The transition of immature B cells from the bone marrow to the periphery is greatly impaired in OBF-1-deficient mice and peripheral lymphoid organs that are usually populated with bone marrow derived B cells, are filled with B cells that are Btk dependent and therefore most likely originated from the fetalrneonatal phase of B lymphopoeisis. In Btk mut mice the maturation to mature B cells is affected moderately. For simplicity the scheme does not cover the developmental aspects taking place after B cells have entered the spleen or the peritoneum and does not indicate the impaired immune response in the absence of OBF-1. Abbreviations: GL, Ig genes in germline configuration; DJ-H, D-J rearrangement in the Ig heavy chain gene; VDJ-H, V-DJ heavy chain Ig gene rearrangement; VJ-L, V-J Ig light chain gene rearrangement.
Acknowledgements I wish to thank all the members of my laboratory for discussions and in particular Patrick Sauter, Steffen Massa, Daniel Schubart and Steffen Junker ŽAarhus University. for help with the figures and critical reading of the manuscript.
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