nubbin/POU domain protein 1


Protein interactions of Pdm homologs

TBP, the TATA box binding protein component of the TFIID factor efficiently associates with Oct1 and Oct2. The interaction is direct and does not depend on the presence of DNA or additional proteins. N- and C-terminal deletions of the different proteins were used to localize the domains involved in the interaction. The POU homeodomain of Oct2 and the evolutionarily conserved C-terminal core domain of TBP are both required and sufficient for the interaction. The Oct1 POU domain, which is highly homologous to the Oct2 POU domain, likewise mediates interaction with TBP. The interaction can also be observed in vivo, as TBP can be co-precipitated with Oct2 from co-transfected Cos1 cells and TBP co-immunoprecipitates with the endogenous Oct1 from HeLa cells. Co-transfection of human TBP and Oct2 expression vectors into B cells results in the synergistic activation of a promoter containing an octamer motif (Zwilling, 1994).

High mobility group (HMG) protein 2 and Oct2 interact via their respective HMG domains and POU homeodomains. This interaction is not restricted to Oct2, as other members of the octamer transcription factor family like Oct1 and Oct6 also interact with HMG2. The interaction with HMG2 results in a marked increase in the sequence-specific DNA binding activity of the Oct proteins. Interestingly, the HMG2 protein is not present in the protein-DNA complex detected by an electrophoretic mobility shift assay. The Oct and HMG2 proteins also interact in vivo. A chimeric protein, in which the strong transactivation domain of VP16 is fused directly to the HMG domains of HMG2, stimulates the activity of an octamer-dependent reporter construct upon cotransfection. Furthermore, the expression of antisense RNA for HMG2 specifically reduces octamer-dependent transcription. These results suggest that one of the functions of HMG2 is to support the octamer transcription factors in their role as transcriptional activators (Zwilling, 1995).

Octamer binding transcription factors (Oct factors) play important roles in the activation of transcription of various genes, however, certain genes require cofactors that interact with the DNA binding (POU) domain. In the present study, a yeast two-hybrid screen with the Oct-1 POU domain as a bait identified MAT1 as a POU domain-binding protein. MAT1 is known to be required for the assembly of cyclin-dependent kinase (CDK)-activating kinase (CAK) (see Drosophila Cyclin-dependent kinase 7), which is functionally associated with the general transcription factor IIH (TFIIH). Further analyses show that MAT1 interacts with POU domains of Oct-1, Oct-2, and Oct-3 in vitro in a DNA-independent manner. MAT1-containing TFIIH also interacts with POU domains of Oct-1 and Oct-2. MAT1 has been shown to enhance the ability of a recombinant CDK7-cyclin H complex (bipartite CAK) to phosphorylate isolated POU domains, intact Oct-1, and the C-terminal domain of RNA polymerase II, but not the originally defined substrate, CDK2. Phosphopeptide mapping indicates that the site (Ser385) of a mitosis-specific phosphorylation that inhibits Oct-1 binding to DNA is not phosphorylated by CAK. However, one CAK-phosphorylated phosphopeptide comigrates with a Cdc2-phosphorylated phosphopeptide previously shown to be mitosis-specific; this suggests that in vitro, CAK is able to phosphorylate at least one site that is also phosphorylated in vivo. These results suggest (1) that interactions between POU domains and MAT1 can target CAK to Oct factors and result in their phosphorylation, (2) that MAT1 not only functions as a CAK assembly factor but also acts to alter the spectrum of CAK substrates, and (3) that a POU-MAT1 interaction may play a role in the recruitment of TFIIH to the preinitiation complex or in subsequent initiation and elongation reactions (Inamoto, 1997).

Steroid hormone receptors are distinguished from other members of the nuclear hormone receptor family through their association with heat shock proteins and immunophilins in the absence of ligands. Heat shock protein association represses steroid receptor DNA binding and protein-protein interactions with other transcription factors and facilitates hormone binding. The hormone-dependent interaction between the DNA binding domain (DBD) of the glucocorticoid receptor (GR) and the POU domains of octamer transcription factors 1 and 2 (Oct-1 and Oct-2, respectively) were examined. The GR DBD binds directly, not only to the homeodomains of Oct-1 and Oct-2 but also to the homeodomains of several other homeodomain proteins. Since these results suggest that the determinants for binding to the GR DBD are conserved within the homeodomain, an examination was carried out to see whether the ectopic expression of GR DBD peptides affect early embryonic development. The expression of GR DBD peptides in one-cell-stage zebra fish embryos severely affects their development, beginning with a delay in the epibolic movement during the blastula stage and followed by defects in convergence-extension movements during gastrulation, as revealed by the abnormal patterns of expression of several dorsal gene markers. In contrast, embryos injected with mRNA encoding a GR peptide with a point mutation that disrupts homeodomain binding or with mRNA encoding the DBD of the closely related mineralocorticoid receptor, which does not bind octamer factors, develop normally. Moreover, coinjection of mRNA encoding the homeodomain of Oct-2 completely rescues embryos from the effects of the GR DBD. These results highlight the potential for DNA-independent effects of GR in a whole-animal model and suggest that at least some of these effects may result from direct interactions with homeodomain proteins (Wang, 1999).

Glucocorticoid receptor (GR) and octamer transcription factors 1 and 2 (Oct-1/2) interact synergistically to activate the transcription of mouse mammary tumor virus and many cellular genes. Synergism correlates with cooperative DNA binding of the two factors in vitro. To examine the molecular basis for these cooperative interactions, the consequences of protein-protein binding between GR and Oct-1/2 have been studied. GR binds in solution to the octamer factor POU domain. Binding is mediated through an interface in the GR DNA binding domain that includes amino acids C500 and L501. In transfected mammalian cells, a transcriptionally inert wild-type but not an L501P GR peptide potentiates transcriptional activation by Oct-2 100-fold above the level that can be attained in the cell by expressing Oct-2 alone. Transcriptional activation correlates closely with a striking increase in the occupancy of octamer motifs adjacent to glucocorticoid response elements (GREs) on transiently transfected DNAs. Intriguingly, GR-Oct-1/2 binding is interrupted by the binding of GR to a GRE. A model is proposed for transcriptional cooperativity in which GR-Oct-1/2 binding promotes an increase in the local concentration of octamer factors over glucocorticoid-responsive regulatory regions. These results reveal transcriptional cooperativity through a direct protein interaction between two sequence-specific transcription factors that is mediated in a way that is expected to restrict transcriptional effects to regulatory regions with DNA binding sites for both factors (Prefontaine, 1998).

Transcriptional synergism between glucocorticoid receptor (GR) and octamer transcription factors 1 and 2 (Oct-1 and Oct-2) in the induction of mouse mammary tumor virus (MMTV) transcription has been proposed to be mediated through directed recruitment of the octamer factors to their binding sites in the viral long terminal repeat. This recruitment correlates with direct binding between the GR DNA binding domain and the POU domain of the octamer factors. In the present study, in vitro experiments have identified several nuclear hormone receptors as having the potential to bind to the POU domains of Oct-1 and Oct-2 through their DNA binding domains, suggesting that POU domain binding may be a property shared by many nuclear hormone receptors. However, physiologically relevant binding to the POU domain appears to be a property restricted to only a few nuclear receptors, since only GR, progesterone receptor (PR), and androgen receptor (AR), interact physically and functionally with Oct-1 and Oct-2 in transfected cells. Thus GR, PR, and AR efficiently promote the recruitment of Oct-2 to adjacent octamer motifs in the cell, whereas mineralocorticoid receptor (MR), estrogen receptor alpha, and retinoid X receptor fail to facilitate octamer factor DNA binding. For MMTV, although GR and MR both induce transcription efficiently, mutation of the promoter proximal octamer motifs strongly decreases GR-induced transcription without affecting the total level of reporter gene activity in response to MR. These results suggest that the configuration of the hormone response element within the MMTV long terminal repeat may promote a dependence for the glucocorticoid response upon the recruitment of octamer transcription factors to their response elements within the viral promoter (Prefontaine, 1999).

Enhancers when functioning at a distance cannot effectively stimulate transcription from core promoters. This is due to the inability of enhancer-bound activators to recruit TBP to a distal TATA box. Surprisingly, binding of a transcriptionally inert Oct-1 POU domain near a core promoter enables an enhancer to function from a distance. POU activity neither requires the coactivator OCA-B nor the interaction of TBP with TFIIA. Instead, the POU domain directly facilitates TBP recruitment to the promoter utilizing a bipartite interaction surface. These results establish that an interaction between the DNA binding domain of an activator and TBP can be used to stimulate transcription. Furthermore, they suggest a mechanism for long-range enhancer function in which a TBP complex is preassembled on a promoter via localized recruitment and then acted upon by distal activators (Bertolino, 2002).

The immunoglobulin (Ig) heavy and light chain genes represent a prototypical system for analyzing the role of a promoter-proximal element in mediating enhancer action at a distance. The promoters of Ig gene segments are relatively simple in structure and contain a conserved octamer element that is positioned at 10-30 bp upstream of the TATA box. After V(D)J recombination, the transcription of a rearranged Ig gene is regulated by one or more distal enhancers that reside 1-100 kbp downstream of the start site. The function of Ig enhancers in stimulating Ig gene transcription is dependent on the octamer motif in the variable gene promoters. The octamer site in B cells is recognized by two related factors, Oct-1 and Oct-2, which are products of distinct genes. Oct-1 and Oct-2 along with Pit-1 and Unc-86 are founding members of the POU family of transcription factors. These proteins recognize DNA by means of a bipartite POU domain (150-160 aa), which consists of a N-terminal POU-specific segment and a C-terminal homeodomain separated by a flexible linker. The Oct-1 and Oct-2 POU domains are highly related (~90% amino acid identity), which accounts for their equivalent DNA binding properties. Both POU domains interact with the lymphoid-specific coactivator OCA-B/OBF1/Bob-1, which cobinds the octamer element to form a ternary complex. The POU domains of Oct-1 and Oct-2 are flanked by activation domains which confer different transactivation properties (Bertolino, 2002).

The Oct-1 POU domain, when bound near a core promoter, enables an enhancer to function from a distance. The POU domain directly facilitates recruitment of TBP to the promoter in the absence of an enhancer or measurable transcription. This step in preinitiation complex assembly is not promoted by the distal activators bound to the enhancer. Conversely, these activators enable recruitment of a Pol II complex, thereby complementing the POU-mediated step. These results establish that an interaction between the DNA binding domain of an activator and TBP can be used to stimulate transcription. Furthermore, they suggest a mechanism in which a TBP complex is assembled independently on a promoter and then acted upon by long-range enhancers. This has important implications for developmentally regulated genes whose spatio-temporal patterns of expression are dependent on multiple enhancers (Bertolino, 2002).

A two-step model for enhancer action at a distance is proposed. In this model, a promoter-proximal regulator initiates PIC assembly, i.e., TBP binding in the absence of transcription or an active enhancer (step 1). In a corresponding manner, activators bound to an enhancer can recruit RNA Pol II/mediator complexes without interaction with promoter-bound factors (step 2). Pol II complexes are then delivered to the preassembled TBP complex via DNA looping. Repeated Pol II recruitment by the enhanceosome and delivery to the promoter enables efficient reinitiation (Bertolino, 2002).

The transcription factor Runx2 is essential for the expression of a number of bone-specific genes and is primarily considered a master regulator of bone development. Runx2 is also expressed in mammary epithelial cells, but its role in the mammary gland has not been established. Runx2 is shown to form a novel complex with the ubiquitous transcription factor Oct-1 to regulate the expression of the mammary gland-specific gene ß-casein. The Runx2/Oct-1 complex forms on a Runx/octamer element that is highly conserved in casein promoters. The Runt domain is a DNA-binding domain that specifically recognizes a consensus binding site (TGT/cGGT) found in the promoters of several cell type-specific genes. Oct1 regulates transcription from a consensus site ATGC(A/T)AAT. Chromatin immunoprecipitation, RNA interference, promoter mutagenesis, and transient expression analyses were used to demonstrate that the Runx2/Oct-1 complex contributes to the transcriptional regulation of the ß-casein gene. Analysis of the complex revealed autoinhibitory domains for DNA binding in both the N-terminal and the C-terminal regions of Runx2. Oct-1 stimulates the recruitment of Runx2 to the ß-casein promoter by interacting with the C-terminal region of Runx2, suggesting that Oct-1 stimulates Runx2 recruitment by relieving the autoinhibition of Runx2 DNA binding. The regulatory element is actually a composite element consisting of a consensus Runx-binding site adjacent to an octamer sequence. These findings demonstrate that Runx2 collaborates with Oct-1 and contributes to the expression of a mammary gland-specific gene (Inman, 2005).

Mutation of Oct-1

The POU domain transcription factors Oct-1 and Oct-2 interact with the octamer element, a motif conserved within Ig promoters and enhancers, and mediate transcription from the Ig loci. Inactivation of Oct-2 by gene targeting results in normal B cell development and Ig transcription. To study the role of Oct-1 in these processes, the lymphoid compartment of RAG-1(-/-) animals was reconstituted with Oct-1-deficient fetal liver hematopoietic cells. Recipient mice develop B cells with levels of surface Ig expression comparable with wild type, although at slightly reduced numbers. These B cells transcribe Ig normally, respond to antigenic stimulation, undergo class switching, and use a normal repertoire of light chain variable segments. However, recipient mice show slight reductions in serum IgM and IgA. Thus, the Oct-1 protein is dispensable for B cell development and Ig transcription (Wang, 2004).

The POU-domain transcription factor Oct-1 is widely expressed in adult tissues and has been proposed to regulate a large group of target genes. Microarray expression profiling was used to evaluate gene expression changes in Oct-1-deficient mouse fibroblasts. A number of genes associated with cellular stress exhibited altered expression. Consistent with this finding, Oct-1-deficient fibroblasts were hypersensitive to gamma radiation, doxorubicin, and hydrogen peroxide and harbored elevated reactive oxygen species. Expression profiling identified a second group of genes dysregulated in Oct-1-deficient fibroblasts following irradiation, including many associated with oxidative and metabolic stress. A number of these genes contain octamer sequences in their immediate 5' regulatory regions, some of which are conserved in human. These results indicate that Oct-1 modulates the activity of genes important for the cellular response to stress (Tantin, 2005).

A transcriptional coactivator, OBF-1 (also known as OCA-B or Bob-1), for Oct-1 and Oct-2

The lymphoid-specific transcriptional coactivator OBF-1 (also known as OCA-B or Bob-1) is recruited to octamer site-containing promoters by interacting with Oct-1 or Oct-2 and thereby enhances the transactivation potential of these two Oct factors. For this interaction, the POU domain is sufficient. By contrast, OBF-1 does not interact with the POU domains of other POU proteins, such as Oct-4, Oct-6, or Pit-1, even though these factors bind efficiently to the octamer motif. The structural requirements for selective interaction between the POU domain and OBF-1 have been examined. Previous data have shown that formation of a ternary complex among OBF-1, the POU domain, and the DNA is critically dependent on residues within the octamer site. Using methylation interference analysis, bases have been identified that react differently in the presence of OBF-1, as compared to the POU domain alone, and several positions influencing ternary complex formation have been identified. Oct-1/Pit-1 POU domain chimeras have been used to analyze the selectivity of the interaction between OBF-1 and the POU domain. This analysis indicates that both the POU specific domain (POUS) and the POU homeodomain (POUH) contribute to complex formation. Amino acids that are different in the Pit-1 and Oct-1 POU domains and are considered to be solvent accessible based on the Oct-1 POU domain/DNA cocrystal structure were replaced with alanine residues and analyzed for their influence on complex formation. Thereby, residues L6 and E7 in the POUS and residues K155 and I159 in the POUH have been identified as critical in vitro and in vivo for selective interaction with OBF-1. Furthermore, in an in vivo assay OBF-1 functionally recruits two artificially separated halves of the POU domain to the promoter DNA, thereby leading to transactivation. These data allow for the proposal of a model of the interaction between OBF-1 and the POU domain, whereby OBF-1 acts as a molecular clamp holding together the two moieties of the POU domain and the DNA (Sauter, 1998).

Molecular dissection of the B-cell-specific transcription coactivator OCA-B has revealed two distinct regions important, respectively, for recruitment to immunoglobulin promoters through interaction with octamer-bound Oct-1 and for subsequent coactivator function. Further analysis of general coactivator requirements has shown that selective removal of PC4 from the essential USA fraction severely impairs Oct-1 and OCA-B function in a cell-free system reconstituted with partially purified factors. Full activity can be restored by the combined action of recombinant PC4 and the PC4-depleted USA fraction, thus suggesting a joint requirement for PC4 and another, USA-derived component(s) for optimal function of Oct-1/OCA-B in the reconstituted system. Indeed, USA-derived PC2 was found to act synergistically with PC4 in reproducing the function of intact USA in the assay system. Consistent with the requirement for PC4 in the reconstituted system, OCA-B interacts directly with PC4. Surprisingly, however, removal of PC4 from the unfractionated nuclear extract has no detrimental effect on OCA-B/Oct-1-dependent transcription. These results lead to a general model for the synergistic function of activation domains in Oct-1 and OCA-B (mediated by the combined action of the multiple USA components) and, further, suggest a functional redundancy in general coactivators (Luo, 1998).

OCA-B is a B-cell-specific coregulator of the broadly expressed POU domain transcription factor Oct-1. OCA-B associates with the Oct-1 POU domain, a bipartite DNA-binding structure containing a POU-specific (POU[S]) domain joined by a flexible linker to a POU homeodomain (POU[H]). OCA-B alters the activity of Oct-1 in two ways. It provides a transcriptional activation domain which, unlike Oct-1, activates an mRNA-type promoter effectively, and it stabilizes Oct-1 on the Oct-1-responsive octamer sequence ATGCAAAT. These properties of OCA-B parallel those displayed by the herpes simplex virus Oct-1 coregulator VP16. OCA-B, however, interacts with a different surface of the DNA-bound Oct-1 POU domain, interacting with both the POU(S) and POU(H) domains and the center of the ATGCAAAT octamer sequence. The OCA-B and VP16 interactions with the Oct-1 POU domain are sufficiently different to permit OCA-B and VP16 to bind the Oct-1 POU domain simultaneously. These results emphasize the structural versatility of the Oct-1 POU domain in its interaction with coregulators (Babb, 1997).

BOB.1/OBF.1 is a transcriptional coactivator that is constitutively expressed in B cells and interacts with the Oct1 and Oct2 transcription factors. Upon activation of Jurkat T cells and primary murine thymocytes with phorbol esters and ionomycin, BOB.1/OBF.1 expression and transactivation function are induced. BOB.1/OBF.1 is phosphorylated at Ser184 both in vivo and in vitro, and this modification is required for inducible activation. Mutation of Ser184 also diminishes transactivation function in B cells, suggesting that the activating phosphorylation that is inducible in T cells is constitutively present in B cells. Thus, BOB.1/OBF.1 is a transcriptional coactivator that is critically regulated by posttranslational modifications to mediate cell type-specific gene expression (Zwilling, 1997).

Many of the key decisions in lymphocyte differentiation and activation are dependent on integration of antigen receptor and co-receptor signals. Although there is significant understanding of these receptors and their signaling pathways, little is known about the molecular requirements for signal integration at the level of activation of gene expression. In primary B cells, expression of the B-cell specific transcription coactivator OCA-B (also known as OBF-1 or Bob-1) is regulated synergistically by the B-cell antigen receptor, CD40L and interleukin signaling pathways. Consistent with the requirement for multiple T cell-dependent signals to induce OCA-B, it has been found that OCA-B protein is highly expressed in germinal center B cells. Accordingly, germinal center formation is blocked completely in the absence of OCA-B expression in B cells, whereas the helper functions of OCA-B-deficient T cells are indistinguishable from controls. The requirement for OCA-B expression in B cells is germinal center specific, because the development of primary B cell follicles as well as the marginal zone and plasma cells remain all intact. Thus, OCA-B is the first example of a transcriptional coactivator that is both synergistically induced by and required for integration of signals that mediate cell fate decisions (Qin, 1998).

The protein Bob1 (also called OCA-B or OBF-1) is a B lymphocyte-specific transcriptional co-activator that can stimulate transcription by interaction with Oct-1 or Oct-2 and the octamer sequence found in the promoter and most enhancer regions of the immunoglobulin genes. The role that Bob1 plays in the maturation and function of B cells was investigated in mice deficient for Bob1. Although early stages of B cell development in the bone marrow do not appear to be affected, these mice show reduced numbers of B cells in peripheral lymphoid organs. Based on staining for B220, heat-stable antigen, and IgD, this reduction is particularly strong in those cells representing the more mature B cell stages. Probably as a consequence of this reduction in mature B cells, Bob1-deficient mice show reduced serum titers of the immunoglobulin isotypes IgG1, IgG2a, IgG2b and IgA, but not IgM. Histological examination of sections from spleen and lymph nodes reveal that while Bob-1-deficient mice have primary follicles, they lack well-developed germinal centers. Interestingly, B1 (Ly1) B cells in the peritoneum do not appear to be affected by the lack of Bob1. Taken together, these results suggest that, at least in conventional B cells, Bob1 plays in important role in the antigen-driven stages of B cell activation and maturation (Nielsen, 1996).

OCA-B was initially identified as a B-cell-restricted coactivator that functions with octamer binding transcription factors (Oct-1 and Oct-2) to mediate efficient cell type-specific transcription of immunoglobulin promoters in vitro. Subsequent cloning studies led to identification of the coactivator as a single polypeptide, designated either as OCA-B, OBF-1 or Bob-1. OCA-B itself does not bind to DNA directly, but interacts with either Oct-1 or Oct-2 to potentiate transcriptional activation. To determine the biological role of OCA-B, OCA-B-deficient mice were generated by gene targeting. Mice lacking OCA-B undergo normal antigen-independent, B-cell differentiation, including appropriate expression of both immunoglobulin genes and other early B-cell-restricted genes. However, antigen-dependent maturation of B cells is greatly affected. The proliferative response to surface IgM crosslinking is impaired, and there is a severe deficiency in the production of secondary immunoglobulin isotypes, including IgG1, IgG2a, IgG2b, IgG3, IgA and IgE in OCA-B-deficient B cells. This defect is not due to a failure of the isotype switching process, but rather to reduced levels of transcription from normally switched immunoglobulin heavy-chain loci. In accord with the defective isotype production, germinal center formation is absent in these mutant mice (Kim, 1996).

The B-lymphocyte-specific transcriptional factor called Oct binding factor (OBF)-1, OCA-B or Bob1 is thought to be involved in the transcription of immunoglobulin genes through recruitment to the highly conserved octamer site of immunoglobulin promoters, mediated by either Oct-1 or Oct-2. To define the in vivo role of OBF-1, gene targeting was used in embryonic stem cells to generate mice lacking the coactivator OBF-1. Such OBF-1-/- mice are born normally, are fertile and seem healthy, and surprisingly, the rearrangement and transcription of immunoglobulin genes is largely unaffected. However, mice deficient in OBF-1 have reduced numbers of mature B cells and a severe reduction in the number of recirculating B cells, but otherwise show normal B-cell differentiation. Serum IgA and particularly IgG levels are greatly reduced. If mutant mice are immunized with either a thymus-independent or a thymus-dependent antigen, their immune responses are dramatically weakened. Strikingly, germinal centers completely fail to develop after immunization with thymus-dependent antigen. These results demonstrate that in vivo OBF-1 is not required for initial transcription of immunoglobulin genes or for B cell development, but instead is essential for the response of B cells to antigens, and is required for the formation of germinal centers (Schubart, 1996).

The chemokine receptor, BLR1, is a major regulator of the microenvironmental homing of B cells in lymphoid organs. In vitro studies identify three essential elements of the TATA-less blr1 core promoter that confer cell type- and differentiation-specific expression in the B cells of both humans and mice: (1) a functional promoter region (-36 with respect to the transcription start site); (2) an NF-kappaB motif (+44), and (3) a noncanonical octamer motif (+157). The importance of these sites was confirmed by in vivo studies in gene-targeted mice deficient of either Oct-2, Bob1, or both NF-kappaB subunits p50 and p52. In all of these animals, the expression of BLR1 is reduced or absent. In mice deficient only for p52/NF-kappaB, BLR1 expression is unaffected. Thus these data demonstrate that BLR1 is a target gene for Oct-2, Bob1, and members of the NF-kappaB/Rel family and provide a link to the impaired B cell functions in mice deficient for these factors (Wolf, 1998).

The 3' flanking sequence of kappa promoter octamers contains either a conserved A or G residue that increases the affinity of the octamer core motif for Octl and Oct2A. By transient transfections it has been shown that decreasing the affinity of an octamer for Oct binding cripples the transcription unit when the octamer is used in a minimal promoter, while it has only marginal effects when analysed in the context of an intact kappa promoter. When the octamer in a kappa promoter is replaced by a TAATGARAT motif with equal affinity for Oct protein binding the latter can still participate in synergistic transcriptional stimulation. Thus, the synergistic interactions involved in kappa promoter transcriptional stimulation are dependent on the presence of Oct proteins but not on the octamer DNA motif per se. Since the transcriptional coactivator OCA-B cannot interact with Oct protein bound to the TAATGARAT motif, the role of OCA-B in these interactions seems to be limited (Liberg, 1997).

The OCA-B (OBF-1, Bob1) coactivator protein functions in immunoglobulin transcription and B-cell differentiation by forming a complex with the Oct-1 or Oct-2 transcription factors and the octamer DNA sequence (ATGCAAAT). OCA-B is expressed almost exclusively in B cells and is essential for high levels of immunoglobulin gene transcription. Absence of OCA-B causes failures in the immune system, including the elimination of germinal centers, the reduction of some immunoglobulin isotypes in serum, and severe deficiency in B-cell-mediated responses to antigen stimulation. The biological role and promoter specificity of transcription stimulated by OCA-B is correlated with binding data showing that it prefers an A in the fifth position of the octamer and suggesting that this peptide makes direct contact with the DNA. OCA-B will not bind to complexes of Oct-1 or Oct-2 when the octamer element contains a T at position 5. In addition, biochemical and mutagenesis studies of ternary complex formation suggest that OCA-B contacts both the POU-specific domain and the POU homeodomain. The crystal structure has been determined at 3.2 Å of a ternary complex containing an OCA-B peptide, the Oct-1 POU domain, and an octamer DNA site. The OCA-B peptide binds in the major groove near the center of the octamer site, and its polypeptide backbone forms a pair of hydrogen bonds with the adenine base at position 5 of the octamer DNA. Numerous protein-protein contacts between the OCA-B peptide and the POU domain are also involved in the ternary complex. In particular, the hydrophobic surface from a short alpha-helix of OCA-B helps to stabilize the complex by binding to a hydrophobic pocket on the POU-specific domain. The structure of this ternary complex is consistent with previous biochemical studies and shows how peptide-DNA and peptide-protein contacts from OCA-B provide structural and functional specificity in the regulation of immunoglobulin transcription (Chasman, 1999).

The expression of immunoglobulin genes is controlled in part by the DNA-binding protein Oct-1 and the B cell-specific transcription co-activator, Bob1 (also known as OCA-B or OBF-1); together, they form a complex on the Igkappa promoter. The assembly of the ternary complex has been characterized using biophysical methods. Bob1 binds specifically as a monomer to the complex of the Oct-1 DNA-binding domain (Oct-1 POU) and the Igkappa promoter, but binds weakly to either Oct-1 POU or the Igkappa promoter alone, indicating that both are required to make an avid complex. Ternary complex formation requires a defined DNA sequence, because the stability of the complex can be strongly affected by either a single base-pair change or by removing 5-methyl groups from selected thymine bases. In isolation, Bob1 appears to have little secondary structure, but may become partially structured upon recruitment into the ternary complex, as demonstrated by circular dichroism spectra and calorimetry. These and other findings suggest that ternary complex formation requires a defined geometry of the POU/DNA complex, and that the co-activator makes stereo-specific contacts to both the POU protein and the major groove of the DNA that induces its fold (Chang, 1999).

POU domain proteins contain a bipartite DNA binding domain divided by a flexible linker that enables them to adopt various monomer configurations on DNA. The versatility of POU protein operation is additionally conferred at the dimerization level. The POU dimer formed on the palindromic Oct factor recognition element (PORE: ATTTGAAATGCAAAT) can recruit the transcriptional coactivator OBF-1, whereas POU dimers formed on the consensus MORE (ATGCATATGCAT) or on MOREs from immunoglobulin heavy chain promoters (AT[G/A][C/A]ATATGCAA) fail to interact (MORE stands for 'More PORE'). An interaction with OBF-1 is precluded since the same Oct-1 residues that form the MORE dimerization interface are also used for OBF-1/Oct-1 interactions on the PORE. Thus one type of POU dimer is formed on the PORE and another is formed on another palindromic DNA motif called MORE. These findings provide a paradigm of how specific POU dimer assemblies can differentially recruit a coregulatory activity with distinct transcriptional readouts (Tomilin, 2000).

The crystal structure of a POU complex in the PORE dimer configuration without OBF-1 is now available. Preliminary crystallographic data reveal an arrangement of the POU subdomains very similar to that predicted by computer modeling, providing an idea of the structural basis of this coactivator interaction in the PORE dimer. Since the PORE structure is based on the monomer configuration in the Oct-1:octamer crystal structure, it is assumed that the observed binding surface of OBF-1 in the monomer is the same in the PORE dimer (Tomilin, 2000).

The ternary monomer complex shows the way the OBF-1 fragment binds to the N-terminal part of helix 1 (residues 6-10) and a segment between helices 3 and 4 (residues 49-60) of the POUS domain. A new structure of the Oct-1:MORE dimer complex provides a rationale for why binding of OBF-1 is inhibited in this dimer configuration. The direct comparison of the Oct-1/ OBF-1:octamer complex and the Oct-1: MORE dimer reveals that the binding site for OBF-1 is identical to the protein-protein POUS/POUH interface site in the MORE dimer, in which the same residues of POUS (helix 1 and the loop between helices 3 and 4) interact with the C terminus of POUH. The most important contact within this interface is a key-lock type interaction: the side chain of Ile159 of POUH fits into a hydrophobic cavity of POUS. The equivalent interaction is observed in the Oct-1/OBF-1:octamer complex where Val28 of OBF-1 fits into the very same pocket of the POUs domain of Oct-1 (Tomilin, 2000).

The analogy can be further extended to specific DNA base binding. The amido group of the Asn151 side chain of POUH makes two specific hydrogen bonds to the A:T base pair in position 5 of the MORE. This hydrogen bond interaction is regarded as a signature for DNA binding of homeo domains. In the Oct-1/OBF-1:octamer complex, the same base is hydrogen-bonded by the amino group and by the carbonyl group of the main chain of Val22 of OBF-1. From this structural comparison, it is concluded that OBF-1 and the POUH domain compete for binding to the same site of the POUS domain where in the MORE dimer, the OBF-1 binding site is blocked by POUH but accessible in the predicted PORE dimer. The specificity of competitive binding of OBF-1 and POUH is further enhanced by the capability of the two competing domains, POUH and OBF-1, to specifically interact with the binding motif of the respective DNA. The data also indicate that the POUS/POUH binding affinity of the examined MORE dimer complexes is superior, when compared to the affinity of POU/OBF-1 interaction (Tomilin, 2000).

A hallmark of the POU domain family transcription factors is their flexibility in DNA recognition. In this study, it has been shown that the flexibility in POU factor functioning can also be extended to dimerization. The binding of Oct factor family members as homo- and hetero-dimers to the two high-affinity regulatory elements, the PORE and the MORE, has been demonstrated. The structural difference between PORE- and MORE-mediated dimerization leads to the differential recruitment of transcriptional coactivators. OBF-1, for example, binds and synergizes in transcriptional activation with the PORE configuration of the Oct-1 dimer, but fails to bind to the MORE-mediated Oct-1 dimer. Thus, the data demonstrate the mechanism by which distinct POU dimer configurations can recruit specific transcriptional coactivators with different effects on gene transcription (Tomilin, 2000).

OBF-1 (also known as Bob1 or OCA-B) is a B-cell-specific transcription coactivator that binds to conserved octamer elements (ATGCAAAT or reverse) in the DNA together with the POU domain transcription factors Oct-1 or Oct-2. OBF-1 is critical for development of a normal immune response and the formation of germinal centers in secondary lymphoid organs. The RING finger protein Siah-1 interacts specifically with OBF-1. This interaction is mediated by the C-terminal part of Siah-1 and by residues in the N-terminus of OBF-1, partly distinct from the residues required for formation of a complex with the Oct POU domains and the DNA. Interaction between Siah-1 and OBF-1 leads to downregulation of OBF-1 protein level but not mRNA, and to a corresponding reduction in octamer site-dependent transcription activation. Inhibition of the ubiquitin-proteasome pathway in B cells leads to elevated levels of OBF-1 protein. Furthermore, in immunized mice, OBF-1 protein amounts are dramatically increased in primary activated B cells, without concomitant increase in OBF-1 mRNA. These data suggest that Siah-1 is part of a novel regulatory loop controlling the level of OBF-1 protein in B cells (Tiedt, 2001).

The BOB.1/OBF.1 coactivator is critically involved in mediating octamer dependent transcriptional activity in B lymphocytes. Mice lacking this coactivator show various defects in B-cell development; most notably, they completely lack germinal centers. Consistent with this phenotype, BOB.1/OBF.1 levels are massively upregulated in germinal center B cells as compared with resting B cells. The mechanism of upregulation has been addressed and it has been found found that only a minor part of this regulation can be attributed to increased levels of BOB.1/OBF.1-specific mRNA. Apparently, BOB.1/OBF.1 is also regulated at the protein level. In support of this suggestion two related BOB.1/OBF.1 interacting proteins, SIAH1 and SIAH2, have been identified in a yeast two-hybrid screen. SIAH1 and SIAH2 are known regulators of protein stability. Coexpression of SIAH results in a destabilization of BOB.1/OBF.1 protein without affecting mRNA levels. Further more, proteasome inhibitors block the degradation of BOB.1/OBF.1 protein. Finally, B-cell receptor cross-linking also results in the degradation of BOB.1/OBF.1 and consequently reduces transcriptional activation of BOB.1/OBF.1-dependent reporters (Boehm, 2001).

Oct-1 interacts with basal transcription factor SNAPc

The RNA polymerases II and III snRNA gene promoters contain an octamer sequence as part of the enhancer and a proximal sequence element (PSE) as part of the core promoter. The octamer and the PSE bind the POU domain activator Oct-1 and the basal transcription factor SNAPc, respectively. Oct-1 (but not Oct-1 with a single E7R mutation within the POU domain) binds cooperatively with SNAPc and, in effect, recruits SNAPc to the PSE. SNAPc recruitment is mediated by an interaction between the Oct-1 POU domain and a small region of the largest subunit of SNAPc, SNAP190. This SNAP190 region is strikingly similar to a region in the B-cell-specific Oct-1 coactivator, OBF-1, which is required for interaction with octamer-bound Oct-1 POU domain. The Oct-1 POU domain-SNAP190 interaction is a direct protein-protein contact as determined by the isolation of a switched specificity SNAP190 mutant that interacts with Oct-1 POU E7R but not with wild-type Oct-1 POU. This direct protein-protein contact results in activation of transcription. Thus, an activation target of a human activator, Oct-1, has been identified within its cognate basal transcription complex (Ford, 1998).

The human RNA polymerase II and III snRNA promoters have similar enhancers [the distal sequence elements (DSEs)], and similar basal promoter elements [the proximal sequence elements (PSEs)]. The DSE, which contains an octamer motif, binds the broadly expressed activator Oct-1. The PSE binds a multiprotein complex referred to as SNAPc or PTF. On DNAs containing both an octamer site and a PSE, Oct-1 and SNAPc bind cooperatively. SNAPc consists of at least four stably associated subunits, three of which are small (SNAP43, SNAP45, SNAP50) and one that is larger (SNAP190). None of the three smaller subunits, which have all been cloned, can bind to the PSE on their own. The isolation of cDNAs corresponding to the largest subunit of SNAPc, SNAP190 is reported. SNAP190 contains an unusual Myb DNA binding domain consisting of four complete repeats (Ra to Rd) and a half repeat (Rh). A truncated protein consisting of the last two SNAP190 Myb repeats, Rc and Rd, can bind to the PSE, suggesting that the SNAP190 Myb domain contributes to recognition of the PSE by the SNAP complex. SNAP190 is required for snRNA gene transcription by both RNA polymerases II and III and interacts with SNAP45. In addition, SNAP190 interacts with Oct-1. Together, these results suggest that the largest subunit of the SNAP complex is involved in direct recognition of the PSE and is a target for the Oct-1 activator. They also provide an example of a basal transcription factor containing a Myb DNA binding domain (Wong, 1998).

snRNA gene transcription is activated in part by the recruitment of SNAPc (snRNA activating protein complex) to the core promoter through protein-protein contacts with the POU domain of the enhancer-binding factor Oct-1. A mini-SNAPc consisting of a subset of SNAPc subunits (consisting of the amino-terminal third of SNAP190, SNAP43, and SNAP50) is capable of directing both RNA polymerase II (Pol II) and Pol III snRNA gene transcription. Mini-SNAPc cannot be recruited by Oct-1, but binds as efficiently to the promoter as SNAPc together with Oct-1 and directs activated RNA Pol III transcription. Thus, SNAPc represses its own binding to DNA, and repression is relieved by interactions with the Oct-1 POU domain that promote cooperative binding. TBP also represses its own binding, and in that case repression is relieved by cooperative interactions with SNAPc. This may represent a general mechanism to ensure that core promoter-binding factors, which have strikingly slow off-rates, are recruited specifically to promoter sequences rather than to cryptic-binding sites in the genome (Mittal, 1999).

Other examples of basal transcription factors with built-in negative control of binding include E. coli sigma70 and the largest TFIID subunit from both Drosophila (dTAFII230) and yeast (yTAFII145). The first example is a case of autoinhibition, in which the amino-terminal region of sigma70 inhibits the binding of the carboxy-terminal domain of the protein to core promoter elements. In the second case, the amino-terminal region of the largest subunit of TFIID interacts directly with the DNA-binding subunit of TFIID, TBP, and inhibits its binding. This amino-terminal region competes with TFIIA for binding to TBP, suggesting that it participates in a mechanism of transcription activation involving TFIIA. However, the mechanism by which the inhibition of binding is relieved, is not known. By homology with TBP and SNAPc in the snRNA promoters, it is suspected that the amino-terminal domain of the largest TFIID subunit becomes engaged in cooperative binding interactions with another transcription factor binding to the same promoter, thus relieving the inhibition and instead increasing TFIID binding. Thus, a number of core promoter binding factors may be similar to SNAPc and TBP in possessing a mechanism that down-regulates their own binding and is reversed through protein-protein contacts with factors binding to the same promoter. Such a partner-activated switch probably serves to ensure that basal transcription factors, which often do not bind DNA with great sequence specificity and have strikingly slow off-rate, are targeted specifically to promoter sequences rather than to random cryptic sites present in the genome (Mittal, 1999 and references).

The human snRNA promoters contain a proximal sequence element (PSE) required for basal transcription and a distal sequence element (DSE) required for activated transcription. The PSE in both the RNA polymerase II and polymerase III snRNA promoters binds a multisubunit complex known as the snRNA activating protein complex SNAPc or PTF, whereas the octamer sequence in the DSE binds the transcription factor Oct-1. SNAPc consists of five types of subunits -- SNAP19, SNAP43, SNAP45, SNAP50, and SNAP190 -- and can be reconstituted from recombinant subunits. On templates containing closely spaced octamer sequence and PSE, Oct-1 and SNAPc bind cooperatively to DNA, and this effect results in transcription activation in vitro. Cooperative binding requires a direct protein-protein contact that involves a glutamic acid at position 7 in the POUS domain and a lysine at position 900 within SNAP190 (Zhao, 2001 and references therein).

In the natural snRNA promoters, the octamer sequence is not located close to the PSE. Instead, the two elements are separated by approximately 150 bp, and this distance is quite conserved in different RNA polymerase II and polymerase III snRNA promoters. On naked DNA probes corresponding to the natural U6 promoter, cooperative binding of Oct-1 and SNAPc is not observed. The striking conservation of a 150 bp spacing between the octamer sequence and the PSE suggests that in their natural context, the two elements might be brought together by a positioned nucleosome so that Oct-1 and SNAPc may bind cooperatively. Indeed, previous studies have shown that chromatin reconstitution of the human U6 gene in vitro results in the positioning of a nucleosome between the DSE and the PSE (Zhao, 2001 and references therein).

Oct-1 and SNAPc bind cooperatively to DNA when their respective binding sites are moved into proximity through a mechanism that involves a defined protein-protein contact. On the natural U6 promoter, cooperative binding of Oct-1 and SNAPc is mediated by a positioned nucleosome that resides between the DSE and the PSE. This cooperative binding requires the same protein-protein contact as cooperative binding to closely spaced sites on naked DNA and mediates transcription activation (Zhao, 2001).

These results suggest a model in which a nucleosome brings into close proximity the U6 PSE and octamer sequence. This proximity allows the direct protein-protein contact, involving the glutamic acid at position 7 in the POUS domain and the lysine at position 900 in SNAP190 that is required for cooperative binding of the Oct-1 POU domain and SNAPc as well as activated transcription. In this model, the octamer sequence is in the opposite orientation relative to the PSE than in naked DNA probes containing closely spaced octamer and PSE. However, on such naked DNA probes, cooperative binding is not affected by the orientation of the octamer sequence, suggesting a highly flexible interaction between SNAPc and Oct-1. The positioning of the nucleosome is independent of Oct-1 or SNAPc; it could depend on an activity present in the S-190 extract, or it could be directed by the sequence itself. The latter possibility is consistent with results that show that efficient transcription of the human U6 gene incubated in chromatin assembly extracts depends not only on maintaining the correct distance but also the natural U6 promoter sequences between the DSE and the PSE. However, it is likely that the Oct-1 POU domain and SNAPc, once bound to the DNA, further lock the nucleosome to its specific location (Zhao, 2001).

Transcriptional activation of the human U1 snRNA genes is dependent on a noncanonical octamer element contained within an upstream enhancer. The U1 octamer only weakly recruits the Oct-1 POU domain, although recruitment is stimulated by a peptide containing the Oct-1-binding domain of SNAP190. Structural analysis of the Oct-1 POU domain/U1 octamer/SNAP190 peptide complex revealed that SNAP190 makes extensive protein contacts with the Oct-1 POU-specific domain and with the DNA phosphate backbone within the enhancer. Although SNAP190 and OCA-B both interact with the Oct-1 POU domain through the same Oct-1 interface, a single nucleotide within the U1 octamer ablates OCA-B recruitment without compromising activator recruitment by SNAP190 (Hovde, 2002).

In humans, the U1 snRNA multigene family maintains a total level of ~106 transcripts per cell. These levels of U1 RNA suggest that preinitiation complex formation and polymerase recruitment to these genes are highly efficient. Activated transcription of most human snRNA genes depends on a distal sequence element (DSE) that serves as an enhancer of transcription. Typically, the DSE contains an octamer element that can recruit the transcriptional activator protein Oct-1, which recognizes the DSE via its POU DNA-binding domain. The POU domain is a bipartite DNA-binding motif consisting of a POU-specific region (POUS) and a POU homeodomain (POUHD) separated by a flexible linker. Both subdomains are important for stable sequence-specific DNA binding to cognate octamer elements and for transcriptional activation. Oct-1 displays an unusual potential for gene activation, activating transcription of certain immunoglobulin genes in lymphocytes in a tissue-specific pattern and transcription of snRNA genes ubiquitously (Hovde, 2002 and references therein).

Oct-1 also contains an activation domain, but surprisingly, the Oct-1 POU DNA-binding domain is sufficient to maintain robust activation of snRNA gene transcription. During a search for potential targets for the Oct-1 POU domain, the SNAP190 subunit of the snRNA activating protein complex (SNAPC) was isolated. SNAPC, also known as PTF, is a general transcription factor that binds to the proximal sequence element (PSE) contained within the core promoters of human snRNA genes. Further analysis demonstrated that Oct-1 cooperates with SNAPC to increase promoter recognition by SNAPC and identified the region within SNAP190 from amino acids 800 to 930 to be required for interactions with the Oct-1 POU domain. Mutational data confirm that direct protein contacts between the Oct-1 POU domain and general transcription factor SNAP190 are required for transcriptional activation by the Oct-1 POU domain (Hovde, 2002 and references therein).

The region of SNAP190 directly targeted by Oct-1 exhibits homology with the B-cell specific coactivator OCA-B. Furthermore, mutations in Oct-1 that abolish activation of human snRNA transcription and interaction with SNAP190 also block Oct-1 synergism with OCA-B, suggesting that Oct-1 may use similar mechanisms to activate transcription of mRNA-type genes via the coactivator OCA-B and of snRNA-type genes via the general transcription factor SNAP190. The OCA-B interaction was revealed in the crystal structure of the Oct-1 POU domain bound to a high-affinity H2B octamer element in a complex with a peptide from OCA-B. In this structure OCA-B binds to the major groove within the octamer element and interacts extensively with the POUS and POUHD domains, which contact the major groove on opposite faces of the DNA helix. Together OCA-B and the Oct-1 POU domain encircle the high-affinity H2B octamer element to form a stable ternary complex. In order to compare the general transcription factor SNAP190 function to that of the OCA-B coactivator as a target for activation by Oct-1, the structure of a complex of the Oct-1 POU domain, a peptide containing the Oct-1-interacting region of SNAP190, and the human U1 octamer sequence was determined to 2.4 Å. It represents the first structure of a transcriptional activator in a complex with its general transcription factor target (Hovde, 2002).

A general mechanism for transcription regulation by Oct1 and Oct4 in response to genotoxic and oxidative stress

Oct1 and Oct4 are homologous transcription factors with similar DNA-binding specificities. This study shows that Oct1 is dynamically phosphorylated in vivo following exposure of cells to oxidative and genotoxic stress. Stress regulates the selectivity of both proteins for specific DNA sequences. Mutation of conserved phosphorylation target DNA-binding domain residues in Oct1, and Oct4 confirms their role in regulating binding selectivity. Using chromatin immunoprecipitation, it was shown that association of Oct4 and Oct1 with a distinct group of in vivo targets is inducible by stress, and that Oct1 is essential for a normal post-stress transcriptional response. Finally, using an unbiased Oct1 target screen a large number of genes were identified that are targeted by Oct1 specifically under conditions of stress; several of these inducible Oct1 targets are also inducibly bound by Oct4 in embryonic stem cells following stress exposure (Kang, 2008).

Oct1 and Oct4 (products of the Pou2f1 and Pou5f1 genes) are members of the POU (Pit-1, Oct1/2, Unc-86) domain transcription factor family. This family is defined by the presence of a bipartite DNA-binding domain in which two subdomains, covalently connected by a flexible linker, typically recognize DNA through major groove interactions on opposite sides of the helix. The classical DNA recognition sequence is known as an octamer motif (5'-ATGCAAAT-3', hereafter called a 'simple' octamer). However, native binding sites for Oct4 frequently exist in complex paired, overlapping, and nonconsensus configurations (Tantin, 2008; Kang, 2008 and references therein).

Oct4 is a master regulator of the stem cell state and has recently been shown to be one of three proteins sufficient to reprogram differentiated adult mouse and human cells to the embryonic stem (ES) cell lineage (Okita, 2007; Takahashi, 2007; Nakagawa, 2008). The biological function of Oct1 is more enigmatic. Oct1 is known to interact with regulatory sites in interleukin, immunoglobulin, and histone genes. Oct1 also moderately stimulates gene expression reporter constructs linked to target sequences in transient transfection assays. However, it has been shown that Oct1 is nonessential for native H2B, IgH, and Igkappa expression. Oct1-deficient cells appear morphologically normal in light microscopy and divide at normal rates. Oct1-deficient mice die in mid-late gestation (embryonic days 12.5-18.5) (Kang, 2008).

Oct1-/- mouse embryonic fibroblasts (MEFs) are hypersensitive to oxidative and genotoxic stress. One explanation for this result is that constitutive products of Oct1-mediated transcription participate in stress response pathways. Support for an alternative hypothesis, namely that Oct1 directly senses cellular stress, comes from the findings that Oct1 interacts with the Ku70 subunit of DNA-dependent protein kinase (DNA-PK) and is phosphorylated in vitro by DNA-PK at physiologically important serine and threonine residues. Oct1 also interacts with BRCA1, and PARP-1, known participants in stress response pathways (Kang, 2008).

In this study, using an affinity purification approach in vivo post-translational Oct1 modification events were identified following treatment of HeLa cells with ionizing radiation (IR) or H2O2. Comparison with a second Oct1 modification data set indicated that two DNA-binding domain Oct1 phosphorylation events have the potential to modulate Oct1 association with DNA. Using two model sequences, termed PORE (palindromic octamer-related element) and MORE (more PORE) (Remenyi, 2001. Full text of article), it was shown that DNA-binding domain modifications alter the in vitro affinity of Oct1 and Oct4 specifically at complex binding sites. Stress-induced binding of both Oct1 and Oct4 to physiological targets in vivo, show that induced Oct1 binding regulates native gene expression, and expand the repertoire of complex site categories to which Oct1 and Oct4 can bind. Using chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIPseq), a large number of constitutive Oct1 targets were identified as well as targets specifically induced in the presence of oxidative stress. These targets frequently contain conserved complex binding sites for Oct1. It was demonstrated that Oct4 inducibly binds two of these targets in vivo using mouse ES cells and ChIP. Together, the data show that Oct1 and Oct4 are potent stress response effectors (Kang, 2008).

A high throughput embryonic stem cell screen identifies Oct-2 as a bifunctional regulator of neuronal differentiation

Neuronal differentiation is a complex process that involves a plethora of regulatory steps. To identify transcription factors that influence neuronal differentiation a high throughput screen was developed using embryonic stem (ES) cells. Seven-hundred human transcription factor clones were stably introduced into mouse ES (mES) cells and screened for their ability to induce neuronal differentiation of mES cells. Twenty-four factors that are capable of inducing neuronal differentiation were identified, including four known effectors of neuronal differentiation, 11 factors with limited evidence of involvement in regulating neuronal differentiation, and nine novel factors. One transcription factor, Oct-2, was studied in detail and found to be a bifunctional regulator: It can either repress or induce neuronal differentiation, depending on the particular isoform. Ectopic expression experiments demonstrate that isoform Oct-2.4 represses neuronal differentiation, whereas Oct-2.2 activates neuron formation. Consistent with a role in neuronal differentiation, Oct-2.2 expression is induced during differentiation, and cells depleted of Oct-2 and its homolog Oct-1 have a reduced capacity to differentiate into neurons. The results reveal a number of transcription factors potentially important for mammalian neuronal differentiation, and indicate that Oct-2 may serve as a binary switch to repress differentiation in precursor cells and induce neuronal differentiation later during neuronal development (Theodorou, 2009).

Oct-2.4 was identified as a transcription factor that induces neuronal differentiation when fused to the VP16 transactivation domain but not when lacking it. It was further demonstrated that Oct-2.4 represses differentiation in a well-characterized EB-based differentiation assay. Interestingly, this is the only Oct-2 isoform that lacks a C-terminal glutamate-rich transcriptional transactivation domain (Theodorou, 2009).

All Oct-2 isoforms containing transactivation domains except Oct-2.6 were shown to be up-regulated during retinoic acid-induced differentiation of EBs. One of these, Oct-2.2, induces neuronal differentiation when overexpressed. Oct-2.1, Oct-2.3, and Oct-2.5, which have putative transactivation domains, were tested for induction of neuronal differentiation. The results from these three isoforms were not definitive and, at most, modest neuronal formation was observed; perhaps cofactors are required for strong neuronal induction with these isoforms. Nonetheless, the knockout results clearly demonstrate that Oct-2 is required for neuronal differentiation as double mutants lacking Oct-2 and its homolog Oct-1 exhibit in a defect in neuronal differentiation (Theodorou, 2009).

The results are consistent with a role for Oct-2 as a bifunctional regulator through its different spliced isoforms. Early in EB formation the Oct-2.4 is one of the more abundant isoforms. This repressive isoform presumably help keep the neuronal differentiation program suppressed. Later during differentiation, the other isoforms containing transcription transactivation domains such as Oct-2.2 become much more abundant and activate neuronal differentiation. In this way a single gene can mediate a switch from repression to activation. Although bifunctional transcriptional regulators have been observed in other contexts (e.g., Max and p53), this is the first time that alternatively spliced isoforms of one transcription factor have been shown to have different functional roles in neuronal differentiation of mES cells (Theodorou, 2009).

It is proposed that Oct-2.4 is an important isoform in undifferentiated cells that repress neuronal differentiation. Later-activating isoforms of Oct-2 such as Oct-2.2 attain much higher levels and stimulate neuronal differentiation (Theodorou, 2009).

These results are consistent with reporter assays that examined the effects of Oct2 isoforms at individual gene promoters. Oct-2.4 was shown previously to repress transcription of the tyrosine hydroxlyase promoter, whereas Oct-2.1 and Oct-2.5 appear to activate expression of this gene promoter in tissue culture cells. This study demonstrates that the Oct-2.4 and 2.2 isoforms are repressors and activators of the process of neuronal differentiation, respectively (Theodorou, 2009).

Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells

It has been shown that defined sets of transcription factors are sufficient to convert mouse and human fibroblasts directly into cells resembling functional neurons, referred to as 'induced neuronal' (iN) cells. For some applications however, it would be desirable to convert fibroblasts into proliferative neural precursor cells (NPCs) instead of neurons. It was hypothesized that NPC-like cells may be induced using the same principal approach used for generating iN cells. Toward this goal, mouse embryonic fibroblasts derived from Sox2-EGFP mice were transfected with a set of 11 transcription factors highly expressed in NPCs. Twenty-four days after transgene induction, Sox2-EGFP(+) colonies emerged that expressed NPC-specific genes and differentiated into neuronal and astrocytic cells. Using stepwise elimination, it was found that Sox2 and FoxG1 are capable of generating clonal self-renewing, bipotent induced NPCs that gave rise to astrocytes and functional neurons. When the Pou and Homeobox domain-containing transcription factor Brn2 to was added Sox2 and FoxG1, it was possible to induce tripotent NPCs that could be differentiated not only into neurons and astrocytes but also into oligodendrocytes. The transcription factors FoxG1 and Brn2 alone also were capable of inducing NPC-like cells; however, these cells generated less mature neurons, although they did produce astrocytes and even oligodendrocytes capable of integration into dysmyelinated Shiverer brain. These data demonstrate that direct lineage reprogramming using target cell-type-specific transcription factors can be used to induce NPC-like cells that potentially could be used for autologous cell transplantation-based therapies in the brain or spinal cord (Lujan, 2012).

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nubbin/POU domain protein 1: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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