Ubiquitous Oct-1 Research Articles

Oct1 cooperates with the Smad family of transcription factors to promote mesodermal lineage specification.

The transition between pluripotent and tissue-specific states is a key aspect of development. Understanding the pathways driving these transitions will facilitate the engineering of properly differentiated cells for experimental and therapeutic uses. Here, we showed that during mesoderm differentiation, the transcription factor Oct1 activated developmental lineage-appropriate genes that were silent in pluripotent cells. Using mouse embryonic stem cells (ESCs) with an inducible knockout of Oct1, we showed that Oct1 deficiency resulted in poor induction of mesoderm-specific genes, leading to impaired mesodermal and terminal muscle differentiation. Oct1-deficient cells exhibited poor temporal coordination of the induction of lineage-specific genes and showed inappropriate developmental lineage branching, resulting in poorly differentiated cell states retaining epithelial characteristics. In ESCs, Oct1 localized with the pluripotency factor Oct4 at mesoderm-associated genes and remained bound to those loci during differentiation after the dissociation of Oct4. Binding events for Oct1 overlapped with those for the histone lysine demethylase Utx, and an interaction between Oct1 and Utx suggested that these two proteins cooperate to activate gene expression. The specificity of the ubiquitous Oct1 for the induction of mesodermal genes could be partially explained by the frequent coexistence of Smad and Oct binding sites at mesoderm-specific genes and the cooperative stimulation of mesodermal gene transcription by Oct1 and Smad3. Together, these results identify Oct1 as a key mediator of mesoderm lineage-specific gene induction.

Transcriptional Coactivator BOB1 (OBF1, OCA-B) in Autoimmune Diseases

Despite significant efforts in biomedicine for several decades, autoimmune diseases continue to remain largely incurable and, moreover, poorly understood in terms of the molecular mechanisms underlying their onset and progression. It is generally accepted that autoimmune pathologies result from a malfunction of the adaptive immune system in genetically susceptible individuals leading to the appearance of autoreactive B- and T-lymphocytes. However, the exact molecular pathways that drive the activation of autoreactive lymphocytes, leading to the amplification and perpetuation of self-directed immune responses are largely unknown. A number of experimental data accumulated over the past few years indicate a key role of BOB1, namely its imbalanced expression, in the onset of autoreactive lymphocytes. It has been postulated that the coactivator BOB1 affects transcription and local chromatin state indirectly, via selective interaction with DNA-binding POU-domain transcription factors – ubiquitous OCT1 and B-cell-specific OCT2, stabilises the binding of the OCT factors to DNA. The review lists the latest evidences of an important role of BOB1 in pathogenesis of autoimmune diseases and positions this protein as a promising target in the treatment of these diseases.

Involvement of the ubiquitous Oct-1 transcription factor in hormonal induction of β-casein gene expression

Transcription of the milk protein beta-casein gene is induced by the lactogenic hormones Prl (prolactin) and glucocorticoids. Multiple transcription factors involved in this induction have been identified, including the STAT5 (signal transducer and activator of transcription 5) and the GR (glucocorticoid receptor). Our previous studies have identified a binding site for the ubiquitous Oct-1 (octamer-binding transcription factor 1) protein in the lactogenic hormonal regulatory region of the mouse beta-casein promoter. In the present study, we report that Oct-1 is indeed expressed and binds to the beta-casein promoter in mammary epithelial cells. Oct-1 activates hormonally induced beta-casein promoter activity in a dose-dependent manner. Hormonal induction of promoter activity was decreased not only by mutating the Oct-1-binding site from ATTAGCAT to GCTAGCAT, which abolishes Oct-1 binding (50% decrease, P<0.01), but also by changing the site to the consensus Oct-1-binding motif ATTTGCAT (40% decrease, P<0.01). Reversing the Oct-1-binding site reduced hormonal induction by 70% (P<0.01), showing that orientation of Oct-1 binding is also critical in hormonal action. In transient transfection experiments, Oct-1 collaboratively transactivated the beta-casein gene promoter with STAT5 and/or GR in the presence of Prl receptor in cells treated with the lactogenic hormones. The C-terminus of Oct-1 was not essential to its function. The results of the present study provide biochemical evidence that the ubiquitous Oct-1 transcription factor may be involved in hormonally regulated, tissue-specific beta-casein gene expression.

Control of myelination in Schwann cells: a Krox20 cis‐regulatory element integrates Oct6, Brn2 and Sox10 activities

Myelination in Schwann cells is governed by several transcription factors, including the POU proteins Oct6 and Brn2, the high mobility group protein Sox10 and the zinc-finger protein Krox20. How the function of these factors is integrated in the control of myelination has not been established. Previously, we identified an enhancer element controlling Krox20 expression throughout myelination in Schwann cells. In this paper, cell culture experiments were combined with transgenesis to identify transcription factors acting directly upstream of Krox20. The results show that during the promyelin-myelin transition, Krox20 expression is directly activated by Oct6 and Brn2 acting on this enhancer. In addition, the enhancer-dependent synergism between these POU proteins and Sox10 suggests that Krox20 expression requires this combination of factors. These results resolve previous controversy concerning the mechanism of action of Oct6 and Brn2 during myelination and provide an explanation for myelin deficiencies in Waardenberg-Hirschsprung disease patients whereby Sox10 mutations could lead to a loss of Krox20 expression.

Functional characterization of the sea urchin sns chromatin insulator in erythroid cells

Chromatin insulators are regulatory elements that determine domains of genetic functions. We have previously described the characterization of a 265 bp insulator element, termed sns, localized at the 3′ end of the early histone H2A gene of the sea urchin Paracentrotus lividus. This sequence contains three cis-acting elements (Box A, Box B, and Box C + T) all needed for the enhancer-blocking activity in both sea urchin and human cells. The goal of this study was to further characterize the sea urchin sns insulator in the erythroid environment. We employed colony assays in human (K562) and mouse (MEL) erythroid cell lines. We tested the capability of sns to interfere with the communication between the 5′HS2 enhancer of the human β-globin LCR and the γ-globin promoter. We found that the sns sequence displays directional enhancer-blocking activity. By the use of antibodies against known DNA binding proteins, in electrophoretic mobility shift assays, we demonstrated the binding of the erythroid-specific GATA-1 and the ubiquitous Oct-1 and Sp1 transcription factors. These factors bind to Box A, Box B, and Box C + T, respectively, in both K562 and MEL nuclear extracts. These results may have significant implications for the conservation of insulator function in evolutionary distant organisms and may prove to be of practical benefit in gene transfer applications for erythroid disorders such as hemoglobinopathies and thalassemias.

Specific Activation of Human Interleukin-5 Depends on de Novo Synthesis of an AP-1 Complex

It is clear from the biology of eosinophilia that a specific regulatory mechanism must exist. Because interleukin-5 (IL5) is the key regulatory cytokine, it follows that a gene-specific control of IL5 expression must exist that differs even from closely related cytokines such as IL4. Two features of IL5 induction make it unique compared with other cytokines; first, induction by cyclic adenosine monophosphate (cAMP), which inhibits other T-cell-derived cytokines, and second, sensitivity to protein synthesis inhibitors, which have no effect on other cytokines. This study has utilized the activation of different transcription factors by different stimuli in a human T-cell line to study the role of conserved lymphokine element 0 (CLE0) in the specific induction of IL5. In unstimulated cells the ubiquitous Oct-1 binds to CLE0. Stimulation induces de novo synthesis of the AP-1 members JunD and Fra-2, which bind to CLE0. The amount of IL5 produced correlates with the production of the AP-1 complex, suggesting a key role in IL5 expression. The formation of the AP-1 complex is essential, but the rate-limiting step is the synthesis of AP-1, especially Fra-2. This provides an explanation for the sensitivity of IL5 to protein synthesis inhibitors and a mechanism for the specific induction of IL5 compared with other cytokines.

An octamer element functions as a regulatory element in the differentiation-responsive CD11c integrin gene promoter: OCT-2 inducibility during myelomonocytic differentiation.

The integrin CD11c/CD18 mediates leukocyte adhesion to endothelium and other cell types and is a receptor for LPS, iC3b, and fibrinogen. CD11c expression is restricted to myeloid and activated B cells, is regulated during leukocyte differentiation, and constitutes a diagnostic tool for hairy cell leukemia. Mapping of in vivo DNA-protein interactions in the CD11c proximal promoter revealed three adjacent myeloid-specific interactions, one of which lies on an octamer consensus sequence, ATTT GCAT (Oct185). Oct185 disruption increased the CD11c promoter activity while decreasing its myeloid differentiation responsiveness, indicating that Oct185 contributes to the activity of the CD11c promoter and suggesting that Oct185 is a negative regulatory element whose function changes during myeloid differentiation. Oct185 is recognized by the ubiquitous Oct-1 factor in all cell lineages and by Oct-2 in B lymphoid lineage cells. Unexpectedly, Oct-2 binding to Oct185 was induced de novo upon monocytic differentiation of U937 and HL-60 cells but not during HL-60 granulocytic differentiation, as determined by electrophoretic mobility shift assays and immunochemical studies, and Oct-2 complexes were also observed in cultured adherent monocytes. Western blotting showed that the pattern of Oct-2 isoforms in myeloid cells is similar to that seen in B cells. The Oct-2 up-regulated expression in differentiating myeloid cells and its binding to the Oct185 negative regulatory element suggests its involvement in the differentiation-regulated activity of the CD11c promoter and might represent an important parameter for the myeloid- and B cell-restricted expression of the CD11c/CD18 integrin and other molecules with similar patterns of expression.

Cellular factors controlling the activity of woodchuck hepatitis virus enhancer II.

Woodchuck hepatitis virus (WHV) efficiently induces hepatocellular carcinoma in chronically infected hosts. A key step in hepatocarcinogenesis by WHV is insertional activation of the cellular N-myc gene by integrated viral DNA. WHV enhancer II (En II) is the major cis-acting element involved in this activation. Here we characterize this viral enhancer element and define the cellular factors involved in its activity. WHV En II activity is strongly liver specific and maps to an 88-nucleotide DNA segment (nucleotides 1772 to 1859) located 5' to the pregenomic RNA start site. Genetic analyses and electrophoretic mobility shift assays indicate that the enhancer contains three subregions important to its activity. The core elements of the enhancer are recognition sites for the liver-enriched factors HNF1 and HNF4; together, these signals account for the bulk of En II activity as well as its strong liver specificity. Multimerization of either recognition site produced strong activity even in the absence of other En II sequences. 5' to these elements is a binding site for the ubiquitous Oct-1 transcription factor, which further augments enhancer activity ca. twofold.

The B cell coactivator Bob1 shows DNA sequence-dependent complex formation with Oct-1/Oct-2 factors, leading to differential promoter activation.

We have shown previously that both octamer binding transcription factors, namely the ubiquitous Oct-1 and the B cell-specific Oct-2A protein, can be enhanced in transcriptional activity by their association with the B cell-specific coactivator protein Bob1, also called OBF-1 or OCA-B. Here we study the structural requirements for ternary complex formation of DNA-Oct-Bob1 and coactivation function of Bob1. In analogy to DNA-bound transcription factors, Bob1 has a modular structure that includes an interaction domain (amino acids 1-65) and a C-terminal domain (amino acids 65-256), both important for transcriptional activation. A mutational analysis has resolved a region of seven amino acids (amino acids 26-32) in the N-terminus of Bob1 that are important for contacting the DNA binding POU domain of Oct-1 or Oct-2. In contrast to the viral coactivator VP16 (vmw65), which interacts with Oct-1 via the POU homeosubdomain, Bob1 association with Oct factors requires residues located in the POU-specific subdomain. Because the same residues are also involved in DNA recognition, we surmised that this association would affect the DNA binding specificity of the Oct-Bob1 complex compared with free Oct factors. While Oct-1 or Oct-2 bind to a large variety of octamer sequences, Bob1 ternary complex formation is indeed highly selective and occurs only in a subset of these sequences, leading to the differential coactivation of octamer-containing promoters. The results uncover a new level in selectivity that furthers our understanding in the regulation of cell type-specific gene expression.

The POU domains of the Oct1 and Oct2 transcription factors mediate specific interaction with TBP.

We had previously shown that the ubiquitous Oct1 and the lymphoid-specific Oct2 transcription factors stimulate transcription at the level of stable preinitiation complex formation. We have therefore investigated whether the octamer binding proteins might physically interact with TBP, the TATA box binding protein component of the TFIID factor. By using several different experimental systems we show that TBP 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. We show that 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 resulted in a synergistic activation of an octamer motif containing promoter.

Primary Brain Tumors Differ in Their Expression of Octamer Deoxyribonucleic Acid-binding Transcription Factors from Long-Term Cultured Glioma Cell Lines

Nervous system-specific transcription factors that bind to the octameric deoxyribonucleic acid sequence motif ATGCAAAT (or ATTTGCAT) are known as N-Oct proteins. Neurons and glia contain the ubiquitous Oct-1 protein and four polypeptide complexes termed N-Oct-2, N-Oct-3, N-Oct-4, and N-Oct-5. Previously, we showed that N-Oct proteins are differentially expressed by human neuroblastoma and glioblastoma cell lines in vitro. We have now extended this work to freshly isolated human primary and metastatic brain tumors. Contrary to brain tumor cell lines, of the five astrocytomas and three glioblastomas analyzed, all but two tumors displayed the complete N-Oct protein profile, irrespective of histopathological tumor grade. Two astrocytomas were negative for N-Oct-4. Ten of 13 ependymomas exhibited N-Oct-2, N-Oct-3, and N-Oct-4 but lacked the N-Oct-5 complex. In contrast, brain metastases of two patients with extracerebral carcinomas contained only Oct-1, and cerebral metastases from two cases of B cell lymphomas showed Oct-1 and Oct-2 complexes, the characteristic Oct protein pattern of B lymphocytes. Thus, metastatic carcinoma and lymphoma expressed a non-nervous system phenotype of Oct proteins.

Regulation of the calcitonin/calcitonin gene-related peptide gene by cell-specific synergy between helix-loop-helix and octamer-binding transcription factors

The calcitonin/calcitonin gene-related peptide (CGRP) gene is transcribed in thyroid C-cells and a subset of neurons. We have localized sequences required for cell-specific enhancement of calcitonin/CGRP transcription in rat thyroid C-cell lines. An 18-base pair element approximately 1 kilobase pair upstream of the transcriptional start site stimulated expression of a luciferase reporter gene 50-fold in 44-2C C-cells. There was less than 2-fold stimulation in HeLa and Rat-1 cells, which do not express the endogenous calcitonin/CGRP gene. The enhancer contains potential binding sites for helix-loop-helix (HLH) and octamer transcription factors based on sequence homologies. The functional significance of these sites was shown by point mutations in the HLH and octamer motifs and by separation of the two motifs, all of which decreased enhancer activity greater than 10-fold. The involvement of HLH proteins was further shown by co-expression of the mammalian achaete-scute homologue-1 HLH protein, which activated the enhancer severalfold in HeLa cells. Electrophoretic mobility shift analyses revealed several DNA-protein complexes containing HLH and octamer-binding proteins. One octamer-binding complex (OB1) most likely contains the ubiquitous Oct-1 protein, whereas a second complex (OB2) was cell-specific. In contrast to OB1, OB2 had lower affinity for a consensus octamer motif, and its DNA binding was not affected by addition of antiserum that recognizes Oct-1 and Oct-2 proteins. In addition, we observed a large complex that appears to contain both an HLH protein and OB2. These results demonstrate that calcitonin/CGRP enhancer activity is controlled by a cell-specific synergistic activation involving HLH and octamer-binding factors.

A Variant Octamer Motif in a Xenopus H2B Histone Gene Promoter Is Not Required for Transcription in Frog Oocytes

Xenopus oocytes, arrested in G2 before the first meiotic division, accumulate histone mRNA and protein in the absence of chromosomal DNA replication and therefore represent an attractive biological system in which to examine histone gene expression uncoupled from the cell cycle. Previous studies have shown that sequences necessary for maximal levels of transcription in oocytes are present within 200 bp at the 5' end of the transcription initiation site for genes encoding each of the five major Xenopus histone classes. We have defined by site-directed mutagenesis individual regulatory sequences and characterized DNA-binding proteins required for histone H2B gene transcription in injected oocytes. The Xenopus H2B gene has a relatively simple promoter containing several transcriptional regulatory elements, including TFIID, CBP, and ATF/CREB binding sites, required for maximal transcription. A sequence (CTTTACAT) in the H2B promoter resembling the conserved octamer motif (ATTTGCAT), the target for cell-cycle regulation of a human H2B gene, is not required for transcription in oocytes. Nonetheless, substitution of a consensus octamer motif for the variant octamer element activates H2B transcription. Oocyte factors, presumably including the ubiquitous Oct-1 factor, specifically bind to the consensus octamer motif but not to the variant sequence. Our results demonstrate that a transcriptional regulatory element involved in lymphoid-specific expression of immunoglobulin genes and in S-phase-specific activation of mammalian H2B histone genes can activate transcription in nondividing amphibian oocytes.

A variant octamer motif in a Xenopus H2B histone gene promoter is not required for transcription in frog oocytes.

Xenopus oocytes, arrested in G2 before the first meiotic division, accumulate histone mRNA and protein in the absence of chromosomal DNA replication and therefore represent an attractive biological system in which to examine histone gene expression uncoupled from the cell cycle. Previous studies have shown that sequences necessary for maximal levels of transcription in oocytes are present within 200 bp at the 5' end of the transcription initiation site for genes encoding each of the five major Xenopus histone classes. We have defined by site-directed mutagenesis individual regulatory sequences and characterized DNA-binding proteins required for histone H2B gene transcription in injected oocytes. The Xenopus H2B gene has a relatively simple promoter containing several transcriptional regulatory elements, including TFIID, CBP, and ATF/CREB binding sites, required for maximal transcription. A sequence (CTTTACAT) in the H2B promoter resembling the conserved octamer motif (ATTTGCAT), the target for cell-cycle regulation of a human H2B gene, is not required for transcription in oocytes. Nonetheless, substitution of a consensus octamer motif for the variant octamer element activates H2B transcription. Oocyte factors, presumably including the ubiquitous Oct-1 factor, specifically bind to the consensus octamer motif but not to the variant sequence. Our results demonstrate that a transcriptional regulatory element involved in lymphoid-specific expression of immunoglobulin genes and in S-phase-specific activation of mammalian H2B histone genes can activate transcription in nondividing amphibian oocytes.

Promoters with the octamer DNA motif (ATGCAAAT) can be ubiquitous or cell type-specific depending on binding affinity of the octamer site and Oct-factor concentration

Immunoglobulin (Ig) gene promoters contain the octamer sequence motif ATGCAAAT which is recognized by cellular transcription factors (Oct factors). Besides the ubiquitous Oct-1 factor, there is also a group of related factors (Oct-2 factors) encoded by a separate gene. The Oct-2 gene is regulated in a cell-type specific manner, and the protein is present in large amounts in B lymphocytes. We have previously shown that simple composite promoters of an octamer/TATA box type are poorly active in non-B cells but are strongly responsive to ectopic expression of Oct-2A factor, a major representative of the lymphocyte Oct-2 factors. In the present study we have tested the activity of a number of composite promoters and natural Ig promoters, and their response to Oct-1 and Oct-2 factors. Unexpectedly, we find that octamer/TATA promoters with a high affinity octamer site direct ubiquitous expression. By contrast, promoter constructions that behave in a B cell-specific manner tend to have a weak octamer binding site. These promoters are responsive to ectopic expression of additional Oct-factor, irrespective of whether it is Oct-1 or Oct-2. Using natural Ig promoters rather than composite promoters, we find that an IgH promoter is well transcribed in non-B cells via the ubiquitous Oct-1 factor, while Ig kappa and Ig lambda light chain promoters require additional Oct factor for maximal expression. It seems therefore likely that during B cell differentiation, Ig heavy chain promoters can be activated by Oct-1, before the appearance of Oct-2 factors. Oct-2 factors then would serve to boost the expression from Ig light chain promoters, which are known to be activated only after successful heavy chain gene rearrangement.

Differential transcriptional activation by Oct-1 and Oct-2: Interdependent activation domains induce Oct-2 phosphorylation

The ubiquitous Oct-1 and lymphoid Oct-2 POU homeodomain transcription factors bind to the same DNA sequence but differ in their activation potential. Oct-2 is a positive, negative, or neutral regulator of β-globin transcription depending on the position and sequence of multimerized binding sites. To activate transcription, Oct-2 relies on two interdependent nonacidic domains, an N-terminal glutamine-rich region and a C-terminal serine-, threonine-, and proline-rich region. Oct-1 also contains a functional glutamine-rich region but fails to activate β-globin transcription in our assay because the Oct-1 C-terminus is inactive, indicating that differential activation by Oct-1 and Oct-2 is determined by the combination of multiple activation domains. Oct-2 displays a unique phosphorylation pattern that is absent from molecules lacking one or the other activation domain, suggesting the activation domains have a role in inducing protein phosphorylation.

Astrocytes and glioblastoma cells express novel octamer-DNA binding proteins distinct from the ubiquitous Oct-1 and B cell type Oct-2 proteins

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

The octamer-binding proteins form multi-protein--DNA complexes with the HSV alpha TIF regulatory protein.

The herpes simplex virus transactivator, alpha TIF, stimulates transcription of the alpha/immediate early genes via a cis-acting site containing an octamer element and a conserved flanking sequence. The alpha TIF protein, produced in a baculovirus expression system, nucleates the formation of at least two DNA--protein complexes on this regulatory element. Both of these complexes contain the ubiquitous Oct-1 protein, whose POU domain alone is sufficient to allow assembly of the alpha TIF-dependent complexes. A second member of the POU domain family, the lymphoid specific Oct-2 protein, can also be assembled into similar complexes at high concentrations of alpha TIF protein. These complexes contain at least two cellular proteins in addition to Oct-1. One of these proteins is present in both insect and HeLa cells and probably recognizes sequences in the cis element. The second cellular protein, only present in HeLa cells, probably binds by protein-protein interactions.