Edgar Schreiber

Identification of a novel lymphoid specific octamer binding protein (OTF-2B) by proteolytic clipping bandshift assay (PCBA)

The octamer sequence ATGCAAAT is found in the promoters of immunoglobulin (Ig) heavy and light chain genes and in the heavy chain enhancer and is a major determinant of the cell type specific expression of Ig genes in B cells. An apparent paradox is that the same sequence serves as an upstream promoter or enhancer element in a variety of housekeeping genes such as the histone H2B and U snRNA genes. The differential usage of this regulatory sequence motif is thought to be mediated by different species of octamer binding proteins. One species of 100 kd, designated OTF‐1, is present in all cell types and may exert its activating function only when it can interact with additional adjacent transcription factors. The lymphoid cell specific octamer binding protein of 60 kd (OTF‐2A) specifically stimulates Ig promoters which consist essentially of a TATA‐box and an octamer sequence upstream of it. Here we present evidence for yet another B cell specific octamer binding protein of 75 kd (OTF‐2B). From several findings, including the absence of OTF‐2B (but not OTF‐2A) from a lymphocyte line that cannot respond to the IgH enhancer, we propose a role of the novel octamer factor in the long range activation by the IgH enhancer. We have used the proteolytic clipping bandshift assay (PCBA) technique to distinguish the three different forms found in B cells. This analysis indicates that the 75 kd‐species OTF‐2B is closely related to the 60 kd species OTF‐2A.

Octamer transcription factors bind to two different sequence motifs of the immunoglobulin heavy chain promoter.

All promoters of immunoglobulin heavy chain genes contain three conserved sequence motifs: a heptamer motif CTCATGA, an octamer motif ATGCAAAT, and a TATA box. We show that, despite their different sequences, both the heptamer and the octamer motif are bound by the same octamer transcription factors (Oct factors, also referred to as OTFs), namely the lymphoid‐specific proteins Oct‐2A and Oct‐2B, as well as the ubiquitous protein Oct‐1. Even though binding to the octamer motif is stronger, a single heptamer motif can bind Oct proteins and mediate transcriptional activity in lymphoid cells. Furthermore, factor binding to the octamer motif facilitates binding to the nearby heptamer motif. We propose that the heptamer element plays a role early in B‐cell differentiation to ensure that the heavy chain promoters are transcriptionally activated before the light chain promoters, which do not contain the heptamer motif.

The protein CDP, but not CP1, footprints on the CCAAT region of the γ-globin gene in unfractionated B-cell extracts

We have identified, by DNase I footprinting, six different factors that interact with the promoter of the human A gamma-globin gene in nuclear extracts of the B-cell line BJA-B. Among them is the vertebrate homologue of the sea-urchin CCAAT displacement protein (CDP) which footprints over the entire duplicated CCAAT region. The CCAAT-binding factor CP1, a potential activator of the gamma-globin promoter, is able to bind to its proximal recognition sequence only once it has been partially enriched and separated from CDP. The factor CDP has an apparent molecular mass of 200 kDa and differs from CP1 by its footprint pattern and competition behavior.

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.

N-Oct 5 is generated by in vitro proteolysis of the neural POU-domain protein N-Oct 3

The neural POU-domain proteins N-Oct 3 and N-Oct 5 were first identified in electrophoretic mobility retardation assays through their ability to bind to the octamer sequence ATGCAAAT. These two N-Oct factors are detected in extracts from tumor-derived and normal neural cells. They are present differentially, however, in extracts from melanocytes and melanoma cells: N-Oct 3 is present in extracts from both melanocytes and melanoma cells, whereas N-Oct 5 is more evident in extracts from metastatic melanoma cells. We show here that a cDNA encoding N-Oct 3 directs synthesis of both the N-Oct 3 and N-Oct 5 proteins and that the N-Oct 5 protein in neural and melanoma-cell extracts is also related to N-Oct 3. N-Oct 5, however, is apparently not expressed in vivo: It is not detected if cells are rapidly lysed in SDS or if extracts are prepared with a cocktail of protease inhibitors that includes the serine-protease inhibitor 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF). These data suggest that N-Oct 5 is a specific in vitro proteolytic cleavage product of N-Oct 3 and is not directly related to melanocyte malignancy.

Long-range activation of transcription by SV40 enhancer is affected by “inhibitory” or “permissive” DNA sequences between enhancer and promoter

The transcriptional enhancer effect is used in many, if not all, organisms for remote control of gene transcription. An enhancer DNA can dramatically stimulate transcription of a linked gene from positions either 5′ or 3′ to the gene. Both the proximal promoter and the distal enhancer sequences are binding sites for transcription factors. Interaction between promoter and enhancer is mediated by these factors, presumably via looping out of the intervening DNA. Here we report that the extent of remote activation by an enhancer depends on characteristics of that intervening DNA. Using Beta-globin and SV40 T-antigen test genes, we show that the effect of an SV40 enhancer is transmitted to the responsive promoter, with little or no loss of efficiency, through certain segments of mammalian DNA derived from rabbit β-globin or mouse α-globin gene regions. By contrast, a strong reduction of enhancer activity is observed with certain spacer segments of prokaryotic DNA (from plasmid pBR322 or phage lambda) or sequences of high (G+C) content from eukaryotic genes. We have analyzed more closely sequences that are more or less permissive for transmission of the transcriptional enhancer effect. It appears that these permissive sequences generally have a high (A+T) content and notably a very low abundance of CpG dinucleotides. By contrast, (G+C)-rich DNA segments with high local densities of CpG were the most deleterious for long-range enhancer action. We note that the latter sequence composition is typical for “CpG islands” of many mammalian genes, including housekeeping genes and the human α-globin gene. This may be related to the finding that promoters of most cell type-specific genes, whose activity depends on a strong enhancer, do not contain CpG islands. Most likely, the spacer DNAs of typical cell type-specific genes have evolved to allow maximal transmission of the enhancer effect.