2.4 5.2 Bacteria! Expression and Purification of His-Tagged Proteins
Chapter 3: The Role of Oct-1 in the Regulation of the Beta-Interferon
Promoter
3.1. Oct-1 Binds to Multiple Regions within the
Beta-lnterferon Promoter
In early EMSA studies in our laboratory, a binding activity was identified that was able to form a com plex on a probe -55/-40, which is derived from the NRD I region (K.V.Visvanathan, and S. Goodboum, unpublished). In competition EMSA experiments, it further became apparent that a complex possessing an identical binding specificity also formed on probes from the NRD II (-108/-95), and TATA box (-33/-20) regions of the human IFN-6 promoter. It was surprising to observe that these probes are not strongly homologous in sequence. A suggestion about the identity of the DNA binding component of the complex was provided by an EMSA analysis using TATA box probes that contained single point mutations. An observation that the mutation -28 A>G, which creates a stronger binding site (ATGTAAAT) for the complex than the wild type TATA box (ATATAAAT), also creates a more complete match with the consensus site (ATGCAAAT; see below) for the previously identified transcription factor Oct-1, prompted us to test the possibility that this complex indeed contains Oct-1.
To confirm that the three probes derived from the IFN-6 prom oter (NRD II, NRD I, TATA) can interact with Oct-1, an antibody-supershift EMSA was performed. Aliquots (Ipl) of an O ct-1-specific polyclonal antiserum (a gift from Dr. P .O ’Hare) or a preimmune serum were incubated with the nuclear extract samples prepared from HeLa cells prior to the addition of a particular radioactively labelled probe, and running the samples on an EMSA gel. While the preimmune sera had no effect on the binding of the activity on any of the IFN-6 probes, the O ct-1-specific antisera raised against the Oct-1 DNA binding domain (the POU domain) completely abolished the complex formation (figure 3.1.). As a positive control, the antiserum against Oct-1 also abolished com plex form ation on a probe containing a consensus octamer binding site (see below). It was thus concluded that the binding activity under study contains, or is indistinguishable from, Oct-1. To further verify this, recombinant Oct-1 was translated in wheat germ extract using mRNA transcribed in vitro from a plasmid containing Oct-1 cDNA under a T7 bacteriophage promoter. When this recombinant product was allowed to associate with the Oct-1 binding site probes, a complex of approximately the same size as the endogenous activity was detected (data not shown), further confirming the identity of the binding factor.
Figure 3.1. A complex forming on multiple probes from the IFN-6 promoter reacts with an antiserum raised against the Oct-1 POU-domain.
An EMSA analysis of the binding activities present in HeLacell nuclear extracts on three probes derived from the IFN-6 promoter [NRD II (-108/-95), TATA (-33/-20), NRD I (- 55/-40)] as well as on a probe containing a perfect octamer m otif (“octa”). The lanes in the panel on the right are from the same autoradiographic exposure of the same gel, while the figure on the left is from a different experiment. The specificity of the anti-POU antiserum for Oct-1 is evidenced by its lack of reactivity with the two higher mobility complexes (U ni and Un2, see chapter 4) that form on the NRD n probe.
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Oct-1 (figure 3.2.) belongs to a family of proteins, which share a highly homologous DNA-binding motif, referred to as the POU-domain (for mammalian proteins Pit-1, Qct-1 and -2, and a nematode protein involved in neural cell lineage determination: U ne-86), according to the proteins in which it was first discovered (Herr et al 1988). Over 20 members o f the POU family have been identified in various organisms from Drosophila to mammals (reviewed in Ruvkun and Finney 1991, Verrijzer and van der Vliet 1993). Many POU proteins are expressed in a tissue-restricted manner, for example, Pit-1 is expressed in the pituitary gland and regulates the transcription of prolactin and growth hormone genes (Nelson et al 1988). In contrast, Oct-1 has a widespread pattern of expression, and has been implicated as having a role in transcription of housekeeping genes, such as small nuclear RNA genes and the histone H2B gene (reviewed in Kemler and Schaffner 1990). The consensus DNA recognition site for Oct-1, as well as for the tissue-restricted members ^ the Oct-protein family, is ATGCAAAT, known as the octamer motif. A selection for an optimal O ct-1 binding site also identified the ATGCAAAT motif, however it also revealed an equal preference for adenine or thymine in position 5 (Verrijzer et al 1992a). Since adenine appears tormore common at this position in naturally occurring octamer sites in cellular prom oters, there may be further constraints for the in vivo functioning of the octamer element.
As judged by an EMSA assay with the four different probes, the relative binding affinity of the O ct-1-containing complex for the three different binding sites in the IFN-6 promoter and for a perfect octamer m otif (octa) is [octa > NRD II > TATA > NRD I] (figure 3.3.). This correlates very well with the degree of sequence similarity between the octamer consensus site and a particular binding site in the IFN-6 promoter. There is no sequence within the NRD I probe that resembles the octamer motif sufficiently to directly deduce the actual O ct-1-DNA contact site; the alignment of the NRD I region with the stronger Oct-1 binding sites in figure 3.3. is derived from an analysis on the effect of point mutations across the NRD I region on the specific binding affinity o f Oct-1 (S.Goodbourn, pers.comm). In any case, it is apparent from this that Oct-1 is able to bind to very degenerate octamer motifs, as previously noted by others (Baumruker et al 1988).
The Oct-1-containing complex appears to be the only specific octamer binding complex we can detect in HeLa cells (see, for example, figures 3.3., 3.9. and 3.10.).