Octamer-binding Proteins Research Articles

Identification and purification of a human lymphoid-specific octamer-binding protein (OTF-2) that activates transcription of an immunoglobulin promoter in vitro

The octamer sequence 5′-ATGCAAAT, in either orientation, serves as an upstream element in a variety of promoters and also occurs as a modular enhancer element. It is of particular interest in immunoglobulin genes since it is found in the upstream regions of all heavy and light chain promoters and in the heavy chain enhancer, both of which are known to be necessary for cell-specific expression. We report here the chromatographic separation of ubiquitous and B cell-specific octamer-binding proteins. The B cell factor was purified to homogenelty using affinity chromatography and consists of three peptides of 62, 61, and 58.5 +/− 1.5 kd. Each of the polypeptides was renatured after SDS-PAGE and shown to bind to the octamer sequence. The specific DNA binding activity of the pure B cell-specific factor was indistinguishable from that of the affinity-purified ubiquitous factor. This B cell-specific octamer-binding factor, in pure form, activated transcription from a κ light chain promoter in vitro, thus demonstrating that it is indeed a B cell-specific transcription factor for this gene. In addition to the ubiquitous and B cell-specific octamer-binding factors, we identified several additional proteins, one of which is B cell-specific, that interact with the κ promoter.

In vitro binding of cell-specific and ubiquitous nuclear proteins to the octamer motif of the SV40 enhancer and related motifs present in other promoters and enhancers.

We have used the gel retardation and DNase I footprinting assays to investigate the in vitro binding of nuclear proteins to the octamer motif present in domain A of the SV40 enhancer and in other enhancer and promoter elements. Three apparently cell-specific (oct-B1A, oct-B1B and oct-B2) and one ubiquitous (oct-B3) proteins were detected in various lymphoid and non-lymphoid cell extracts. We show that the previously described 'ubiquitous' NF-A1 factor may correspond in fact to two proteins, oct-B1A in HeLa cells and oct-B1B in lymphoid cells. Interestingly, the HeLa cell protein oct-B1A formed a complex with the SV40 octamer, which could be detected in gel retardation, but not in DNase I footprinting assays. This absence of protection from DNase I digestion correlates with the inactivity of the SV40 octamer in HeLa cells in vivo. We have also found that the in vitro interaction between the SV40 octamer motif and the lymphoid cell-specific protein oct-B2 was negatively modulated by a component present in the nuclear extracts from several lymphoid cell lines. The interactions between the multiple octamer-binding proteins and the related octamer motifs present in other promoter and enhancer elements were systematically compared and the possible role of these proteins in the control of transcription is discussed.