RFX1, a Single DNA-binding Protein with a Split Dimerization Domain, Generates Alternative Complexes

Abstract

The transcription of various viral and cellular genes is regulated by palindromic and nonpalindromic DNA sites resembling the EP element of the hepatitis B virus enhancer, which generate similar DNA-protein complexes. The upper EP complex contains homodimers of the transcription regulator RFX1. We show that RFX1 possesses a split, extended dimerization domain composed of several evolutionarily conserved boxes, one of which was previously shown to mediate dimerization. Such an unusually long and complex dimerization domain could potentially serve for generating multiple complexes. In addition to the previously characterized complex, RFX1 generated a novel DNA-protein complex of extremely low mobility, formed only with palindromic DNA sites. Different deletions within the dimerization domain altered the relative abundance of the two complexes, suggesting an interplay between them. Formation of the low mobility complex correlated with transcriptional repression, in that both activities were mediated by several portions of the conserved region. Our results propose a mechanism by which the extended dimerization domain mediates the formation of alternative homodimeric complexes, which differ in the nature of the intersubunit interaction. By participating in different types of interactions, this domain may regulate the relative abundance of the different complexes, thus affecting transcriptional activity.EP is a regulatory element found in several viral enhancers, such as those of the hepatitis B virus (HBV), 1 polyomavirus, and equine infectious anemia virus (1-5). EP-homologous sites are also present in cellular genes, including the MIF-1 binding site (MIE) in the first intron of the human c-myc gene (6, 7), the X box of MHC class II promoters (8, 9), the ␣ element in the mouse rpL30 ribosomal protein gene promoter (10, 11), and a binding site in the proliferating cell nuclear antigen promoter (12, 13). These different sites can be divided into two major groups. One group includes palindromic or partially palindromic sites, such as the EP elements of the HBV and polyomavirus enhancers. Members of the other group, e.g. the MHC promoter X box, are nonpalindromic and contain only a single EP-homologous half-site. While the EP elements of the HBV and polyomavirus enhancers, as well as the X box and rpL30␣ element, are positively acting sites within their natural DNA context (3, 4, 8 -10, 14 -17), a multimerized EP site cannot stimulate transcription significantly (4). Moreover, EP and MIE multimers can silence transcription (Refs. 18 -20), 2 demonstrating that the activity of EP is context-dependent.The EP element is bound by a ubiquitous nuclear protein complex, generating a typical pattern of several slowly migrating bands in gel shift essays (1-4). These EP complexes were independently characterized by several groups studying seemingly unrelated DNA-binding factors, which were later found to represent the same nuclear complex, referred to as EP, EF-C, MDBP, MIF, or 7,8,19,[21][22][23]. The EP complex contains homo-and...

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