Interaction of EF-C/RFX-1 with the inverted repeat of viral enhancer regions is required for transactivation.
The hepatitis B virus (HBV) and polyomavirus (Py) enhancer regions contain multiple cis-acting elements that contribute to enhancer activity. The EF-C binding site was previously shown to be an important functional component of each enhancer region. EF-C is a ubiquitous binding activity that interacts with an inverted repeat sequence in the HBV and Py enhancer regions. Although the EF-C binding site is required for optimal enhancer function, the EF-C site does not possess intrinsic enhancer activity when assayed in the absence of flanking elements. With both the HBV and Py enhancer regions, EF-C stimulates the activity of adjacent enhancer elements in a synergistic manner. EF-C corresponds to RFX-1, a protein that binds to a conserved and functionally important site in major histocompatibility complex (MHC) class II antigen promoter regions. Interestingly, the RFX-1 binding site in MHC class II promoters only contains an EF-C half-site, maintaining one arm of the inverted repeat in an EF-C binding site. We have investigated the binding of purified EF-C and RFX-1 to sites in the Py and HBV enhancer regions that carry mutations that either disrupt one arm of the EF-C inverted repeat, or alter the spacing between the repeats. Our results show that the interaction of EF-C and RFX-1 with an intact inverted repeat is required for functional activity of these viral enhancer regions. Chemical footprinting and modification interference assays show that the interaction of EF-C and RFX-1 with the DRA MHC class II promoter truly represents half-site interaction, and that this binding is unstable. In contrast, the binding of EF-C and RFX-1 to the viral inverted repeats is stable. These results suggest that an additional activity may be required to stabilize EF-C/RFX-1 interaction with the MHC class II promoter, and that viral enhancer regions have evolved high affinity binding sites to sequester dimeric EF-C/RFX-1.
David
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Department of Molecular Genetics and Microbiology, State University of New York, Stony Brook 11794, USA.
Garcia
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AD
Hearing
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D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004279:DNA, Viral; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005802:Genes, MHC Class II; D006367:HeLa Cells; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011120:Polyomavirus / Q000235:genetics*; D011401:Promoter Regions, Genetic; D000071701:Regulatory Factor X Transcription Factors; D000071716:Regulatory Factor X1; D012091:Repetitive Sequences, Nucleic Acid; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation
D004279:DNA, Viral; D004268:DNA-Binding Proteins; C000605537:RFX1 protein, human; D000071701:Regulatory Factor X Transcription Factors; D000071716:Regulatory Factor X1; C000605538:Rfx1 protein, mouse; D014157:Transcription Factors