Interaction of EF-C/RFX-1 with the Inverted Repeat of Viral Enhancer Regions Is Required for Transactivation

Ebenezer David,Alonzo D Garcia,Patrick Hearing

doi:10.1074/jbc.270.14.8353

Ebenezer David, Alonzo D Garcia + Show 1 more

Open Access

https://doi.org/10.1074/jbc.270.14.8353

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Abstract

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.

Highlights

From the Department of Molecular Genetics and Microbiology, Health Sciences Center, State University of New York, Stony Brook, New York 11794
Our results show that the interaction ofEF-C and RFX-l with an intact inverted repeat is required for functional activity of these viral enhancer regions
The binding of EF-C and RFX-l to the viral inverted repeats is stable. These results suggest that an additional activity may be required to stabilize EF-CIRFX·l interaction with the major histocompatibility complex: (MHC) class II promoter, and that viral enhancer regions have evolved high affinity binding sites to sequester dimeric EF-CIRFX-l

Summary

Introduction

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. Our results show that the interaction ofEF-C and RFX-l with an intact inverted repeat is required for functional activity of these viral enhancer regions. The binding of EF-C and RFX-l to the viral inverted repeats is stable These results suggest that an additional activity may be required to stabilize EF-CIRFX·l interaction with the MHC class II promoter, and that viral enhancer regions have evolved high affinity binding sites to sequester dimeric EF-CIRFX-l. :j:Supported by United States Public Health Service Training Grant CA09176 from the NCI, National Institutes of Health

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