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. 2000 Nov;1(5):422-7.
doi: 10.1093/embo-reports/kvd091.

An enhanceosome containing the Jun B/Fra-2 heterodimer and the HMG-I(Y) architectural protein controls HPV 18 transcription

I Bouallaga  1 , S Massicard, M Yaniv, F Thierry
Affiliations

An enhanceosome containing the Jun B/Fra-2 heterodimer and the HMG-I(Y) architectural protein controls HPV 18 transcription

I Bouallaga et al. EMBO Rep. 2000 Nov.

Abstract

Recent studies have reported new mechanisms that mediate the transcriptional synergy of strong tissue-specific enhancers, involving the cooperative assembly of higher-order nucleoprotein complexes called enhanceosomes. Here we show that the HPV18 enhancer, which controls the epithelial-specific transcription of the E6 and E7 transforming genes, exhibits characteristic features of these structures. We used deletion experiments to show that a core enhancer element cooperates, in a specific helical phasing, with distant essential factors binding to the ends of the enhancer. This core sequence, binding a Jun B/Fra-2 heterodimer, cooperatively recruits the architectural protein HMG-I(Y) in a nucleoprotein complex, where they interact with each other. Therefore, in HeLa cells, HPV18 transcription seems to depend upon the assembly of an enhanceosome containing multiple cellular factors recruited by a core sequence interacting with AP1 and HMG-I(Y).

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Figures

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Fig. 1. Deletion analysis of the HPV18 enhancer. (A) Schematic representation of the HPV18 long control region (LCR). Predicted binding sites for cellular and viral proteins were represented along the LCR of HPV18, which is composed of three functionally distinct regions: a proximal region, containing the early promoter P105 that is devoid of intrinsic transcriptional activity; an upstream tissue-specific enhancer of 230 nucleotides between nucleotides 7510 and 7740; and a 5′ region dispensable for the P105 transcriptional activation (Garcia-Carranca et al., 1988). The enhancer was cloned upstream of the heterologous minimal tk promoter in front of the CAT reporter gene, as well as 3′ or 5′ deletion mutants (B) and stereo-alignment mutants (C). Internal deletions of half a turn (five nucleotides) or a complete turn (10 nucleotides) of the DNA helix were made either upstream, between nucleotides 7594 and 7598 for Δ5(I), or 7589 and 7598 for Δ10(I), or downstream, between nucleotides 7677 and 7681 for Δ5(II), or 7672 and 7681 for Δ10(II), of the 109 bp core fragment. Structural effects of these deletions were schematized on the right, underlining the loss of synergy. CAT activities are the mean of at least three independent transfection experiments.
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Fig. 2. Cooperative formation of a higher-order complex containing AP1 and HMG-I(Y). (A) The 109 bp fragment was labelled by Klenow filling and incubated with increasing concentrations of HeLa nuclear extract (1, 2, 4, 6, 8, 10, 12, 15, 18, 20 and 24 µg, respectively, in lanes 3–13; no extract in lane 1), or with 1 µl of an in vitro-translated AP1 heterodimer (Jun B/c-Fos), before loading onto a 5% native polyacrylamide gel. (B) Competition experiments were performed with 10 µg of nuclear extract in the presence of an 80-fold excess of oligonucleotides containing either the wild-type or mutated AP1 binding site, and either a consensus or non-consensus (Oligo 7644-57) Oct binding site. (C) The graph presents quantification of the radioactivity found in the two bound complexes relative to the total radioactivity of the probe, plotted against the amount of nuclear proteins used in the assays shown in (A). (D) Presence of HMG-I(Y) in the slower migrating complex was assessed by use of two specific antibodies prepared either against the full-length HMG-I(Y) protein, or its DNA binding domain (ATX).
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Fig. 3. A model for the structure of the HPV18 enhanceosome. (A) The AP1 factor binding the HPV18 enhancer is a Jun B–Fra-2 heterodimer. Eight micrograms of nuclear extract were incubated with the 109 bp fragment in the absence, or in the presence, of pre-immune serum (PI) or of specific antibodies against Fos B, c-Fos, Fra-1, Fra-2, Jun B, c-Jun or Jun D, as indicated. (B) HMG-I(Y) interacts specifically with Jun B and Fra-2. The GST–HMG-I(Y) protein was incubated with the 35S-labelled in vitro-translated proteins JunB, Fra-2 or the Jun B–Fra-2 fusion protein, the HPV18 E1 protein as a negative control, or cJun as a positive control. One tenth of the input proteins were loaded (I) as well as reactions with the GST (G) alone and the GST–HMG-I(Y) fusion (GH) proteins. (C) The core enhancer sequence containing nucleotides 7566–7674 binds the Jun B/Fra-2 heterodimer and the architectural factor HMG-I(Y), forming a cooperative complex that may include other factor(s). This core sequence cooperates in a stereo-specific manner with at least two distal factors.
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