Biomedical subjects

F Thierry

Publications and source records attributed to F Thierry.

At least 19 recordsLinked to original sources

PubMed2001

Stability of the human papillomavirus type 18 E2 protein is regulated by a proteasome degradation pathway through its amino-terminal transactivation domain.

The E2 proteins of papillomaviruses regulate both viral transcription and DNA replication. The human papillomavirus type 18 (HPV18) E2 protein has been shown to repress transcription of the oncogenic E6 and E7 genes, inducing growth arrest in HeLa cells. Using HPV18 E2 fused to the green fluorescent protein (GFP), we showed that this protein was short-lived in transfected HeLa cells. Real-time microscopy experiments indicated that the E2-dependent signal increased for roughly 24 h after transfection and then rapidly disappeared, indicating that E2 was unstable in HeLa cells and could confer instability to GFP. Similar studies done with a protein lacking the transactivation domain indicated that this truncation strongly stabilizes the E2 protein. In vitro, full-length E2 or the transactivation domain alone was efficiently ubiquitinated, whereas deletion of the transactivation domain strongly decreased the ubiquitination of the E2 protein. Proteasome inhibition in cells expressing E2 increased its half-life about sevenfold, which was comparable to the half-life of the amino-terminally truncated protein. These characteristics of E2 instability were independent of the E2-mediated G(1) growth arrest in HeLa cells, as they were reproduced in MCF7 cells, where E2 does not affect the cell cycle. Altogether, these experiments showed that the HPV18 E2 protein was degraded by the ubiquitin-proteasome pathway through its amino-terminal transactivation domain. Tight regulation of the stability of the HPV 18 E2 protein may be essential to avoid accumulation of a potent transcriptional repressor and antiproliferative agent during the viral vegetative cycle.

Cysteine Endopeptidases↗

PubMed2000

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

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).

Binding Sites↗

PubMed1999

Papillomavirus E2 induces p53-independent apoptosis in HeLa cells.

We have previously shown that expression of the papillomavirus E2 protein in HeLa cells induces p53 accumulation and causes both cell cycle arrest and apoptosis. In contrast to growth arrest, onset of apoptosis was not correlated with an increase of p53 transcriptional activity. In the present study, we conducted biochemical and genetic experiments in order to determine whether E2-induced apoptosis was independent of p53 induction. We showed that E2 did not alter the transcription of Bax, a known p53-activated cell death inducer. The time course of apoptotic cell death preceded p53 induction by several hours. Overexpression of the HPV18 E6 oncogene prevented E2-mediated p53 accumulation, but did not alter the rate of cell death. Finally, point mutants of the HPV18 E2 transactivation domain induced apoptosis, although they were unable to induce high p53 accumulation or cell cycle arrest. In addition, the results obtained with these mutants indicated that both transcriptional activation and replication functions of E2 were dispensable for the induction of cell death. These observations show that E2-induced apoptosis is an early event, independent of p53 accumulation and unrelated to downstream p53-dependent transcriptional events.

Animals↗

PubMed1998

The human papillomavirus type 18 (HPV18) replication protein E1 is a transcriptional activator when interacting with HPV18 E2.

The human papillomavirus type 18 E1 and E2 proteins are both required for the initiation of viral DNA replication. Whereas E2 is the major viral transcription regulator, E1 is the replication initiator protein. They interact with each other and with the origin sequences to initiate viral DNA replication. We show that the HPV18 E1 and E2 proteins, when bound to an origin sequence cloned upstream of a heterologous promoter, synergistically activate transcription. This synergy required binding of E2 to at least two binding sites, but was partially independent of E1 binding to the origin of replication. Transcriptional activation was observed even in the absence of replication of the target DNA. Only homologous E1 and E2 proteins binding to homologous origin sequences from BPV1 or HPV18 viruses could synergistically activate transcription. We show that the HPV18 E1 protein can activate transcription when targeted to the DNA by fusion of the complete polypeptide with the BPV1 E2 C-terminus dimerization/DNA binding domain, implying that HPV18 E1 is an intrinsic transcriptional activator, though less potent than E2. The interaction between E1 and E2 may form a transcriptionally active complex during initiation of viral DNA replication.

Chloramphenicol O-Acetyltransferase↗

PubMed1997

Expression of the papillomavirus E2 protein in HeLa cells leads to apoptosis.

The papillomavirus E2 protein plays a central role in the viral life cycle as it regulates both transcription and replication of the viral genome. In this study, we showed that transient expression of bovine papillomavirus type 1 or human papillomavirus type 18 (HPV18) E2 proteins in HeLa cells activated the transcriptional activity of p53 through at least two pathways. The first one involved the binding of E2 to its recognition elements located in the integrated viral P105 promoter. E2 binding consequently repressed transcription of the endogenous HPV18 E6 oncogene, whose product has been shown previously to promote p53 degradation. The second pathway did not require specific DNA binding by E2. Expression of E2 induced drastic physiological changes, as evidenced by a high level of cell death by apoptosis and G1 arrest. Overexpression of a p53 trans-dominant-negative mutant abolished both E2-induced p53 transcriptional activation and E2-mediated G1 growth arrest, but showed no effect on E2-triggered apoptosis. These results suggest that the effects of E2 on cell cycle progression and cell death follow distinct pathways involving two different functions of p53.

Apoptosis↗

PubMed1997

Different mechanisms contribute to the E2-mediated transcriptional repression of human papillomavirus type 18 viral oncogenes.

Transcription of the human papillomavirus type 18 (HPV18) E6 and E7 oncogenes is repressed by the viral E2 protein. In C33 cells, we have previously shown that of the four E2 binding sites (E2 BS) present in the HPV18 long control region (LCR), only the binding site adjacent to the TATA box (E2 BS 1) was involved in E2-mediated repression. In the present study, we sought to determine whether this phenomenon was conserved in other cell lines. We first showed that all three E2 BS proximal to the P105 promoter were required for full repression of its activity in HeLa and HaCaT cells. Repression by E2 at E2 BS 2 occurred through the displacement of Sp1. Second, a truncated E2 product, lacking the N-terminal transactivation domain, repressed transcription more efficiently than the full-length protein. Repression was abolished when the N-terminal domain of E2 was replaced by the activation domain of VP16. The VP16-E2 chimeric protein could activate transcription from an LCR mutated in its TATA box. DNA-protein binding studies showed that E2 associates with its four binding sites in the LCR with similar affinities. However, challenge of such complexes with excess binding sites demonstrated that interaction with E2 BS 4 was the most stable while interaction with E2 BS 1 was the least stable. Furthermore, complexes with the full-length E2 were less stable than those formed with the N-terminally truncated protein.

Binding Sites↗

PubMed1996

A single element mediates glucocorticoid hormone response of HPV18 with no functional interactions with AP1 or hbrm.

We determined that the human papillomavirus type 18 (HPV18) regulatory region contains one functional GRE sequence that interacts with the glucocorticoid receptor. This sequence conferred a moderate hormonal activation to the HPV18 P105 promoter. Two modulators of glucocorticoid hormone activity, AP1 and hbrm, both involved in P105 transcription, were found not to interfere with this hormonal activation.

Base Sequence↗

PubMed1995

Control of HPV 18 DNA replication by cellular and viral transcription factors.

Papillomavirus replication in vivo requires the interaction of the virally encoded proteins E1 and E2 with the origin of replication which is localised in the regulatory region (long control region or LCR) of the viral genome. In genital human papillomaviruses (HPVs), the origin overlaps promoter elements of early transcription. In this study, we analysed the replication of HPV18 DNA using the complete LCR containing mutations in transcription regulatory elements. We found that each of the three E2 binding sites proximal to the AT-rich sequence of the origin contributes to the replication rate of DNA, although not identically. In addition, two sequences important for early transcription, an Sp1 binding site and the TATA box, were also found to play a role in replication. In contrast, two AP1 binding sites required for the enhancer-mediated activation of early transcription did not affect the replication, while other upstream sequences in the LCR did contribute to the replication efficiency. Our results indicate that besides a core origin of replication containing an AT-rich sequence and three E2 binding sites, auxiliary elements affect HPV18 DNA replication in the context of the full length LCR, some of which are important for transcription.

Adenovirus E2 Proteins↗

PubMed1994

The E2 transcriptional repressor can compensate for Sp1 activation of the human papillomavirus type 18 early promoter.

The E6/E7 early promoter (P105) of genital human papillomavirus type 18 contains binding sites for the viral regulator E2, tandemly repeated and closely flanked by two crucial promoter elements; the TATA box downstream and an Sp1 binding site upstream. We showed that binding of purified E2 and Sp1 proteins in vitro to their neighboring sites is mutually exclusive and that Sp1 is displaced by E2. However, this displacement did not result in repression of P105 transcription. In contrast, binding of E2 to its site overlapping the Sp1 binding site activated transcription of P105 derivatives lacking the E2 site most proximal to the TATA box. Surprisingly, a truncated form of E2, deleted of part of the transactivation domain and known as the E2 transcriptional repressor, as well as the E2 DNA-binding domain alone also supported transcription of these P105 derivatives. In the context of P105, the viral E2 protein can thus activate P105 transcription in place of Sp1, even in the absence of its transactivation domain.

Base Sequence↗

PubMed1994

The bovine papillomavirus type 1 (BPV1) replication protein E1 modulates transcriptional activation by interacting with BPV1 E2.

The study of bovine papillomavirus type 1 (BPV1) DNA replication has shown that E1 and E2 are the only viral proteins required for this process. Both E1 and E2 interact with the viral origin of replication (ori). The BPV1 E2 protein is also a well-characterized transcriptional regulator. We show in this report that E1 can modulate transcription by interactions with E2. At low concentrations, E1 enhanced the E2-mediated transactivation of heterologous promoters containing the BPV1 ori by promoting cooperative binding of both E1 and E2 to the DNA. In contrast, in the presence of excess E1, transactivation by E2 is repressed. This last process, however, does not require cooperative DNA binding of the two proteins. These results imply that the balance between these two distinct types of interaction is crucial both for control of replication and for early viral transcription.

Base Sequence↗

PubMed1992

Two AP1 sites binding JunB are essential for human papillomavirus type 18 transcription in keratinocytes.

The activity and epithelial tropism of the human papillomavirus type 18 P105 early promoter, which directs the synthesis of the E6 and E7 transforming genes, are controlled by cis elements included in the viral long control region. To identify potential cellular regulators of this promoter, we mutagenized one or both of the 5'-TGACTAA-3' cis elements capable of interacting with the AP1 transcription factor, which is composed either of homodimers or heterodimers of the Jun products or of heterodimers of Jun and Fos. Mutation of both elements completely abolished P105 promoter activity in human keratinocytes. We show that either AP1 site can interact efficiently in vitro with any of the three different Jun products as heterodimers with c-Fos. However, in nuclear extracts prepared from human keratinocytes, JunB was the predominant Jun component bound to the DNA probe containing this cis element. These results implicate JunB as an important factor in human papillomavirus type 18 transcription in keratinocytes and strongly suggest a potential role of this Jun gene product in the tissue-specific transcription of the genital papillomaviruses.

Base Sequence↗

PubMed1991

Functional analysis of E2-mediated repression of the HPV18 P105 promoter.

Transcription of the transforming genes E6 and E7 of the human papillomavirus type 18 (HPV18) can be repressed by the product of the E2 open reading frame. Mutations were introduced in the cis-responsive elements upstream from the E6 and E7 transcriptional promoter, P105. The effect of these mutations in the absence or presence of E2 was examined in the human cervical carcinoma cell line C33, transfected with expression plasmids. In the presence of HPV18 E2, repression was relieved only when both of the two E2 binding motifs, immediately proximal to the P105 TATA box, were mutated. Mutation of a third E2 binding site, 110 nucleotides upstream of the TATA box, led to a slight but consistent activation in the presence of the full-length E2. Therefore, three E2 binding sites appear to be involved in the negative regulation of the P105 promoter by HPV18 E2. In the presence of the BPV1 E2 protein, only the E2 binding site most proximal to the promoter TATA box was involved in the P105 repression. The comparative analysis of the in vitro and in vivo properties of the human and bovine E2 proteins on the HPV18 P105 promoter provides new insights into the mechanism of transcriptional repression, which appears to involve steric hindrance at the site of transcriptional initiation.

Base Sequence↗

PubMed1990

Mutational analysis of cis elements involved in E2 modulation of human papillomavirus type 16 P97 and type 18 P105 promoters.

cis-Acting elements involved in E2 modulation of human papillomavirus type 16 (HPV-16) P97 promoter activity and HPV-18 P105 promoter activity were examined. In transfected primary human keratinocytes, each promoter had a basal activity that could be repressed by the bovine papillomavirus type 1 E2 gene product. Mutational analysis of the E2-binding sites in the long control region upstream of each promoter revealed that E2 repression was mediated through the E2-binding sites proximal to each promoter. In the context of a mutated E2-binding site at the promoter proximal position, the HPV-16 P97 and HPV-18 P105 promoters could be transactivated by E2. E2-mediated repression of HPV-18 P105 promoter activity was shown to be a transcriptional effect. The interaction of E2 with promoter-proximal E2-binding sites is likely to be important for the controlled expression of viral genes transcribed from the HPV-16 P97 promoter and the HPV-18 P105 promoter in infected human genital epithelial cells.

Base Sequence↗

PubMed1990

Cooperative activation of transcription by bovine papillomavirus type 1 E2 can occur over a large distance.

The viral transcriptional factors encoded by the E2 open reading frame bind to the specific DNA sequence elements ACCGNNNNCGGT, allowing activation or repression of transcription. We have analyzed bovine papillomavirus type 1 E2 transactivation using recombinant genes containing E2-binding sites inserted at either 3' or 5' positions relative to the heterologous transcriptional initiation site of the herpes simplex virus thymidine kinase gene. In these hybrid plasmids, strong transactivation required the presence of a minimum of two E2-binding sites in close proximity to the promoter or five binding sites at a distance. The presence of a single E2-binding motif 5', close to the initiation site, increased the efficiency of E2 transactivation from a distance in a more-than-additive manner. Since each E2-binding site bound a dimer of the E2 protein, these experiments suggest that transactivation by E2 requires the interaction between several E2 dimers with other essential transcription factors. This interaction may be facilitated by DNA looping, which would bring E2 molecules close to the promoter.

Base Sequence↗

PubMed1989

The human papillomavirus type 18 (HPV18) E2 gene product is a repressor of the HPV18 regulatory region in human keratinocytes.

The human papillomavirus type 18 (HPV18) long control region (LCR) harbors transcriptional promoter and enhancer elements. Recombinant plasmids bearing all or part of the HPV18 LCR cloned in enhancer or promoter configuration upstream of the chloramphenicol acetyltransferase (CAT) gene were transfected into human fibroblasts and keratinocytes. Although the HPV18 enhancer can function in the absence of E2 gene products in both fibroblasts and keratinocytes, the promoter activity of the HPV18 LCR is detectable in keratinocytes but not in fibroblasts, suggesting that it is tissue specific. This promoter activity was repressed in human keratinocytes not only by the bovine papillomavirus type 1 E2 gene product but also by the homologous HPV18 E2 gene product. The promoter involved in the HPV18 E2 repression is located within a 230-base-pair domain directly upstream of the E6 open reading frame of the HPV18 LCR and is probably the previously identified E6 promoter. Although one cannot rule out the possibility that this repressing effect is mediated by a truncated form of HPV18 E2 protein, as was previously demonstrated for bovine papillomavirus type 1, a more likely explanation would be that the full-length HPV18 E2 protein behaves as a repressor. Indeed, at the same doses at which it inhibits transcription from the homologous HPV18 LCR, the HPV18 E2 gene product activates transcription from constructs bearing E2-binding palindromes cloned in enhancer configuration upstream of a heterologous promoter. The fact that the homologous HPV18 E2 gene product acts as a transcriptional repressor of the HPV18 LCR suggests a possible explanation for the overexpression of E6 and E7 open reading frames in cervical carcinoma cells and in cell lines derived from them.

Base Sequence↗

PubMed1988

A dimer of BPV-1 E2 containing a protease resistant core interacts with its DNA target.

The E2 proteins encoded by papillomaviruses interact with the viral DNA to regulate its transcription. In the present study, we have constructed bacterial vectors expressing the full-length or N-terminal truncated BPV-1 E2 proteins under the control of an inducible promoter. By UV cross-linking experiments we show that a dimer of the intact or truncated E2 protein interacts with a single palindromic site ACCGNNNNCGGT. The DNA-binding domain of E2 can be reduced to a small protease resistant core. Methylation interference studies show that this C-terminal domain interacts with the major groove of the DNA by contacting two consecutive guanine residues in both halves of the palindrome. Although one binding site is sufficient for high affinity binding in vitro or in vivo, two E2 binding sites are required for transcriptional activation in eukaryotic cells.

Binding Sites↗

PubMed1988

Interplay of viral and cellular proteins along the long control region of human papillomavirus type 18.

The long control region of human genital papillomavirus type 18 harbors transcription regulatory elements, such as the E6 promoter and a cell type-specific enhancer independent of the E2 protein. By performing DNase I footprint experiments in vitro with protein extracts from different cell lines and tissues we searched for cellular factors interacting with the totality of the control region. We detected a total of eight different protected sites; most of them were found with all the extracts. Two of these sites, one in the enhancer and the other in the sequences proximal to the E6 cap site, interact with a member of the activator protein 1 family. However, in the absence of fine mutational analysis, we cannot readily discern an exact functional role for the different binding sites. We also characterized two regions, which are protected only in the presence of E2, corresponding to the perfect palindromes ACCGN4CGGT and present either 500 nucleotides or tandemly repeated about 70 nucleotides upstream of the E6 cap site. This last protected area covers a large part of the putative TATA box of the E6 promoter and could explain its repression by bovine papilloma virus type 1 E2, which could interfere with binding of the TFII D ubiquitous transcription factor to the TATA sequence of the promoter.

Base Sequence↗

PubMed1987

The BPV1-E2 trans-acting protein can be either an activator or a repressor of the HPV18 regulatory region.

The human papillomavirus 18 (HPV 18) long control region contains promoter and enhancer elements whose activity is restricted to several human cell lines of epithelial origin. This enhancer possesses a considerable constitutive activity which is further stimulated in the presence of the E2 trans-activating protein of bovine papillomavirus 1 (BPV1). Surprisingly the same BPV1 protein strongly repressed transcription from the genuine HPV18 enhancer-promoter DNA sequences. We suggest that binding of several molecules of E2 protein between the viral CAAT and TATA elements sterically hinders transcription initiation from this promoter, while the same DNA--protein assembly stimulates the SV40 promoter when cloned in an enhancer configuration upstream of this heterologous promoter. Unlike BPV1-E2 the homologous E2 gene product does not seem to strongly modulate viral transcription. Finally the BPV1-E2 gene product may repress some essential viral or host genes, since we failed to isolate HeLa cells expressing BPV1-E2.

Cell Line↗