Ets-2 Repressor Factor (ERF) mediates repression of the human cytomegalovirus major immediate-early promoter in undifferentiated non-permissive cells

Mark Bain, Marc Mendelson{dagger} and John Sinclair

Department of Medicine, University of Cambridge, PO Box 157, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QQ, UK

Correspondence
John Sinclair
js{at}mole.bio.cam.ac.uk


   ABSTRACT
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ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
The repression of human cytomegalovirus immediate-early (IE) lytic gene expression is crucial for the maintenance of the latent viral state. By using conditionally permissive cell lines, which provide a good model for the differentiation state-dependent repression of IE gene expression, we have identified several cellular factors that bind to the major immediate-early promoter (MIEP) and whose expression is down-regulated after differentiation to a permissive phenotype. Here we show that the cellular protein Ets-2 Repressor Factor (ERF) physically interacts with the MIEP and represses MIEP activity in undifferentiated non-permissive T2 embryonal carcinoma cells. This factor binds to the dyad element and the 21 bp repeats within the MIEP – regions known to be important for the negative regulation of MIEP activity. Finally, we show that following differentiation to a permissive phenotype ERF's repressive effects are severely abrogated.

{dagger}Present address: Infectious Diseases Clinical Research Unit, The Lung Institute, University of Cape Town, Cape Town, South Africa


   Introduction
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ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Human cytomegalovirus (HCMV) is a betaherpesvirus whose sero-prevalence can vary from 50 to 90 % depending on the socio-economic status of the population. Following primary infection, which rarely causes disease in the immunocompetent, HCMV maintains a latent infection throughout the lifetime of the infected host. However, infection or reactivation in the immunocompromised, such as transplant recipients or AIDS patients, is associated with morbidity and mortality (reviewed in Alford & Britt, 1993).

Productive HCMV infection and reactivation require an ordered cascade of gene expression and are dependent on the expression of the immediate-early (IE) gene products IE72 and IE86 (also known as IE1 and IE2 respectively). These IE proteins are potent and promiscuous transactivators of both cellular and viral genes, and serve to activate the viral early (E) gene promoters (Pizzorno et al., 1988; Malone et al., 1990), ultimately leading to viral DNA replication, expression of the viral late (L) genes and virus assembly. The expression of IE72 and IE86 is under the control of the major immediate-early promoter (MIEP), a highly complex region comprising a TATA box, an upstream imperfect dyad symmetry element (also termed the modulator), and an extremely powerful enhancer region made up of a series of 17, 18, 19 and 21 bp repeat motifs (reviewed in Meier & Stinski, 1996). MIEP activity is regulated by a myriad of positively and negatively acting cellular and viral proteins. For example, there are numerous nuclear factor-1 (NF-1) sites within the MIEP and the 18 and 19 bp repeat sequences contain NF-{kappa}B and cyclic AMP-responsive element binding protein (CREB) binding sites respectively (Meier & Stinski, 1996). Conversely, several studies using non-permissive cells have shown that the modulator and the 21 bp repeats are responsible for the inhibition of MIEP activity in such cells (Nelson et al., 1987; Lubon et al., 1989; Kothari et al., 1991).

In vivo, monocytes have been identified as an important site of HCMV latency (Taylor-Wiedeman et al., 1991); these cells carry the viral genome in the absence of lytic (productive) gene expression. Experiments in our laboratory and those of others have shown that the differentiation of peripheral blood monocytes to macrophages (monocyte-derived macrophages; MDMs) results in the induction of endogenous HCMV IE gene expression and reactivation of virus (Taylor-Wiedeman et al., 1994; Soderberg-Naucler et al., 1997). Therefore, it is clear that the differentiation state of the cell is critical for the silencing of IE gene expression and thus the maintenance of latency. In vitro, many studies of the differentiation-specific regulation of IE gene expression have used the conditionally permissive T2 embryonal carcinoma and the THP-1 myelomonocytic cell lines as model systems (Gonczol et al., 1984; Weinshenker et al., 1988). These cells, which are non-permissive to HCMV infection due to, at least in part, a block in IE expression, become permissive when differentiated to a neuronal (T2 cells) or macrophage-like (THP-1 cells) phenotype. Moreover, in both cell lines this repression of IE expression is apparently mediated by cellular factors that bind to specific regulatory regions within the MIEP, such as the modulator and the 21 bp repeats, in a differentiation-state dependent manner (Nelson et al., 1987; Shelbourn et al., 1989; Kothari et al., 1991; Huang et al., 1996; reviewed in Sissons et al., 2002). We have previously shown that one of these factors is YY1 (Liu et al., 1994), a ubiquitous cellular transcription factor that is involved in both the activation and the repression of numerous cellular and viral genes (reviewed in Shi et al., 1997). YY1 binds to both the dyad symmetry element and the 21 bp repeats within the MIEP. However, it is evident that YY1 alone is not sufficient for the full repression of the MIEP in non-permissive cells. For example, fibroblasts are fully permissive for HCMV infection yet contain relatively high levels of YY1 protein (data not shown).

In this report we extend our characterization of the cellular factors negatively regulating MIEP activity in non-permissive cells. Previously, we identified an MIEP-binding factor which we named modulator binding factor 1 (MBF1; Shelbourn et al., 1989; Kothari et al., 1991). Analysis by gel shift indicated that MBF1 was a member of the ets family of transcription factors – a family which, certainly in simian CMV, play a role in MIEP regulation (Chan et al., 1996). Here we show that in undifferentiated non-permissive T2 embryonal carcinoma cells the Ets-2 Repressor Factor (ERF), an ets protein which functions as a transcriptional repressor of target promoters, physically interacts with, and represses, the MIEP in transient transfection assays.


   Methods
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ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Cell culture.
The NTera2 D1 (T2) human teratocarcinoma cell line (Andrews et al., 1984) was maintained in EMEM supplemented with 10 % FCS. Differentiation of T2 cells to permissive T2RA cells (during which they develop a neuronal-like phenotype) was induced by retinoic acid (Sigma) over a period of 5 days.

Plasmids.
The reporter plasmids pEScat and pIEP1cat have been described previously (Shelbourn et al., 1989). Briefly, pEScat contains the chloramphenicol acetyltransferase (cat) gene under the control of the HCMV MIEP (the -2100 to +72 region of the MIEP from the AD169 strain of HCMV; Fig. 1). The shorter pIEP1cat construct contains cat under the control of sequences from -302 to +72 of the MIEP and thus lacks the imperfect dyad symmetry, the 21 bp repeat elements and the NF-1 cluster (Fig. 1). pESd12cat, which was derived from pEScat, has the region from -985 to -935 removed by digestion with EcoRV and thus lacks the dyad symmetry (Shelbourn et al., 1989). 2x21cat (a kind gift from T. Stamminger) contains two copies of the 21 bp repeat motif ligated to the 5' end of the basal HCMV IE promoter (position -65 to +72).



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Fig. 1. HCMV MIEP reporter plasmids used in this study. Numbers refer to the base position with respect to the transcription start site (+1) within the MIEP of the AD169 strain of HCMV. The imperfect dyad symmetry within the modulator region, the enhancer (which contains several copies of the 21 bp motif at its 5' end) and the TATA box are shown. refers to two copies of the 21 bp repeat motif ligated upstream of the basal IE promoter. In all cases cat is the reporter gene.

 
pHK3 contains the SV40 early promoter upstream of a multiple cloning site and was a kind gift from T. Kouzarides. pSV2Ets68-1 (from J Ghysdael), pSV2PU.1 (from T. Kouzarides) and pSV2PEA3 (from J. Hassell) contain the ORFs for the Ets factors Ets68-1, PU.1 and PEA3 respectively, under the control of the SV40 early promoter. pSG5-ERF and pSK-ERF (both kind gifts from G. Mavrothalassitis) contain the 1·9 kb ERF cDNA cloned into pSG5 (Stratagene) or pBluescript SK+ (Stratagene), respectively. pcDNA3-ERF contains the ERF coding sequence cloned into pcDNA3 (Invitrogen). pHK3-YY1 includes YY1 under the control of the SV40 promoter (Liu et al., 1994). pcDNA3-YY1 contains the YY1 coding sequence cloned as an EcoRI fragment from pHK3-YY1 into EcoRI-digested pcDNA3. pFM564 contains the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA and was a kind gift of R. Watson.

Transfections and CAT assays.
Transfections typically used 8x105 T2 cells or 1·2x106 T2RA cells. Cells were transfected by calcium phosphate co-precipitation with cat reporter plasmid and variable quantities of expression vector. Cells were harvested 48 h post-transfection and lysed by three cycles of freeze/thawing. Reactions to determine CAT activity were performed using equivalent amounts of protein. All T2RA cell extracts were diluted (1 : 5–1 : 10) because of the much higher levels of CAT activity seen in these cells. Acetylated/non-acetylated CAT species were separated by TLC and spots quantified on an InstantImager (Packard). Results shown are the averages of at least three independent experiments.

Cloning and purification of GST-fusion proteins.
pGEX-3X-P-ERF was produced by transferring ERF from SmaI-digested pSG5-ERF into pGEX-3X-P (Pharmacia). This cloning strategy resulted in the loss of carboxy-terminal ERF sequences but the Ets-binding domain remained intact. The induction, purification and quantification of GST-ERF and GST-p27 (used as a negative control and a kind gift from X. Lu) have been described previously (Hagemeier et al., 1993).

Electrophoretic mobility shift assays (EMSAs).
Purified GST-fusion protein (50 ng) was assayed for its ability to retard 10 ng of [32P]dCTP-labelled probe. Binding reactions contained 50 ng GST-fusion protein, 1 µg non-specific competitor [poly(dI·dC); Stratagene], 4 µl 5x times; binding buffer [100 mM HEPES (pH 7·9), 5 mM MgCl2, 2·5 mM DTT, 250 mM NaCl and 20 % (v/v) Ficoll], 10 ng radio-labelled probe and dH2O to 20 µl. Where indicated, approximately 100-fold molar excess of additional cold competitor was added to the reaction for 10 min prior to the addition of the probe. Table 1 shows the sequences of the probes used in these studies. The PEA3 probe has been described previously (Kothari et al., 1991).


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1 Sequences of the oligonucleotide probes used in EMSAs

Underlining indicates bases which were ‘filled in’ Klenow.

 
Northern blot analysis.
Northern blots were carried out using standard procedures (Sambrook et al., 1989). Total RNA from undifferentiated or differentiated T2 cells was prepared using a single-step protocol and the RNAzol B reagent (Biogenesis, UK). 10 µg of each RNA sample was denatured in RNA loading buffer and resolved on a 1 % agarose–formaldehyde gel. Following electrophoresis, gels were stained with ethidium bromide to ensure equal sample loading and then soaked in 20x times; SSC for 1 h at room temperature. RNA was transferred to positively charged nylon membranes (Hybond-N+; Amersham) by overnight capillary transfer. Following cross-linking using a UV Stratalinker (Stratagene), filters were prehybridized for 30 min at 65 °C in 20 ml of Rapid-Hyb hybridization buffer (Amersham) before the addition of labelled probe. Probes were prepared as follows: a 735 bp YY1 probe was generated by digestion of pcDNA3-YY1 with PstI/HindIII. The 781 bp ERF probe was produced by digestion of pSK-ERF with PstI and EcoRI. As a further control for RNA quantity and integrity, a 1·2 kb GAPDH fragment was produced by digestion of pFM564 with PstI. In all instances fragments were gel purified and then 25 ng of each probe was random-primer labelled according to the manufacturer's instructions (Amersham) using [32P]dCTP as label. The entire reaction mix was added to the filter and hybridization allowed to proceed for 1·5 h at 65 °C. Filters were then washed twice in 2x times; SSC, 0·1 % SDS at room temperature (30 min per wash) and then returned to 65 °C for higher stringency washes. These washes were: 20 min with 1x times; SSC, 0·1 % SDS, 20 min with 0·5x times; SSC, 0·1 % SDS and finally 20 min with 0·1x times; SSC, 0·1 % SDS. Filters were wrapped in Saran wrap and bands detected following autoradiography at -70 °C.


   Results
Top
ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
ERF physically interacts with the MIEP in vitro
In previous work we showed that MBF1 binds an ets consensus sequence and which, in undifferentiated non-permissive cells, binds to regions of the MIEP known to be important for repressing its activity, and whose expression and/or binding activity is down-regulated in permissive differentiated T2RA cells (Shelbourn et al., 1989; Kothari et al., 1991). Therefore, we have proposed that MBF1 is a cellular repressor factor whose activity is abrogated in permissive cells. To date, most mammalian ets family members function as transcriptional activators (for reviews see Dittmer & Nordheim, 1998; Mavrothalassitis & Ghysdael, 2000), which is not in keeping with the proposed role of MBF1 in the repression of the MIEP. However, Sgouras et al. (1995) have identified a factor, ERF, as an ets family member that functions as a transcriptional repressor. Consequently, we asked whether this ets repressor factor may be MBF1. Because we have previously shown that MBF1 binds to the 21 bp repeats and the 3' half of the dyad symmetry within the MIEP (see Fig. 1 for the relative locations of these domains; Shelbourn et al., 1989; Kothari et al., 1991), we determined whether ERF also binds to these motifs by performing a series of EMSAs. Using purified GST–ERF protein, we showed that ERF protein bound specifically to the 21 bp repeats (Fig. 2A) and to both the 3' and 5' halves of the dyad symmetry (Fig. 2B). This binding is abolished in the presence of specific cold competitor (100-fold molar excess), but not by a similar concentration of the non-specific competitor poly(dI·dC). In addition, binding of ERF to the 21 bp repeat element is efficiently competed by the ets protein-binding PEA3 probe (Wasylyk et al., 1989; Wasylyk & Wasylyk, 1992; Fig. 2). To ensure that the ERF : MIEP interaction is not occurring through the GST moiety we used GST–p27, a fusion protein of GST and the cyclin-dependent kinase inhibitor p27, as a control in these assays. Fig. 2 shows that GST–p27 did not retard any of the probes used in this study, and so illustrates the specificity of the ERF : MIEP interaction.



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Fig. 2. ERF binds to the 21 bp repeats and the 3' and 5' halves of the dyad symmetry in vitro. GST–ERF specifically retards [32P]dCTP-labelled probes encompassing the 21 bp repeat (A) and the 3' and 5' halves of the dyad symmetry (B). Where indicated, 100-fold molar excess of specific cold competitor (CC) was added to the reaction. As an additional control, GST fused to the non-DNA binding cyclin-dependent kinase inhibitor p27 (GST–p27) was used to show that binding of GST–ERF was not occurring non-specifically through the GST moiety. PEA3 indicates the addition of the Ets protein-binding PEA3 motif.

 
ERF represses transcription from the MIEP
We have carried out transient transfection assays in undifferentiated T2 cells using the full-length MIEP reporter plasmid pEScat (see Fig. 1), together with either empty vector control (pHK3) or SV40 promoter-driven ERF, Ets68-1, PEA3 or PU.1 expression vectors. Fig. 3 clearly shows that ERF significantly and reproducibly repressed MIEP activity, indicating a functional interaction between ERF and the MIEP. In contrast, the ets proteins Ets68-1, PEA3 and PU.1 all transactivated the MIEP, which is in keeping with their previously identified roles as transactivators (Wasylyk et al., 1990; Martin et al., 1988; Klemsz et al., 1990). Although the level of repression mediated by ERF is modest it should be noted that a particular problem with this type of assay is that pEScat is, by definition, repressed in this cell type and hence has a very low basal activity (typically 5–15 % conversion). In addition, unless properly controlled, we have found that it is possible to titrate out any repressive effects by increasing the amount of pEScat added to the transfection (Shelbourn et al., 1989; our unpublished results).



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Fig. 3. ERF represses expression from the full-length MIEP reporter plasmid in undifferentiated T2 cells. Non-permissive T2 cells were transfected, by calcium phosphate precipitation, with 100 ng of the pEScat reporter plasmid and either 5 µg empty vector control plasmid (pHK3) or 5 µg SV40 promoter-driven expression vectors encoding the ORFs for ERF, Ets68-1, PEA3 or PU.1. Cell extracts were prepared 48 h post-transfection by three cycles of freeze–thawing. CAT reactions were performed using equal amounts of protein. Acetylated/non-acetylated CAT species were separated by TLC and spots quantified using an InstantImager (Packard). Activities are shown relative to those obtained using an equivalent amount of empty vector (pHK3) which was arbitrarily set at 100 %.

 
Mapping of ERF-responsive regions within the MIEP
Studies have shown that the imperfect dyad symmetry and the 21 bp repeat elements within the MIEP are important for the negative regulation of MIEP activity (Nelson et al., 1987; Shelbourn et al., 1989; Kothari et al., 1991), and it has been demonstrated that it is with such sequences that MBF1 interacts (Nelson et al., 1987; Shelbourn et al., 1989; Kothari et al., 1991). Therefore, it follows that if ERF is MBF1 then ERF-mediated repression should occur via these motifs. Transient co-transfections using an ERF expression vector and the truncated MIEP reporter plasmid pIEP1cat, which lacks both the dyad symmetry and 21 bp repeats (see Fig. 1), clearly showed that ERF had no significant effect on this promoter (Fig. 4A). This is in contrast to the results obtained with the full-length promoter reporter plasmid. These data indicate that not only are the 21 bp repeats and the dyad symmetry crucial for the repression mediated by ERF, but also that this repression is not due to ERF non-specifically squelching or quenching MIEP activity. Co-transfection of an ERF expression vector with the pESd12cat reporter plasmid, which lacks the dyad symmetry but still contains ERF-binding sites within the enhancer (Fig. 1), showed that ERF also repressed this promoter construct (Fig. 4A) albeit not as the full-length promoter construct – consistent with the loss of ERF-responsive sequences. These data are in good agreement with our observations that MBF1 interacts with several regions of the MIEP (Kothari et al., 1991). Finally, a synthetic promoter plasmid, consisting of two copies of the21 bp repeat ligated directly upstream of the basal HCMV immediate-early promoter (2x21cat; Fig. 1), is also efficiently repressed by ERF (Fig. 4A). Consequently, it is clear that both the 21 bp repeats and the dyad symmetry mediate the interaction between ERF and the MIEP.



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Fig. 4. ERF represses MIEP activity by interacting with the dyad symmetry and the 21 bp repeat elements, and this repression is specific to undifferentiated T2 cells. 100 ng of various MIEP reporter plasmids (5 µg in the case of 2x21cat; Fig. 1) were transfected into undifferentiated T2 cells (A) or differentiated T2RA cells (B) along with either 10 µg empty vector (pcDNA3) or 10 µg pcDNA3-ERF. CAT assays were as described above. Results shown are the averages of at least three independent experiments. In order to ensure the reaction was in the linear range, cell extract from T2RA cells was diluted to take into account the increased level of CAT activity seen in these cells.

 
ERF-mediated repression is abrogated in differentiated permissive T2RA cells
The experiments described above were done with undifferentiated, non-permissive T2 cells. Because we have previously demonstrated that repression of the MIEP by YY1 is much reduced in differentiated permissive T2RA cells (Liu et al., 1994) we have assessed whether ERF is able to repress the MIEP in permissive T2RA cells. Fig. 4(B) shows that ERF has little-to-no effect on the full-length reporter construct in such differentiated cells. Although repression is sometimes observed, it is much reduced compared to that observed in T2 cells. Similarly, pIEP1cat and pESd12cat are not significantly affected by ERF. Interestingly, the 2x21cat construct is still reproducibly repressed in these cells by co-transfection with ERF (although slightly less efficiently than in T2 cells). This suggests that ERF is still functional as a repressor in T2RA cells. Its apparent lack of effect on pEScat, pIEP1cat and pESd12cat may be due to the action of numerous induced activators of the MIEP (see below) in T2RA cells which simply overcome the repression mediated by ERF. Thus, a basal reporter construct lacking the sites for these activators but bearing ERF-binding sequences, i.e. 2x21cat, is repressed. Alternatively, these experiments may indicate that a co-factor which is required for ERF's repressor function is simply absent or down-regulated in T2RA cells.

The level of ERF mRNA is unaltered during differentiation of T2 cells
It is clear that the level of MIEP activity is likely to be due to a balance of positively and negatively acting factors. Thus, in undifferentiated non-permissive cells the action of numerous cellular repressors results in the repression of the MIEP. However, during differentiation numerous known activators and putative activators (e.g. MBF2 and MBF3; Shelbourn et al., 1989) are induced and MIEP activity is greatly enhanced. Differentiation may be accompanied by a reduction or disappearance of repressors of the MIEP. Indeed, we have previously shown that MBF1 activity, as determined by EMSA, decreased during differentiation (Shelbourn et al., 1989; Kothari et al., 1991). Therefore we have analysed the level of ERF in undifferentiated and differentiated cells. Because of the lack of suitable reagents it has not yet been possible to assess directly the relative ERF protein levels in undifferentiated/differentiated cells. However, Northern blot analysis of multiple independent RNA preparations from undifferentiated and differentiated cells clearly showed no change in the abundance or distribution of ERF mRNA during differentiation of T2 cells (Fig. 5A). Interestingly, the decrease in YY1 protein that we have shown to accompany differentiation of T2 cells (Liu et al., 1994) is not reflected by changes in the steady state level of YY1 mRNA in such cells (Fig. 5B), arguing that any differentiation-specific decreases in ERF and YY1 are likely to be post-transcriptional events.



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Fig. 5. The level of ERF and YY1 mRNA is unaltered during differentiation of T2 cells. (A) Northern analysis of multiple independentpreparations of total RNA from undifferentiated and differentiated cells (numbered 1, 2 and 3) were probed with an ERF-specific 32P-labelled cDNA probe. - indicates undifferentiated non-permissive T2 cells; + indicates differentiated permissive T2RA cells. (B) The previously observed decrease in the level of YY1 during differentiation (Liu et al., 1994) is a post-transcriptional phenomenon. Northern blot analysis of undifferentiated/differentiated T2 cells, probed using a YY1-specific probe. The small arrowhead indicates the 2·4 kb YY1 species. Also shown is a large abundant species which, because of the high stringency washes used in these studies (see Methods) is presumed to be a bona fide YY1 transcript. (C) A representative GAPDH blot to ensure equal loading and integrity of the RNA samples.

 

   Discussion
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ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
It is clear that the silencing of HCMV MIEP activity is critical for the maintenance of the latent viral state and that this repression is differentiation state-dependent. Here we extend our analyses of the cellular factors which directly repress MIEP activity in non-permissive cells. Physical, functional and biological characterization studies strongly suggest that ERF, a recently characterized ets protein family member, is the previously unidentified MBF1 (Figs 2, 3 and 4). To our knowledge this is the first demonstration of a role for ERF in the control of viral gene expression.

We suggest that differentiation to a permissive phenotype causes: (i) an increase in the number and activity of transcriptional activators of the MIEP and (ii) a concomitant reduction in the levels of the MIEP repressors which are active in the undifferentiated non-permissive state. This is consistent with our Western blot data which show that YY1 is down-regulated during differentiation to a permissive phenotype (Liu et al., 1994). Although it is clear that the absolute level of YY1 protein does decrease in permissive cells, it is likely that, in addition, such factors are also inactivated by post-transcriptional modification during differentiation. Although the mechanisms regulating the activity of these factors are unclear, phosphorylation/dephosphorylation has been suggested as modulating the DNA-binding and/or repressor activities of both YY1 and ERF (Austen et al., 1997; Sgouras et al., 1995; Le et al., 1999) and recently it has been shown that YY1 activity is also regulated by acetylation/deacetylation (Yao et al., 2001). However, there is accumulating evidence that the ubiquitously expressed YY1 protein is subject to proteolytic regulation. For example, YY1 specifically represses muscle-specific gene expression in undifferentiated myoblasts. However, during differentiation to myotubes, this repression is lifted due to the proteolytic degradation of YY1 (Lee et al., 1994; Galvagni et al., 1998; Walowitz et al., 1998). It is interesting to note, therefore, that there is at least one report which suggests that the decrease in YY1 protein during the differentiation of T2 cells is due to proteolysis (Pizzorno, 2001).

It is intriguing that both of the factors that we have characterized (YY1 and ERF) are ubiquitous cellular transcription factors, which have promoters that resemble those of house-keeping genes (Yao et al., 1998; Liu et al., 1997). Indeed, the importance of YY1 is evident by its ability to regulate the activity of numerous cellular promoters, interact with members of the basal transcription complex, its function as an initiator of transcription in the absence of TBP and its required presence for embryo viability (Seto et al., 1991; Usheva & Shenk, 1994; Shi et al., 1997; Donohoe et al., 1999). ERF has been proposed as being a regulator of cell proliferation during the G0–G1 transition (Le et al., 1999). Therefore, the level of expression and/or activity of such important molecules is likely to be exquisitely regulated within the cell. This is in keeping with observations that the decrease in the level of YY1 protein during differentiation is extremely subtle (Liu et al., 1994; Lee et al., 1994; Galvagni et al., 1998). We think it likely, therefore, that the processes of HCMV latency/reactivation are acutely responsive to the intracellular milieu. We have attempted to test this model by generating clones which either stably or inducibly overexpress YY1 and/or ERF irrespective of their state of differentiation. However, we have found that although it is possible to select for such antibiotic-resistant clones, careful analysis of protein or mRNA levels shows no increase in YY1 or ERF expression (M. Bain & J. Sinclair, unpublished results). We believe this may reflect the vital nature of YY1 and ERF (both presumed housekeeping factors) and that the cell will simply not tolerate such overexpression.

Although the repression domains of YY1 and ERF have been at least partly mapped and characterized it is unclear how these factors specifically repress HCMV MIEP activity. YY1 apparently represses transcription by diverse mechanisms such as DNA bending, interaction with basal transcription factors and association with a histone deacetylase activity (reviewed in Shi et al., 1997; Thomas & Seto, 1999). Observations which show that YY1 can not only block the recruitment of the general transcription factor TFIIB to a pre-initiation complex on the MIEP, but can also recruit the chromatin re-modelling factor histone deacetylase 3 (HDAC3), offer at least two possible mechanisms for the specific repression of MIEP activity by this factor (Sinclair, 1999; Thomas & Seto, 1999). Indeed, we have recently shown that chromatin acetylation/deacetylation plays a major role in the control of the NIEP in permissive and non-permissive cells (Murphy et al., 2002).

Our results reiterate that there is a degree of redundancy with respect to the sites through which a number of candidate repressors can prevent expression from the MIEP. Thus YY1 and ERF interact not only with the dyad repeat element, but also with the repeated 21 bp motif (Liu et al., 1994; Figs 2 and 4). These data are in agreement with observations which show that, although the dyad motif is important for mediating the repression of the MIEP in non-permissive cells, it is not the only region mediating this repression. Thus, both YY1 and ERF efficiently repress MIEP reporter constructs which lack the dyad motif (Liu et al., 1994; Fig. 4A).

It has not escaped our attention that other known ets-like factors appear to activate the MIEP in transfection assays (Fig. 3). Whilst we do not know if these factors mediate their activation via the 21 bp repeat motifs, it is possible that these sites act as repressors in undifferentiated non-permissive cells but activators in differentiated permissive cells. We are currently analysing whether the expression of these ets-like factors are differentially regulated in undifferentiated and differentiated cell types.

The role of the 21 bp repeats and the modulator in the context of viral infection is still a matter of debate. HCMV recombinants in which the modulator is deleted are still inhibited at the level of immediate-early gene expression in undifferentiated non-permissive cells (Meier & Stinski, 1997; Meier, 2001). This is in good agreement with a certain degree of redundancy in the MIEP. Factors which can no longer bind to the modulator simply mediate their effects through the 21 bp repeats (for example see Fig. 4). However, more recently a recombinant virus has been produced which lacks both the 21 bp repeats and the modulator. Surprisingly this virus is not ‘de-repressed’ with respect to IE gene expression in non-permissive cells when compared to wild-type virus (Meier, 2001), questioning the role of the 21 bp repeats and the modulator in the repression of the MIEP. However further, careful, analysis by these same workers has shown that such a mutant virus also lacks a critical distal enhancer region (-300 to -579 with respect to the transcription start site) which is crucial for the correct expression of the major IE genes (Meier & Pruessner, 2000; Meier et al., 2002). Consequently, as this virus appears compromised in the correct regulation of the IE genes in permissive cells, the use of such a virus in a comparison of the levels of IE expression in permissive and non-permissive cells may be problematic. Clearly, definite evaluation of the role of the 21 bp repeats and the modulator awaits the production of a more precisely mutated virus.

Because of the importance of monocytes in the carriage of the latent HCMV genome (Taylor-Wiedeman et al., 1991, 1994; Soderberg-Naucler et al., 1997), we are extending our analyses of these factors to the more biologically relevant THP-1 monocytic cell line. In addition, it will be interesting to note whether these putative repressor factors are similarly regulated in primary peripheral blood monocytes and monocyte-derived macrophages.


   ACKNOWLEDGEMENTS
 
We thank George Mavrothalassitis for the ERF expression plasmids. This work was supported by an MRC Programme Grant. M. Bain and M. Mendelson contributed equally to this work.


   REFERENCES
Top
ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
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Received 5 June 2002; accepted 4 September 2002.