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
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ABSTRACT |
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Present address: Infectious Diseases Clinical Research Unit, The Lung Institute, University of Cape Town, Cape Town, South Africa
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Introduction |
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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-
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.
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Methods |
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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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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 : 51 : 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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Results |
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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. 5
A). 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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Discussion |
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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 G0G1 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.
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ACKNOWLEDGEMENTS |
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Received 5 June 2002;
accepted 4 September 2002.