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ª Oncogene (2002) 21, 5388 – 5393 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc Gene silencing in mammalian cells and the spread of DNA methylation Mitchell S Turker*,1,2 1 Center for Research on Occupational and Environmental Toxicology, Oregon Health and Science University, Portland, Oregon, OR 97201, USA; 2Department of Molecular and Medical Genetics, Oregon Health and Sciences University, Portland, Oregon, OR 97201, USA Aberrant gene silencing in mammalian cells is associated with promoter region methylation, but the sequence of these two events is not clear. This review will consider the possibility that gene silencing is not a single event, but instead a series of events that begins with a dramatic drop in transcription potential and ends with its complete cessation. This transition will be portrayed as a chaotic process that ensues when transcription levels drop and DNA methylation begins spreading haltingly towards the diminished promoter. According to this view, silencing is stabilized when the promoter region is ‘captured’ by the spread of DNA methylation near or into its transcription factorbinding sites. Oncogene (2002) 21, 5388 – 5393. doi:10.1038/sj.onc. 1205599 Keywords: DNA methylation; methylation spreading; silencing Introduction This review is meant to be speculative. It will focus on methylation-associated gene silencing in mammalian cells and it will take the view that a decrease in transcription is just the first step in a gradual process that involves a feedback loop between diminishing transcription and the spread of DNA methylation. In my opinion normal methylation patterns represent a balancing act between competing forces; open chromatin established by an active promoter and the spread of DNA methylation from a pre-existing focus (Turker, 1999). If so, a significant diminishment in transcription would lead to a loss of this balance allowing DNA methylation to spread towards the promoter. The presence of variegated methylation patterns near silenced promoters will be used to build a model in which transcription smolders before being extinguished. Methylation patterns result from opposing forces that promote and block DNA methylation Many regions of the mammalian genome are methylated at one or more CpG sites (Bird, 2002) and exhibit *Correspondence: MS Turker, CROET, L606, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201, USA; E-mail: [email protected] allele specific methylation patterns (Chandler et al., 1987; Silva and White, 1988; Turker et al., 1989). These patterns are formed during embryonic development (Reik et al., 2001) beginning with genome wide demethylation that commences shortly after fertilization (Monk et al., 1987; Howlett and Reik, 1991; Santos et al., 2002). Remethylation of most of the genome occurs after the blastocyst implants (Monk et al., 1987; Okano et al., 1999; Santos et al., 2002) and continues at a slower pace during the remainder of development. It is believed that this demethylation/ remethylation process converts gamete specific methylation patterns to those specific to somatic cells. Remethylation of the genome can be divided into two steps involving different forms of de novo methylation (Turker, 1999). The first is de novo methylation that occurs at discrete CpG sites throughout the genome due to the presence of cis-acting methylation signals (Figure 1a). Once CpG sites within cis-acting signals are methylated they can act as foci for a second form of de novo methylation, which is spreading to distal CpG sites (Figure 1b,c). After a CpG site becomes methylated via spreading this modification is preserved via maintenance methylation, which involves the targeting of DNMT1 to hemimethylated DNA shortly after replication (Leonhardt et al., 1992). Most spreading of methylation ceases about the time of birth, although low level spreading may continue during an individual’s lifetime (Issa, 2000; Ahuja and Issa, 2000). While the phenomenon of spreading has not been defined mechanistically, I suggest that it represents a self-perpetuating interaction between chromatin modifying proteins and DNA methylation. According to this view, DNA methylation closes chromatin via the attraction of repressive complexes (Bird and Wolffe, 1999). The presence of these complexes will, in turn, alter nearby chromatin making it accessible to the spread of methylation. The net effect of this interaction is the gradual spreading of methylation along the DNA molecule until it run into a counteracting force in the form of open and active chromatin (Figure 1c). Based on our work with the mouse Aprt gene region, I have proposed that the initial de novo methylation events are signaled by cis-acting elements that are dispersed throughout the genome (Turker, 1999) (Figure 1a). For Aprt, we have identified two upstream B1 repetitive elements as providing a significant de novo methylation signal (Yates et al., 1999). They reside Silencing and DNA methylation spreading MS Turker 5389 Figure 1 Dynamic processes shape methylation patterns. Methylation is established at CpG sites (closed circles) by cis acting sequences (striped rectangle) (a) and then begins spreading to unmethylated CpG sites (open circles) in both directions (only one direction is shown) (b) until it reaches an open chromatin domain (c) established by the expressed promoter (flag, ‘+’ represents relative level of expression). The junction between the open chromatin domain and methylation spreading is considered to be a boundary between the repressive complexes that bind methylated CpG sites and open chromatin. Shifting of the boundary can move methylated CpG sites into the open chromatin domain, causing them to become demethylated (d) within a larger DNA fragment that we have termed a methylation center (Mummaneni et al., 1993). Other retroposons that are also suspected of signaling de novo DNA methylation are B2 (Hasse and Schultz, 1994), Alu (human equivalent of B1) (Magewu and Jones, 1994; Graff et al., 1997), LINE-1 (Liang et al., 2002), and mouse IAP (Walsh et al., 1998). De novo methylation of retroposons may be a protective function to block their transposition (Yoder et al., 1997). Once the B1 elements upstream of Aprt become methylated, methylation spreads towards the promoter (Figure 1b,c), which is comprised of three consensus Sp1 binding sites. It has also been shown in transgenic mice that DNA methylation can spread from an integrated retrovirus to flanking sequences in the mouse genome (Jähner and Jaenisch, 1985). Similarly, it has been shown in plants that short retroposons can act as nucleation centers for de novo DNA methylation and that DNA methylation then spreads from these centers to distal sites (Arnaud et al., 2000). The Aprt promoter provides a counteracting force to spreading as demonstrated by two types of studies. One used embryonic cells and developing embryos to show that removal or site directed mutation of one or more of the Sp1 binding sites allows methylation to spread into the promoter (Mummaneni et al., 1995, 1998; Brandeis et al., 1994; Macleod et al., 1994). Because the site directed mutations were designed to eliminate transcription factor binding it was assumed that protein binding plays an important role in blocking the spread of DNA methylation, presumably by maintaining an open chromatin domain around the promoter region. A second set of studies has shown that the addition of Sp1 binding sites can prevent transgene (Siegfried et al., 1999) and retroviral (Hejnar et al., 2001) silencing. Additional studies provided evidence that DNA methylation patterns are not static in somatic cells, as often assumed, but instead can vary at individual CpG sites (Silva et al., 1993; Turker et al., 1989). For mouse Aprt, we showed that a CpG site located between the Aprt promoter and the methylation center is methylated on approximately 25% of alleles, and that this level persisted even when the cells were subcloned (Turker et al., 1989). These observations can best be explained by assuming that methylation for CpG sites on a given allele can be either gained or lost. By combining the results from the above studies, I proposed a model depicting a ‘dynamic equilibrium’ between forces that support the spread of DNA methylation and those that actively block this spread (Turker, 1999). The frontline where these forces meet (Figure 1c) can be considered a boundary that separates repressive complexes from those that keep chromatin in an open and expressed conformation (Gerasimova and Corces, 2001). An A-rich sequence in the human GSTP1 gene has also been shown to form a boundary between methylated and unmethylated DNA (Millar et al., 2000). For mouse Aprt, the upstream methylation center and the promoter provide the opposing forces of DNA methylation spreading and blocking. According to the model, the boundary can shift back and forth along the DNA molecule and methylated CpG sites caught behind the boundary are at risk to switch from being methylated to being unmethylated (Figure 1d). If this model is correct, it follows that the protective force of the Aprt promoter will extend at least several hundred bases upstream from the end of its associated nuclease sensitivity (Cooper et al., 1993) and nucleosome positioning (Macleod et al., 1994) regions. Although a mechanism for direct removal of methyl groups from DNA has been proposed (Bhattacharya et al., 1999) and debated (Bird, 2002), the boundary model is more consistent with a replication dependent mechanism (Santos et al., 2002; Rougier et al., 1998) in which maintenance methylation is blocked during S phase (Bird, 2002). A final aspect of this model is that methylation of demethylated sites will occur anew via spreading when the front line shifts again towards the promoter (not shown). Fortunately for Aprt, the frontline of the battle is located several hundred base pairs away from the promoter, and while some shifting of this frontline can occur, it rarely moves close enough to the promoter to jeopardize gene expression. The last section of this review will deal with what happens when silencing begins, and the blocking force is weakened thereby allowing the spread of methylation towards the promoter. First I will consider some things we know and do not know about silencing. Gene silencing in mammalian cells A simple definition of gene silencing is the conversion of an actively expressed allele to one that is not Oncogene Silencing and DNA methylation spreading MS Turker 5390 expressed. This term usually denotes aberrant loss of expression. In mammalian cells, gene silencing is most often associated with hypermethylation of the promoter region and a closed chromatin conformation (Robertson and Jones, 2000; Rountree et al., 2001; Baylin et al., 2001; Jones and Wolffe, 1999). Chromatin conformation is controlled to a large extent by modification of lysine residues on histones tails (Jenuwein and Allis, 2001). These modifications include acetylation/deacetylation and methylation/ demethylation at lysine positions 4, 9 and 14. Histone acetylation, deacetylation and methylation are enzymatically catalyzed, but histone demethylation may require a more complex process. In general, histone acetylation is associated with active chromatin and deacetylation is associated with closed chromatin. DNA methyl binding proteins attract histone deacetylases, which appears to explain the relationship between DNA methylation and gene repression (Bird and Wolffe, 1999; Wade, 2001). For histone methylation the picture is more complex because methylation of lysine 4 is associated with active chromatin and methylation of lysine 9 is correlated with inactive chromatin (Jenuwein and Allis, 2001). The term ‘histone code’ (Jenuwein and Allis, 2001) has been used to describe the various potential combinations of histone modifications. Because many different combinations of histone tail modifications can potentially exist, it is possible that the level of gene expression for a given allele can vary from very high to undetectable. If so, the specific histone modifications that are present near the promoter on a given allele could determine the probability that the promoter will be able to bind to transcription factor, the strength of this interaction, and perhaps for how long it will last. This interaction, in turn, could dictate how far the boundary is located away from the promoter. As discussed in the previous section, DNA outside the boundary would be methylated and histones outside the boundary would have repressive modifications. However, these modifications would pose little threat to the promoter as long as the boundary is present. But what would happen if transcription were disrupted transiently for a given allele? During that time the boundary would be weakened significantly or disappear, which would initiate a battle for the allele between repressive forces that are trying to silence expression permanently and a promoter that is struggling to regain high level transcription. The outcome of a battle for a given allele is not predetermined as evidenced by observations such as position-effect-variegation (PEV) for eye color in Drosophila (Wakimoto, 1998; Weiler and Wakimoto, 1995). Eye color PEV is caused by the translocation of an active gene region to a heterochromatic (i.e. repressive) region, leading to cell-to-cell variation for expression of an eye pigment gene. This gene is termed white because the normally red eye cells appear white when expression is lost. Since each cell inherits exactly the same translocation event, the cell-to-cell difference in color demonstrates the lack of a predetermined Oncogene outcome for each translocated allele. DNA methylation occurs at extremely low levels in the fly (Gowher et al., 2000; Lyko et al., 2000) and thus is unlikely to be involved in gene repression. This suggests that allelic battles for expression can occur in the absence of DNA methylation, and by extension that chromatin modifications play the central role. Not surprisingly, it has been shown that altered expression of a variety of chromatin modifying proteins (Wakimoto, 1998; Weiler and Wakimoto, 1995) can influence the percentage of red versus white cells in a given eye. Cell to cell variation has also been shown in the mouse, perhaps best visualized as variegated coat color due to an insertion of an IAP retroviral element into the agouti locus that causes cell-to-cell variation in silencing of this allele (Duhl et al., 1994; Michaud et al., 1994). In these cases, DNA methylation of the IAP element has been correlated with loss of agouti expression. An important component for the silencing process is the initiation event, which unfortunately is a part of the process that has not been defined mechanistically (Baylin and Herman, 2000). If DNA methylation initiates silencing in mammalian cells by spreading past the protective boundary and recruiting repressive complexes, loss of transcription should be stable once it occurred. Consistent with this notion are numerous studies showing that the methylated promoters are stably inactivated and not expressed at detectable levels, presumably because methyl-binding proteins attach to the methylated CpG sites and attract repressive complexes (Bird and Wolffe, 1999; Rountree et al., 2001; Wade, 2001). The problem with methylation spreading being the trigger for silencing in mammalian cells is that evolving processes have been observed in those instances where it has been possible to follow silencing. In a study of p16 inactivation in human mammary epithelial cells that escape senescence after stable transfection with the E6 papillomavirus gene, it was shown that escape from senescence was correlated initially with a decrease but not complete loss of p16 expression (Wong et al., 1999). Significantly, at this early time point some p16 alleles were found to be unmethylated in the promoter region. Eventually, as the cells continued to be passaged, promoter region methylation increased and the levels of p16 expression became undetectable. Additional evidence to support a gradual silencing process came from a study of silencing of a retroviral LTR (long terminal repeat) fused to a GFP reporter gene in mouse leukemia cells (Lorincz et al., 2000). In this study, silenced GFP constructs were reactivated by treatment with the deacetylating agent trichostatin A (TSA) soon after construct silencing occurred. The reactivated alleles were then found to gradually silence again, but for many cellular passages the silenced alleles could be reactivated anew by treatment of the cells with either TSA or the demethylating agent 5-azadC. However, with continued passage the silenced alleles eventually reached a point where they became refractory to either TSA or 5-aza-dC exposures by themselves. Increased methylation levels were corre- Silencing and DNA methylation spreading MS Turker 5391 lated with loss of responsiveness to TSA or 5-aza-dC. Similarly, in a recently completed study (submitted) we have found that mouse Aprt constructs exhibited at least two stages of silencing in a differentiated cell type. The first stage was characterized by ‘leaky’ (i.e. readily detectable) low-level transcription in most cells and very high spontaneous reactivation frequencies. At this stage many of the silenced alleles exhibited methylation patterns virtually identical to those observed for expressing alleles. The second stage, which apparently required the first stage as an intermediate, was characterized by loss of transcription and loss of spontaneous reactivation. Increased levels of construct methylation correlated with stabilization of the silencing process. In all three studies mentioned above the observed methylation patterns were variegated rather than complete. Variegated patterns are defined by alternating regions of methylated and unmethylated CpG sites (see Figure 2 for an unpublished example from our work). Moreover, in all three studies the variegated patterns were different from allele to allele, even though clonally derived cell populations were examined. These observations can only be explained by assuming that DNA methylation patterns are evolving on silenced alleles, and that silencing is a process not a single event. Similarly, a study that correlated transcription and DNA methylation levels for the human p15 gene in acute leukemias also led to the suggestion of an evolving process, at least for methylation changes (Cameron et al., 1999). An evolving silencing process would be in marked contrast to a wide spectrum of mutational events, which require no more than two cell divisions to become established (Turker, 1998). The next section describes how an initial decrease in transcription can start a process that causes the spread of methylation and the formation of alleles with variegated methylation. Figure 2 Example of a variegated methylation patterns for unstably silenced alleles. Unpublished work from the author’s group showing bisulphite sequencing analysis for 19 CpG sites (circles) on 14 Aprt alleles isolated from a clonally derived cell population expressing low levels of activity and high spontaneous reversion frequencies. In these constructs methylation initiates mostly at CpG sites 1 – 3 and methylation spreads from these CpG sites to the promoter region, which contains 2 Sp1 binding sites. CpG sites 13 and 16 are within these binding sites. Closed and open circles represent methylated and unmethylated CpG sites, respectively. The distances separating CpG sites represents relative distances that they are apart on the Aprt plasmid that was examined. Typical of silenced alleles obtained from a clonal population, only two of the 14 alleles exhibit identical methylation patterns Silencing and the spread of methylation There are two ways to consider the relation between silencing and the spread of DNA methylation. One is that the spread of DNA methylation into the promoter causes silencing via the attraction of chromatin modifying factors. This, in turn, would lead to a loss in transcription factor binding. According to this scenario silencing, as defined as loss of transcription, would be a rapid and relatively stable process. However, the presence of silenced alleles with variegated methylation patterns in tumor material (Melki et al., 2000; Song et al., 2001; Dodge et al., 1998; Nosaka et al., 2000; Cameron et al., 1999) and cultured cells (Wong et al., 1999; Lorincz et al., 2000; Domann et al., 2000; Liang et al., 2000) suggests an alternative scenario in which silencing is a gradual and evolving process that involves promoter region methylation as a very late event. I speculate that silencing initiates with a dramatic, albeit incomplete and still undefined, drop in transcription that could result from spontaneous, environmentally-induced, or malignancy-related perturbations in expression acting at the level of individual alleles. Silencing then progresses via series of mutually reinforcing steps that gradually reduce the probability of transcription factor binding. This creates a situation in which a given promoter is bound by transcription factor for periods of time interspersed with increasing periods of time when the promoter is unoccupied. Because the boundary that acts to prevent methylation from spreading towards the promoter is a product of transcription factor binding, a chaotic scenario will ensue in which the boundary to methylation spreading will alternately disappear and reappear (Figure 3). As a result methylation will spread towards the promoter in a halting process, slowed or even reversed occasionally when transcription factor binding occurs and the boundary is formed anew. At the same time, because of the interrelationship between histone modification and DNA methylation, repressive modifications will move towards the promoter, also in a halting and reversible process. Moreover, because the location of a boundary should be a function of the strength and longevity of transcription factor biding, which in turn could be affected by the proximity of DNA methylation, the boundary would develop closer and closer to the promoter each time it is reformed. This evolving situation will create interesting circumstances, such as when a boundary forms upstream of methylated CpG sites (Figure 3). Such a transient event would allow methylated CpG sites to become unmethylated if transcription and hence the open domain, is restored and maintained during replication. However, because boundaries are forming and disappearing continuously, there is no certainty that an unmethylated CpG site caught outside the protective domain will become methylated, or alternatively that a methylated site caught inside the protective domain will become unmethylated. Instead, because the boundary is being formed, lost, and formed again, there are Oncogene Silencing and DNA methylation spreading MS Turker 5392 Figure 3 Silencing as a process. An expressed allele (a) with a boundary between DNA methylation spreading and the open chromatin region of the expressed promoter (flag with ‘+’) undergoes a dramatic loss in transcription (flag with ‘7’), which eliminates boundary allowing methylation to spread closer to promoter. A series of restoration and loss of promoter expression (c – g) ensues, with transcription levels decreasing as methylation encroaches. Finally, a sufficient density of methylation and/or methylation of critical CpG sites (filled in flag) occurs leading to stabilization of silencing (h). A by-product of this process is the formation of variegated methylation patterns continue inexorably because the spread of DNA methylation and closing of chromatin due to histone modification will feedback upon each other. During malignant progression this process will be hastened by selection for alleles with progressively lower levels of expression because loss of normal regulation of cell growth and sometimes DNA repair pathways are critical for the development of cancerous cells (Turker, 1998). While it may be possible for a rare allele to reverse the process, particularly if selection for expression is present, for most alleles DNA methylation will continue spreading towards the promoter as the interaction between the promoter and transcription factor weakens in strength and decreases in duration. Moreover, by influencing chromatin conformation via the attraction of methyl-binding proteins, the spread of methylation will play a role in decreasing this interaction, further facilitating its spread. The battle finally ends when a sufficient number of CpG sites near or within the promoter and/or critical CpG sites become methylated to preclude further interaction between the promoter and transcription factor, though there may be remaining unmethylated CpG sites that will eventually succumb to methylation. Omissions and conclusion increasing probabilities for each CpG site being methylated as the boundary is pushed gradually towards the promoter, but no guarantees. The net result of this ongoing process is the gradual advance of DNA methylation towards the promoter and the formation of alleles with variegated methylation patterns. The reader is reminded that Figure 3 shows the CpG methylation history for only a single allele and that each allele could potentially have its own unique history. Therefore, countless variegated patterns will be observed when ‘sampling’ the methylation pattern for a silenced gene (Figure 2). Finally, by combining the concept of shifting boundaries shown in Figure 1 with the concept that CpG sites caught in these shifting regions will sometimes, but not always, change methylation status (Figure 3), it is also possible to explain variegated DNA methylation patterns that have been reported for non-silenced regions of the genome (Liang et al., 2002; Millar et al., 2000; Hansen et al., 2000; Bourc’his et al., 2001; Hanel and Wevrick, 2001). Unlike position effect variegation, which is established and fixed during development, the process described above for a dividing cell population will To keep this review focused, I omitted or discussed minimally a number of potential additional players. These include maintenance methylation, de novo methylation, the different DNA methyltransferases and their functions, non-CpG methylation, demethylation mechanisms, the aberrant mechanisms that cause the initial diminishment of transcription, genomic damage, and selective processes. 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