It is widely recognized that DNA methylation inhibits transcription mainly by recruiting methyl-binding proteins that, associating with histone deacetylase and chromatin remodeling complexes, stabilize a condensed chromatin conformation (5
). In accordance with this, transfection and microinjection experiments of differentially methylated templates have demonstrated that transcriptional silencing often does not require modification of promoter DNA, suggesting that methylation effects are transmissible in cis
). In this work, microinjections of regionally methylated vectors into Xenopus
oocytes were used to address some basic yet unanswered questions regarding CpG methylation and to analyze the parameters influencing diffusion of methylation-mediated gene silencing and the importance of histone deacetylation in this spreading effect.
The human aprt
CpG island is characterized by a natural distribution and density of methylatable dinucleotides, and the HSV thymidine kinase promoter has been previously demonstrated to be inhibited by DNA methylation by an indirect mechanism (29
). Using a protocol that we optimized, we were able to obtain patch-methylated constructs characterized by having only a portion of this CpG island as the methylated region and the HSV tk
The major conclusions from this work are that (i) a certain number of modified cytosines are required to organize a stable, diffusible chromatin structure (Fig. to ), (ii) histone deacetylation contributes significantly to gene repression only when the number of modified sites is insufficient to exert repression over a long distance (Fig. ); and (iii) because of competition between transcriptional factors and methyl-binding proteins, promoter modification does not lead to a greater repressive effect (Fig. ).
DNA methylation can inhibit gene expression at different levels. Even though direct interference of modified CpG can block binding of transcriptional factors, this level of repression does not represent the main mechanism by which methylation-mediated gene silencing is exerted. In most cases, DNA methylation seems to repress gene expression by recruiting binding proteins specific for methylated DNA (5
). MBD proteins can, per se, block transcriptional factor association or mediate the formation of a repressive, inaccessible chromatin structure. Since it has been demonstrated that promoter methylation is often not required for inhibition, it is possible to foresee that a particular chromatin structure is seeded at methylated DNA and diffuses on flanking, unmodified sequences. Different parameters, such as the number or density of modified dinucleotides, might influence the composition of chromatin structure, its stability, and, therefore, its capability of spreading on the fiber.
The results shown in Fig. demonstrate that 11 modified CpGs upstream of the tk
promoter can silence its expression. Since, as demonstrated previously, this repression requires time to occur (Fig. ) and correlates with chromatin assembly (29
) (data not shown), we suggest that inhibition is not mediated simply by recruitment of a deacetylase activity which modifies transcription factors and/or the transcriptional apparatus. Therefore, nucleation of a repressive chromatin structure at the methylated DNA and its diffusion on the unmodified tk
sequences must occur. Decreasing the number of modified dinucleotides (Fig. ) reveals that even three methyl groups are able to modulate tk
expression, and a nonlinear relationship between the number of methylated sites and repression level is observed.
However, the importance of the number of methylcytosines shows up when the ability of the repressive effect to spread over a long distance is investigated (Fig. ). In fact, our results demonstrate that inhibition can propagate for several hundreds of base pairs 3′ or 5′ from the modified DNA only when a sufficient number of CpGs are methylated. In contrast, a short region of methylated DNA silences only an immediately adjacent tk promoter, suggesting that a few methylated sites cannot seed the formation of the repressive chromatin structure, nor can they guarantee its stability.
The analysis of the contribution of histone deacetylation to this repression (Fig. ) seems to reinforce these observations. In fact, our experiments show that histone deacetylation is of fundamental importance to the silencing mechanism only when the number of modified sites does not reach the threshold sufficient for an effect over a long distance. We propose (see the model in Fig. ) that when only a limited number of modified dinucleotides are close to a promoter, they recruit MBD proteins and their associated histone deacetylation activity; histone deacetylation occurs, remaining localized to a small number of nucleosomes, and transcriptional repression is observed. In this situation, trichostatin A treatment allows bypassing the main mechanism by which methylated DNA silences gene expression, and therefore inhibition is relieved.
FIG. 8. Model for the molecular mechanisms of gene silencing mediated by DNA methylation. The left side of the model represents the repressive mechanism determined by a small number of modified dinucleotides; on the right, the hypothetical mechanism by which (more ...)
The repression mechanism is significantly different when the number of methylated sites is increased and reaches the threshold that leads to diffusion of gene silencing on the DNA fiber. We propose that in these conditions, a specialized chromatin structure, formed not only by MBD proteins but also by other structural and remodeling activities, is organized on the modified DNA. Afterward, nucleation of this chromatin conformation and propagation on the flanking, unmodified DNA occur (Fig. , right panel). The contribution of histone deacetylation to transcriptional inhibition in these conditions is of secondary importance; in fact, even in the presence of trichostatin A, transcriptional levels remain significantly lower than in the unmethylated controls (Fig. ).
Obviously, we cannot exclude that even a limited number of methylated CpGs can recruit on modified DNA proteins other than histone deacetylases; however, if this is the case, the chromatin structure nucleated is not strong enough to propagate itself, and transcriptional repression is mainly due to histone deacetylation anyway (Fig. and ).
We believe that our data and the proposed model can explain the apparently contradictory data existing in the literature about the response of methylated DNA to trichostatin A treatment. In fact, while methylated transfected genes can be reactivated by tricosthatin A (reviewed in reference 46
), naturally densely methylated endogenous genes cannot be reinduced with trichostatin A alone. Remarkably, this drug does lead to a strong reexpression of several hypermethylated tumor suppressors only following minimal demethylation by 5-aza-2′deoxycytidine treatment (10
Even though the model was obtained from experiments performed with the tk
promoter injected into Xenopus
oocytes, we think that it might be considered a general mechanism of methylation-dependent gene silencing. In fact, the oocyte system has proven generally useful in defining transcriptional regulation in a chromatin context, permitting confirmations of earlier results obtained with different experimental systems (1
). In particular, in studying DNA methylation, Xenopus
oocytes were used to confirm and extend existing data deduced from microinjections of methylated templates into mammalian cells (8
). Molecular characterization of Xenopus
MeCP2 and other MBD proteins demonstrated almost identical behavior in mammalian and frog cells (5
). Moreover, since, as already mentioned, it has been reported that DNA methylation inhibits gene expression mainly by an indirect mechanism and that methylation effects are transmissible in cis
, our data seem to be in perfect accord with previous reports.
The response of differentially methylated templates to trichostatin A also seems to confirm previous observations obtained with different promoters (10
). Therefore, we believe that this model of methylation-dependent gene silencing can be applied to other promoters; the final level of repression as well as the number of mCpGs useful to establish a stable repressive structure will vary depending on the promoter strength and transcription factor concentration present in the particular cell type.
It is generally assumed that methylation effects are more effective when the regulative gene sequences are modified (42
); therefore, changes in promoter methylation are usually investigated as causes of loss of gene function in epigenetic events (4
). The results shown in Fig. seem to contradict this assumption; in fact, injection of regionally methylated constructs clearly demonstrates that when the tk
promoter is modified, despite the high number of methylated CpGs, transcriptional inhibition is much weaker than that seen on templates characterized by a smaller number of methylated sites positioned upstream of the promoter. Moreover, the ability of exogenous activators to overcome these methylation effects (Fig. ) demonstrates the existence of competition between transcription factors and MDB proteins in vivo. This effect is not restricted to a particular transcriptional factor; in fact, any activator analyzed (TBP, GAL4VP16, TR, OCT1, and OCT2) is able to reinduce tk
expression. However, the final transcriptional levels are a function of the factor examined.
It is important to note that in these experiments, exogenous proteins are highly expressed; we believe that in a more physiological context, transcription from a methylated promoter is a function not only of the strength but also of the concentration of the specific activator. The experiment performed using only the GAL4 DNA-binding domain indicates that an increase in the abundance of a DNA-binding protein does not eliminate the silencing effect; an activation domain seems to be required. In the future, it will be interesting to compare the nucleoprotein structure organized over the methylated promoter in the presence of GAL4 or GAL4VP16.
Finally, the experiment shown in Fig. demonstrates that an increase in the concentration of a general transcription factor, such as TBP, cannot block the silencing effect imposed by the methylation of a nonregulatory region. This result confirms all previous experiments (Fig. to ), in which it was evident that even though the general transcription machinery is present in oocytes and able to sustain transcription from a tk promoter, it cannot impede spreading of the silencing effect. Therefore, in this context, DNA methylation exerts a dominant transmissible repression. As already demonstrated, expression of a strong activator, such as GAL4VP16, can, however, reinduce expression.
In conclusion, when a promoter is methylated, its expression will depend mainly on transcriptional factor abundance and, of course, on the ability to bind to methylated recognition elements. On the contrary, when DNA sequences lacking transcription factor binding sites are modified, MBD proteins can easily bind and seed a repressive chromatin structure involving nonmethylated flanking regulatory sequences. In this situation, only specific activators can overcome the inhibitory effect.