In this historical paper, Antequera et al. (1989) demonstrated that methylated CpGs were specifically associated with MeCP, a protein that was the first characterized from the MBD family. They started with a simple -but very powerful- hypothesis: If specific proteins are bound to methylated CpG rich sequences, this DNA should be resistant to the restriction enzyme cleavage, in contrast with unmethylated CpGs that would be fully accessible. To test this, they employed two methylation sensitive (HpaII and HinPI) enzymes, and MspI, that cleaves CpGs irrespective of its methylation status. They first cleaved liver nuclear DNA with the three enzymes, and then the digested DNA was end-labelled with radioactive dCTP and the Klenow fragment of the DNA Polymerase I. Electrophoretic separation showed a similar pattern for the three enzymes, a ladder of ~200 bp fragments, that corresponded to the nucleosomal repeat. These results were surprising since the difference in sensitivity to methylation between these enzymes. They evaluated if DNA- associated proteins were responsible of this, by repeating the experiment in naked DNA, and this time MspI cut more frequently than the other enzymes. This showed that MspI was insensitive when naked DNA was used as substrate, but it became indistinguishable from methyl-CpG sensitive enzymes when chromatin is cleaved. This particularity was confirmed by digestion with enzymes that contained no CpGs in their recognition sites (HaeIII and Sau3A). Also, it was observed that this resistance to cleavage could not be overcome by the addition of more restrictase. Confirmation that MspI was cutting only in unmethylated DNA was seen by thin layer chromatography of the 5' terminal Cs, after full DNase I digestion of the 32P-ATP labelled DNA. Using another enzyme (Tth) proved that the cleavage blockage was not unique to MspI. However, artificially methylated DNA was cleaved by MspI, thus showing that the methylation alone is not enough to inhibit the cleavage, but it was a consequence of the exclusion by 5mCpG- associated proteins. As this results predicted, analysis of specific sequences (here they used a 16 Kb region of the alpha-globin gene) showed the same effect, as seen by Southern blot. They considered that MeCP, a methyl-CpG binding protein, could be responsible of the protection. To evaluate this possibility, they used PC13 cells, which have very low levels of MeCP, and measured the amount of cleaved methylated CpG by thin layer chromatography. This time, although some protection was still visible, the amount of cleavage in methylated sites was considerably greater than in brain cells. In vitro protection assays gave the same results. Taken together, their results demonstrated that, although nucleosomes protected the DNA from restriction endonucleases, as seen by the ~200 bp ladders formation, they could be associated with methylated sequences, but because the reduced protection of 5mCpGs in PC13 cells, these could not be responsible of the less efficient digestion; however, MeCP is a better candidate, as it binds CpGs in several sequence contexts, and enzymatic cleavage protection was reduced in PC13 (MeCP deficient) cells and in vitro. From this, the authors postulated that MeCP binds to the 5mCpG sequences, and could function as a transcriptional repressor.
Antequera, F. et al. 1989. Cell 58(3):509-517
epigenetics
Antequera, F. et al. 1989. Cell 58(3):509-517
epigenetics
