Sometime between the age of 6 and 18 months, after a period of seemingly normal development, girls affected with Rett Syndrome lose interest in play; they gradually become withdrawn and anxious, develop autistic-like behaviors, and acquire specific symptoms like repetitive teeth-grinding and hand-wringing. This devastating neurological disease affects one in 15,000 female children.
Just five years ago, Rett Syndrome was tracked to mutations in a gene on the X chromosome, MECP2 . But how this gene, not previously associated with the brain or nervous system, could cause a neurological developmental disorder remained a puzzle.
Now, a team of scientists with the U.S. Department of Energy's Lawrence Berkeley National Laboratory has developed new methods and overturned mistaken assumptions to discover how the product of this gene, the protein MeCP2, can remodel chromatin, the material that makes up chromosomes. For the first time a human disease — Rett Syndrome, the first identified epigenetic disease — has been linked to specific defects in the three-dimensional folding of chromatin.
The research was supervised by Terumi Kohwi-Shigematsu, a biochemist with Berkeley Lab's Life Sciences Division; it reveals how mutated MeCP2 protein represses genes, and identifies some of the most important of those genes. Kohwi-Shigematsu and her colleagues, Shin-ichi Horike, Shutao Cai, Masaru Miyano, and Jan-Fang Chen, report their results in advanced online publication of the January issue of Nature Genetics.
How MECP2 works
So-called CpG "islands" are found at the promoter regions of many housekeeping genes, which code for proteins essential to cell function. They contain high densities of cytosine and guanine base pairs, called CpG dinucleotides. MECP2 stands for "methyl CpG-binding protein 2;" as its name indicates, it can bind to these base pairs when methyl groups (CH3) are attached to them.
Normally, CpG dinucleotides in CpG islands are not methylated and the genes are active. If CpG islands are methylated, however, they attract MeCP2 proteins, which bind additional proteins that repress gene transcription and turn the promoter off. Thus MeC2P is thought to be a key player in assembling the protein factors that silence transcription. Because MeCP2 binds to methylated CpG dinucleotides, its effects are not dependent on the primary sequence of DNA.
"One proposal for how defective or absent MeCP2 protein might cause Rett Syndrome was that, by failing to attach to methylated CpG dinucleotides, it would fail to repress inappropriate gene expression in the brain," says Kohwi-Shigematsu.
For some genes, called imprinted genes, their expression status depends on whether the gene came from the maternal or paternal allele, with the two forms often having differently methylated CpG islands at or near their promoters. A leading hypothesis of how mutated MECP2 could produce Rett Syndrome is that the mutation disrupts this imprinting mechanism.
An imprinted gene, one with a methylated promoter, is usually silent. If defective or missing MeCP2 protein were to fail to silence an imprinted allele, the expression of the gene would double. Failure to repress imprinted alleles has been implicated in several neurological disorders.
"MeCP2, the protein coded for by the MECP2 gene, is expressed in many tissues, including brains," says Kohwi-Shigematsu. "People thought it was a general repressor that regulates gene expression throughout the body. Yet the main syndrome of Rett patients pointed to neurodevelopmental problems after birth. So our first challenge was to find out which genes MeCP2 directly regulates in the brain, and how it regulates them."
Searching for targets
The researchers examined hundreds of MeCP2 binding sequences in the brains of mice. In wild-type mouse brains they found that the MeCP2 protein binds in the vicinity of some five dozen genes, several of which reside in a cluster of imprinted genes on mouse chromosome 6 (corresponding to a region of human chromosome 7).
When the binding sites in wild-type mouse brains were compared to the same sites in MeCP2-null mice — "knockout" mice bred with no MecP2 gene and thus no MeCP2 protein — one region in particular stood out: expression of the adjacent genes Dlx5 and Dlx6 almost doubled in the knockout mice. The identification of the Dlx5 gene in mice was highly suggestive, since in humans the DLX5 protein plays an important role in the synthesis of GABA, gamma-aminobutyric acid, an important neurotransmitter.
The researchers sought similar effects in cells from Rett Syndrome patients, substituting cultured lymphoblasts (immune-system cells) for inaccessible brain cells. In humans, normally only the maternal allele of DLX5 expresses, because the paternal allele is imprinted. But lymphoblasts from many Rett Syndrome patients exhibited a much higher rate of DLX5 expression. When an imprinted gene nevertheless continues to express, the phenomenon is called "loss of imprinting."
The researchers had now identified at least one gene targeted by MeCP2 where, if the protein were missing or defective, the result might lead to misregulation in the production of the neurotransmitter GABA. But the mechanism by which normal MeCP2 acts to regulate the DLX5 gene and how this regulation goes awry were still to be determined.
One thing was clear: MeCP2's propensity to bind to methylated CpG islands played no part. CpG islands near Dlx5 and Dlx6 were found — quite unexpectedly — to be completely unmethylated in wild-type mice, knockout mice, and the human lymphoblast cell line. Even where individual CpG base pairs in the region outside CpG islands were methylated, there were no differences in methylation patterns between the maternal and paternal alleles.