Close examination of the genes that MeCP2 repressed also revealed a counterintuitive finding. These sequences had heavily methylated islands, even though there were not many islands in total. That odd combination seemed to convince MeCP2 to turn off the gene. Research continues on many such genes in an attempt to understand the specific actions of this talented protein’s global reach (Figure).
But what about cognition? Rett syndrome and autistic spectrum disorders are phenomena that affect the ability to process specific kinds of information. Were any molecular interactions observed in these experiments that might give hints to these effects on cognitive behaviors? The answer to this question was yes, and it was the most exciting part of the work.
Narrowing the choices
The MeCP2 protein was certainly binding things in island-specific ways. But to what molecules, among hundreds of thousands possible, is it associating? Could the characterization of its partners lead to a greater understanding of the role of MeCP2 in Rett syndrome and autism?
It is possible to answer at least the binding parts of these questions in 2 ways. First, you can use a mass spectrometer to identify proteins associated with your target (as long as you are clever about stabilizing native molecular associations in specific tissues in your sample). Mass spectrometry can identify molecules based on their mass-to-charge ratios (in essence, you chemically fragment your sample into ions and then calculate the mass-to-charge ratio by passing them through a series of electric and magnetic fields). The researchers uncovered a whopper—MeCP2 was associating with CREB. A molecule whose mutations were known to be involved in autistic behaviors was actually binding to a molecule like CREB, known to be involved universally with information processing!
Researchers confirmed this using the chromatin immunoprecipitation protocol. This technique (described previously) involves isolating the histone-DNA-regulatory protein combinations by using antibodies that are capable of binding to ordered chromatin structures. The researchers were able to show that CREB was involved in half the promoters to which MeCP2 was binding. Specifically, CREB was associated with MeCP2 at the promoters where MeCP2 was an activator. Remarkably, CREB was completely absent at promoters that MeCP2 was known to be repressing. In other words, MeCP2 was specifically associating with molecules that normally associated with learning and was activating the genes to which both bind! Not only did this confirm the data from the mass spectrometry, it also extended and refined the results (see Figure).
Clearly, MeCP2 interacts with a broad swath of the transcription regulatory machinery available inside brain cells, both to activate and repress. It specifically interacts with CREB, a protein whose role in learning is preserved over a broad phylogenetic range.
Like all good data, however, these raise as many questions as they answer. At what level are these interactions occurring? Do they interact directly with RNA polymerase II at the promoter, thus exerting more local effects? Do they help in remodeling the higher-order chromatin structure, thus exerting more global effects? Does it do both? Most important, how do the deficits associated with Rett syndrome and autism come from the mutations observed in MeCP2? These questions will represent the next generation of experiments.
A number of caveats must be mentioned, of course. First, these findings have been shown in laboratory animals, not in humans, and the normal grumpy cautions are in order (these are somewhat assuaged because of the remarkable phylogenetic conservation of CREB-mediating learning responses). Second, these data come from the examination of the hypothalamus. But what about medial temporal lobe structures and other brain regions, such as the forebrain?
None of these questions tarnish the data. They simply contextualize the first—and, in my opinion, most remarkable—association between a known learning protein and a mutation involved in a known cognitive pathology.
And with such boundaries, we have come full circle. I hope that after reading both columns, you can see more clearly the edge of our understanding regarding autism and autism-related disorders. The purse seiner approach described in Part 1, combined with the narrower fishing pole approach described here, represents some truly amazing progress in the attempts to understand these baffling diseases.
It is a heck of a time to be dropping lines in these waters!