Did you know an extra copy of MECP2 sleeps within each of our cells? Females have two X chromosomes (XX), while males only have one (XY). In healthy females, a phenomenon called X inactivation randomly shuts off one X chromosome in order to avoid twice as much product from X-linked genes.
A person with Rett syndrome has one mutated MECP2 but the other copy is healthy. What if we could reawaken this healthy copy and allow every cell to produce normal MeCP2 protein? It’s a question whose answer could cure Rett syndrome.
The potential power of this approach has lead RSRT to allocate significant funding ($4.6 million) and effort in this direction by creating the Reactivating MECP2 Consortium. The Consortium members consist of scientists who “walk the walk.” They meet in person annually and throughout the year via conference calls, during which they collaborate and exchange information. Most importantly, they share tangible things such as compounds and cell lines.
As a result of this RSRT-funded research, two studies were recently published in the journal Proceedings of the National Academy of Sciences (PNAS). Both used cell lines to screen for ways to reactivate the X chromosome and identified different regulatory pathways.
Why do we think X reactivation is a promising strategy? We already know restoration of MeCP2 reverses symptoms in mice -- even in adulthood. The challenge is that X inactivation is designed to be permanent. Many parallel mechanisms are set in place to ensure the other X chromosome stays silent. Fortunately, scientists are continually learning more about these mechanisms and developing ingenious tools to disrupt them, making X chromosome reactivation a reality.
Is it risky to reactivate all the other genes on the X chromosome alongside MECP2? It is still too early to definitively answer, and scientists have differing opinions. Some are encouraged by work in mice that demonstrates it is possible to be healthy with two activated X chromosomes, and suggests that compensation prevents doubling of protein levels. Others believe that activating the back up X could be detrimental. For now, RSRT is pursuing both approaches in parallel: activating MECP2 specifically and activating the entire X chromosome.
In December Jeannie Lee’s group at Harvard Medical School published a paper describing a screen for drugs that reactivate the X chromosome.
- Method: They screened approximately 300,000 small molecules. The X chromosome was tagged with green fluorescent protein, enabling cells in which the chromosome was turned back on to glow.
- Results: Several drugs reactivated the X chromosome by inhibiting the Aurora kinase and DNA methylation pathways. A combination of drugs was most effective, indicating the two pathways work together to inhibit reactivation. Two drugs in particular worked in synergy to activate MECP2 (5azadC and VX680).
- Conclusions: Different treatments turned on distinct sets of genes on the inactive X chromosome, opening the possibility of targeted reactivation of certain genes or regions of interest.
Another paper was published this month from Antonio Bedalov’s group at the Fred Hutchinson Cancer Research Institute, describing a screen for genes that normally repress MECP2 expression. They used artificial RNA molecules called small hairpin RNAs (shRNAs) to silence many different genes and then looked for reactivation of MECP2.
- Method: They screened approximately 60,000 shRNAs. MECP2 was tagged with green fluorescent protein, enabling cells in which MECP2 was turned back on to glow.
- Results: 30 genes were identified as MECP2 repressors. Six of these genes are members of a well-known pathway called BMP/TGF-b that had never been implicated in regulating X chromosome inactivation.
- Conclusions: This study found a novel role for this well-known pathway in X inactivation. In addition, two-thirds of the identified genes code for proteins that can be targeted by drugs, expanding options for therapy.
It is important to keep in mind these studies were conducted in cultured mouse cells, which differ from human neurons in many ways. Work remains to be done before clinically applying these findings. However, it is encouraging that many different labs are working collaboratively using different strategies. Each newly identified gene or pathway involved in MECP2 inactivation brings us closer to our goal of curing Rett syndrome.