Research Awards
Weidong Wang, Ph.D.
National Institute of Aging, NIH
"Characterization of the MeCP2 Complex Involved in Rett Syndrome"
2-Year Award: $100,000
Research Sponsor: Family and Friends of Gabriela Southren
Final Report (November 2004)
The Majority of Rett syndrome patients are defective in the MeCP2 gene. This gene encodes a protein that plays a key role in regulating gene expression. Elucidating the mechanism of how MeCP2 controls gene expression is highly critical for understanding of the Rett syndrome. The objective of our proposal is to study this mechanism.
It is known that many proteins in cells do not function by themselves. Instead, they associate with other proteins to work together like a concerted machine. The overall function of the machine is determined not by one protein, but by all the protein components. Thus, to fully understand function of a given protein, one must identify and investigate the machines which the investigated protein is part of. Because the cell usually expresses thousands of genes, there will be thousands of proteins and protein machines present in cells. Technically, it is quite a challenge to isolate a specific machine away from thousands of others. Our lab has recently developed a highly efficient method to isolate such machines from cells. For example, we have isolated and characterized protein machines involved in a different mental retardation disease, ATRX-syndrome, as well as those involved in Bloom syndrome, Rothmund-Thomson syndrome and Fanconi anemia.
Previous studies have suggested that MeCP2 could be part of a multi-protein machine that regulates gene expression through a specific enzyme (called histone deacetylase). But this machine has not been isolated by unbiased biochemical approaches to a degree of purity that would allow us to understand its structure or function. We have applied our established method and isolated MeCP2 from human cell extract and mouse brain extract under very mild conditions, with the hope to retain the potential MeCP2 machines as intact as possible. The purity of our MeCP2 preparation is very high, and we were able to unequivocally identify MeCP2. To our surprise, we found that MeCP2 does not appear to form a stable machine with any other proteins. In particular, we found no evidence that MeCP2 associates stably with the previously described enzyme. Another report this year from Adrian Bird,s paper reached a similar conclusion. Based on these studies, we suggest that MeCP2 probably does not directly work through this enzyme as many people have believed.
Interestingly, we found that our biochemically isolated MeCP2 is modified by a mechanism called phosphorylation. It has been well established that phosphorylation is a common mechanism for cells to reversibly regulating activity of a protein. A report from Mike Greenberg,s lab has shown that phosphorylation of MeCP2 plays a critical role in regulating its activity in neurons. We were able to identify a single phosphorylation site on MeCP2 protein isolated from several different sources, including human cells, mouse and rat brain. We show that the level of this phosphorylation is regulated when cells are at different stages of growth: it is very low when cells synthesize their DNA, and becomes very high when cells divide. Our study suggest that phosphorylation of MeCP2 at this particular site is important to regulate its function during cell growth.
Weidong Wang, Ph.D.National Institute of Aging, NIH
"Characterization of the MeCP2 Complex Involved in Rett Syndrome"
2-Year Award: $100,000
Research Sponsor: Family and Friends of Gabriela Southren
Final Report (November 2004)
The Majority of Rett syndrome patients are defective in the MeCP2 gene. This gene encodes a protein that plays a key role in regulating gene expression. Elucidating the mechanism of how MeCP2 controls gene expression is highly critical for understanding of the Rett syndrome. The objective of our proposal is to study this mechanism.
It is known that many proteins in cells do not function by themselves. Instead, they associate with other proteins to work together like a concerted machine. The overall function of the machine is determined not by one protein, but by all the protein components. Thus, to fully understand function of a given protein, one must identify and investigate the machines which the investigated protein is part of. Because the cell usually expresses thousands of genes, there will be thousands of proteins and protein machines present in cells. Technically, it is quite a challenge to isolate a specific machine away from thousands of others. Our lab has recently developed a highly efficient method to isolate such machines from cells. For example, we have isolated and characterized protein machines involved in a different mental retardation disease, ATRX-syndrome, as well as those involved in Bloom syndrome, Rothmund-Thomson syndrome and Fanconi anemia.
Previous studies have suggested that MeCP2 could be part of a multi-protein machine that regulates gene expression through a specific enzyme (called histone deacetylase). But this machine has not been isolated by unbiased biochemical approaches to a degree of purity that would allow us to understand its structure or function. We have applied our established method and isolated MeCP2 from human cell extract and mouse brain extract under very mild conditions, with the hope to retain the potential MeCP2 machines as intact as possible. The purity of our MeCP2 preparation is very high, and we were able to unequivocally identify MeCP2. To our surprise, we found that MeCP2 does not appear to form a stable machine with any other proteins. In particular, we found no evidence that MeCP2 associates stably with the previously described enzyme. Another report this year from Adrian Bird,s paper reached a similar conclusion. Based on these studies, we suggest that MeCP2 probably does not directly work through this enzyme as many people have believed.
Interestingly, we found that our biochemically isolated MeCP2 is modified by a mechanism called phosphorylation. It has been well established that phosphorylation is a common mechanism for cells to reversibly regulating activity of a protein. A report from Mike Greenberg,s lab has shown that phosphorylation of MeCP2 plays a critical role in regulating its activity in neurons. We were able to identify a single phosphorylation site on MeCP2 protein isolated from several different sources, including human cells, mouse and rat brain. We show that the level of this phosphorylation is regulated when cells are at different stages of growth: it is very low when cells synthesize their DNA, and becomes very high when cells divide. Our study suggest that phosphorylation of MeCP2 at this particular site is important to regulate its function during cell growth.