Research Awards
Christopher L. Woodcock, Ph.D.
University of Massachusetts, Amherst
"3D Conformation of Chromatin-MeCP2Complexes"
2-Year Award: $90,000
Research Sponsor: Provident Bank
Final Report (November 2005)
We know that most cases of Rett Syndrome are caused by a defect in the MeCP2 protein that can be as small as a single amino acid change, resulting in interference with proper binding of the protein to methylated DNA. In living cells, MeCP2 encounters not naked DNA but the DNA-protein complex known as chromatin, and we have found that MeCP2 induces dramatic increases in chromatin compaction which may well be connected with its in vivo function. Our work has focused on the details of the interaction between MeCP2 and chromatin, especially the architecture of the compact state and the molecular interactions that lead to this compaction.
Our analyses have included both biochemical and structural measurements of the interactions between purified recombinant human MeCP2 (and its key mutations) and defined chromatin arrays using direct imaging by electron microscopy in combination with molecular tools, has produced the following major results:
Christopher L. Woodcock, Ph.D.University of Massachusetts, Amherst
"3D Conformation of Chromatin-MeCP2Complexes"
2-Year Award: $90,000
Research Sponsor: Provident Bank
Final Report (November 2005)
We know that most cases of Rett Syndrome are caused by a defect in the MeCP2 protein that can be as small as a single amino acid change, resulting in interference with proper binding of the protein to methylated DNA. In living cells, MeCP2 encounters not naked DNA but the DNA-protein complex known as chromatin, and we have found that MeCP2 induces dramatic increases in chromatin compaction which may well be connected with its in vivo function. Our work has focused on the details of the interaction between MeCP2 and chromatin, especially the architecture of the compact state and the molecular interactions that lead to this compaction.
Our analyses have included both biochemical and structural measurements of the interactions between purified recombinant human MeCP2 (and its key mutations) and defined chromatin arrays using direct imaging by electron microscopy in combination with molecular tools, has produced the following major results:
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1. Like most proteins that bind specifically to DNA, MeCP2 also binds non-specifically (in this case to unmethylated DNA and chromatin containing unmethylated DNA). This property is thought to be advantageous in vivo since it speeds the protein to its target methylation sites. In our experiments, an excess of unmethylated competitor DNA enables us to observe specific binding to the target methylated DNA and chromatin.
2. Under these conditions, there is, as predicted, no interaction between DNA and MeCP2 carrying mutations that disable the domain that binds to methylated DNA (MBD) and cause the most severe RTT symptoms. However, with chromatin we observe three levels of interaction. The first two levels occur whether or not the DNA is methylated. The weakest is observed with MeCP2 mutations that disrupt the MBD and cause the most severe RETT symptoms. The next level is also non methylation specific and occurs with normal MeCP2. As expected, the third and most pronounced interaction occurs between normal MeCP2 and methylated chromatin. All three interaction levels result in chromatin compaction, and imaging experiments to define the changes in chromatin architecture that accompany each level are in progress. These findings indicate a new and unexpected role of the MBD in chromatin binding and compaction which may have important implications for MeCP2 function in normal and RTT patients.