This web page was produced as an assignment for Genetics 677, an undergraduate course at UW-Madison.
Summary
The goal of this project was to learn about the role of Methyl CpG Binding Protein 2 in Rett Syndrome. Throughout the semester, I used a variety of genomic and bioinformatics tools to accomplish this. I began by collecting information about the MECP2 gene. I found that it was located on the X chromosome and was about 76 Kb long. This shows why Rett Syndrome is only found in girls, assuming that at least one wildtype copy of MECP2 must be present to produce a viable organism. I also found that six organisms (Chimp, Cow, Dog, Mouse, Rat, and Zebrafish) have MECP2 genes with similar sequences, suggesting that MECP2 is highly conserved in other organisms, mainly mammals. I was able to create a phylogenetic tree that confirmed that mammalian MECP2 homologs were very similar. Any of these should be very effective model organisms to learn more about MECP2 and its function in Rett Syndrome.
I found more information about the processes MECP2 is involved in and its molecular function using gene ontology data. MECP2 is involved with negative regulation of transcription from the RNA polymerase II promoter. I also found that it binds methylated DNA and acts as a transcriptional co-repressor. MECP2 localizes to the nucleus and mitochondrion. By looking at mouse MECP2 gene ontology terms, I was able to find more information about the biological processes it is involved with. I found that MECP2 is involved with adult locomotary behavior and socialization, two areas where girls with Rett Syndrome are impaired. I also found that MECP2 plays a role in chromatin silencing via histone modifications as well as several areas in neuron development and functioning.
Then, I analyzed the protein domains found in human MECP2 and compared them to the 6 homologs. I found that MECP2 contains an MBD domain which binds to methylated DNA at CpG sites and recruits a histone deacetylase complex. This complex condenses chromatin and silences associated genes. This domain was conserved in every homolog. I also found that MECP2 has 2 AT hook domains. This domain also binds DNA with a preference for A/T rich regions, but the function was not explained. The AT hook domains were not found in Danio rerio. I was able to confirm that MECP2 is localized to the nucleus using Uniprot, which supports the idea that MECP2 interacts with DNA. I also discovered that MECP2 is phosphorylated on Ser-423 in the brain upon synaptic activity. This attenuates its repressor activity and seems to regulate dendritic growth and spine maturation.
I was able to determine that MECP2 interacts with several interesting proteins. It interacts with HDAC1, HDAC2, and SIN3A, all of which are part of the histone deacetylase complex. This supports MECP2's role in transcriptional repression. I also found that MECP2 interacts with CDKL5, a protein that causes a severe form of Rett Syndrome. MECP2 also interacts with BDNF and may interact with FXDY1. These proteins are both involved with neuron development and synaptic transmission. These interactions further suggest that MECP2 regulates proteins involved in brain development and neurological functions.
I was able to find limited information about phenotypes in organisms with MECP2 mutations. Several studies have been conducted in mice, but not in other model organisms. In mice with MECP2 knockouts, several behavioral phenotypes were observed including: abnormal stress response, hyperphagia, aggression, locomotor activity, and respiration. These phenotypes are consistent with the gene ontology terms and the symptoms seen in girls with Rett Syndrome.
In conclusion, my data supports the hypothesis that MECP2 binds to methylated DNA at CpG sites and recruits a histone deacetylace complex. This turns these regions into heterochromatin and silences gene expression. This seems to play a role in brain development and neuron function.
Future Directions
1. Create a comprehensive list of genes that are differentially expressed in the presence and absence of MECP2.
Based on my findings, it seems that the least amount of information is known about which specific genes MECP2 silences. I believe the next step in discovering MECP2's role in Rett Syndrome would be creating a comprehensive list of genes that of which MECP2 alters the expression. One method would be to use a gel shift to determine which genes MECP2 interacts with. Then, a microarray could be use to determine which genes have changes in expression in the presence and absence of MECP2. This would focus the group of genes that the experiment would be testing, allowing for analysis of slighter changes in expression. An alternative method would be to use mass spectrometry to analyze differences in protein expression in brain tissue in the presence and absence of MECP2. Both of these experiments could also be done while overexpressing MECP2.
2. Utilize organisms with similar MECP2 proteins to discover more detailed information on the function of MECP2 in the brain of patients with Rett Syndrome.
It would be very beneficial to use homologs as model organisms, other than mice. The Zebrafish homolog would be especially interesting to pinpoint the phenotypes that MECP2 deficiencies cause. Morpholino experiments would be very effective for this. Xenopus may also be a useful model organism, if it can be determined that it has a MECP2 protein with a similar sequence.