7. Rett syndrome and MeCP2 Flashcards
What are neurodevelopmental disorders?
Neurodevelopmental disorders - conditions that arise from atypical development of brain + NS - affect cognitive, behavioral, and motor functions
Characteristics:
- heterogenous
- genetic + env causes
- early onset
- lifelong condition
Examples: Rett syndrome, Fragile X, Angelman syndrome, SCZ
Why and how neurodevelopmental disorders are studied?
Neurodevelopmental disorders observed in humans -> create disease models (ex mice, in vitro organoids) -> study causes at molecular level - to understand pathology -> develop pharmaceutical for treatment -> treatment
Explain Rett syndrome
Rett syndrome - a genetic neurodevelopmental disorder - mainly in girls - caused by MeCP2 gene mutation on X chromosome - crucial for brain development
Symptoms:
- motor impairment
- cognitive impariment
- physical symptoms: seizures
What is the underlying cause of rett syndrome?
MeCP2 gene mutation on X chromosome - found to be mosaic in females - random X-inactivation - mutated copy silenced in some cells but not in others -> Rett phenotype mosaicism - different levels
MeCP2 XY males don’t survive - full MeCP2 expression - not silenced in some cells as in females (X inactivation)
How was MeCP2 mapped to cause Rett syndrome?
In genetic studies for Rett - MeCP2 candidate -> used rarer “carrier” mothers - observed offspring -> Rett inheritance was skewed - all children had Rett even with different fathers -> linked to MeCP2 mutation on X chrm
What is the function of MeCP2?
MeCP2 gene produces MeCP2 protein - binds methylated DNA - methylated cytosine at C5 in CpG islands
Investigated by Southwestern assay - MeCP2 assocated only with methylated DNA
Explain how MeCP2 interacts with methylated DNA
MeCP2 protein binds methylated C in CpG islands - via its methyl-CpG binding domain (MBD)
in Rett syndrome - most missense mutations map onto the MBD domain of MeCP2 - make it dysfunctional
Explain experiments done to determine that MeCP2 binds methylated DNA
Experiments done to test if MeCP2 binds methylated DNA:
- Southwestern blot - MeCP2 seen in methylated DNA but not in unmethylated DNA
- A. if binds DNA at all: looked ta fluorescence colocalization with DAPI in cells
- B. with which Xi / Xa associates: Xist promoter region taken from Xa and Xi - MeCP2 associated with methylated Xi - H3Ac associated with Xa unmethylated
- C. ChIP of MeCP2 (crosslink protein-DNA, fragment, immunoprecipitation, uncrosslink, sequence, observe DNA methylation) - MeCP2 associated sequences were methylated
- D. Methylation process impaired by Dnmt2a cKO line - less MeCP2 binding observed when less methylated genome compared to WT MeCP2 levels
=> MeCP2 binds methylated DNA in vitro and in vivo
What is the function of MeCP2?
Two theories for MeCP2 function once boudn to methylated DNA:
- transcriptional repressor
- transcriptional activator
Disagreement in the field - still debated - different evidence from experiments
What is the evidence that MeCP2 is a transcriptional repressor?
Reporter gene experiment - observed GAL4 DBD-MeCP2 fusion protein repressive function
Upon DMSO treatment - induce cellular stress - repression upregulated at methylated sites / increased methylation??- ?? MeCP2 recruited?
What is the Rett syndrome model of mice?
MeCP2 KO mice - model for Rett syndrome (however, still not equivalent to human Rett)
What is the evidence that MeCP2 is a transcriptional activator?
Microarray analysis of gene expression in hypothalamus - compared over-expression vs KO of MeCP2:
- when MeCP2 removed in KO - more genes repressed than activated + MeCP2 found to interact with other co-activators
- opposite expression patterns observed in over-expression vs KO - good functional experiment (valid evidence)
- in MeCP2 mutant stem cell-derived neurons - less transcription observed than in WT when overal RNA levels compared
(iPSCs / ES cells -> CRISPR to edit MeCP2 gene -> differentiation into neurons -> gene expression / phenotype analysis => less overall RNA levels observed in MeCP2 edited cells -> MeCP2 a transcriptional activator
Explain how in vitro experiments can be used to determine if a protein is a transcriptional repressor or activator?
Take stem cells - edit the target gene - can differentiate cells into target cell type - observe overall RNA levels in mutants
- if in target gene mutation less RNA than WT -> activator
- if in target gene mutation more RNA than WT -> repressor
How was it identified that MeCP2 repressed long highly metylated genes in genomes?
MeCP2 prefers long, highly methylated gene binding - tested by ChIP:
- gene length: MeCP2 enrichment increased with gene legth
- methylation level: MeCP2 enrichment increased with higher methylation genes
What was observed when Rett brain tissue was investigated for MeCP2?
Post-mortem MeCP2 +/- brain samples dissociated - scRNAseq - notice different excitatory vs VIP neuron patterns for methylation:
- excitatory neurons - increase in methylation drives higher RNA expression (??)
- VIP neurons - variation but no significant increase of expression overall as methylation changes
Methylation expressed as a fraction methylated/unmethylated sites
Read paper
When a MeCP2 mutation impairs its interaction with its co-proteins, what could be an example of such outcome
MeCP2 mutation - impaired ability to recruit NCoR co-repressor complex to chromatin
IN R306C mutation NCoR no longer colocalized with MeCP2 in Western blot
What is usually the effect of MeCP2 mutations on the protein?
MeCP2 mutations usually:
- affect its binding site
- affect its interaction with other co-proteins
How was it determined that MeCP2 acts in a complex rather than on its own
Protein interaction studies - co-immunoprecipitation - identify proteins interacting at higher levels than others -> more strongly associated proteins form a complex with MeCP2 for the function + mutations in MeCP2 which lead to loss of interaction induce non-functional MeCP2 and Rett syndrome phenotype
Ex. one such Rett syndrome mutation impairs MeCP2 ability to recruit NCoR co-repressor complex to chromatin
What is another syndrome besides Rett also associated with MeCP2 gene?
MeCP2 duplication syndrome (M2DS) - rare genetic disorder caused by an extra copy (duplication) of MECP2 on X chrm - excessive MeCP2 protein production - disrupts normal gene regulation -> disrupts neurological and developmental function - primarily affects males - one X chrm
Earlier symptom onset than Rett - neonatal hypotonia, more prominent seizures, life expectancy ~25yrs
What is the underlying toxicity mechanism behind excess MeCP2?
Excess MeCP2 disrupts target gene regulation - MeCP2 quantity is crucial in the toxic phenotype - 2 experiments:
1.
- MeCP2 duplication - toxic, MeCP2 duplication but one of the two is an inducable transgene -> when transgene removed survive
2.
- When MeCP2 fused with Tau (string neuronal marker) - leads to x3 more copies of MeCP2 -> investigated genotypes WT homoz, WT+Tau-MeCP3 heteroz, Tau-MeCP2 homoz => WT MeCP2 control, heteroz survive but develop M2DS, homoz Tau-MeCP2 lethal
- When MeCP2 transgene mutated that can’t interact with its co-proteins - same homoz/hetero/homoz experiment repeated => Tau-MeCP2 homoz survived because the copy was dysfunctional - even tho MeCP2 was expressed at lethal levels but non-functional
What are the arguments for MeCP2 being a transcriptional activator vs repressor?
Explain how Cre-loxP can be used to study MeCP2 function
Cre-loxP uses in MeCP2 study:
1. Make MeCP2 deletion tissue specific
2. Male MeCP2 deletion in whole organism
3. Make MeCP2 induction in whole organism
Discuss how the mechanisms for these are different ??
Different Cre-loxP studies done for MeCP2 function
No need to learn, just examples
How was it determined if MeCP2 is required only in development or throughout life?
MeCP is required throughout life, not only in development:
- MeCP2 KO mice don’t survive past 25 days, conditional KO also decreased survival
- Cre-loxP experiment - MeCP2 expression upon +Tam -> Rett syndrome somewhat reversible when functional MeCP2 introduced in adult Rett mice - improvement in symptoms - improvement both when MeCP2 introduced before onset of Rett syndromes and after the onset => potential for human therapy even after disease development
Explain how gene therapy can be used to treat Rett syndrome
Shown that Rett symptoms can be reversed in functional MeCP2 introduced even after rett symptom onset -> gene therapy:
- introduction of functional MeCP2 in adenovirus (AAV9) - not pathogenic, no insertional mutations introduced, good for CNS transduction
BUT: immune repsonse, DNA payload must be tightly controlled - if several MeCP2 copies introduced - can induce M2DS -> death
Current several start-ups working on gene therapy for Rett syndrome - have already started clinical trials - very careful not to introduce too many copies - at this point too conservative amounts to actually treat Rett
What is a danger of Rett gene therapy?
MeCP2 dosage - narrow therapeutic window - if too many copies introduced can induce M2DS
What are the alternatives to gene therapy for Rett treatment?
Gene therapy - inserts functional MeCP2 copy - instead for replacing the defective copy witha functional -> alternative approaches:
- activate existing MeCP2 copy in Xi - epigenators to recruit endogenous copy activation
- DNA editing - edit the mutation within MeCP2 to produce functional protein
- RNA editing - edit the mutation in RNA transcript into functional base (idk look up how RNA editing works if this)
Further reading
“The Molecular Basis of MeCP2 Function in the Brain” Rebekah Tillotson, Adrian Bird
https://www.sciencedirect.com/science/article/pii/S0022283619305959
