5 - DNA methylation Flashcards
Give the key concepts for DNA methylation
- Can be inherited without sequence change
- Common in plants, vertebrates (10%) and absent or rate in yeast, flies and nematodes
- Occurs predominantly at 5’-CpG-3’ dinucleotide positions
What percent of CpG sites are methylated in mammals?
60-90%
How big are typical CpG islands? Are they hypermethylated or hypomethylated?
300-3000 base pairs
Hypomethylated
They are near approximately 70% of human promoters and are correlated with tissue-specific gene expression
How does DNA methyltransferase (DNMT) methylate cytosine?
Removes a methyl from SAM and puts it on the cytosine (then called 5-methyl-cytidine)
List four methyltransferases been identified in mammals
- DNMT1 (maintenance)
- DNMT3L (inactive on own, but critical)
- DNMT3A (de novo)
- DNMT3B (de novo)
What are the two types of DNA methylation?
Maintenance methylation (DNMT1) - Methylation of newly synthesized DNA strand at positions opposite methylated sites on the parent strand (occurs after DNA replication)
De novo methylation (DNMT3A, 3B)
- Methylation of totally new positions, changes the pattern of methylation in a localized region of the genome during gametogenesis and early development.
Give one method for DNA methylation analysis
Sodium bisulfite sequencing
- Converts unmethylated cytosine to uracil during PCR (which converts to thymine). The subsequent ratio of cytosine and thymine present at each CpG site is quantified and reflects the methylation level of that site in genomic DNA
What are two major biological functions of DNA methylation?
- Imprinting
- Heterochromatin formation
How do regions of pericentromeric heterochromatin cause targeting for de novo DNA methyltransferase activity?
They have PWWP domains (pro-trp-trp-pro motifs)
Give the mechanisms of DNA methylation mediated repression.
Methylation in binding regions of some transcription factors can result in inhibition. Methyl binding proteins (MBPs) directly recognize methylated DNA and recruit co-repressor molecules to silence transcription and to modify surrounding chromatin
- MBPs may be involved in inhibiting elongation, either directly or by their effects on the surrounding chromatin structure.
- DNMT enzymes are physically linked to histone deacetylases and histone methyltransferase (HMT) activities
What is the most signicant methyl-CpG-binding protein (MECP)?
MECP2
It is a transcriptional repressor, x linked and able to bind a single methyl CpG. Expression correlates with maturation of the neural system. Mutations are associated with Rett syndrome and perhaps autism.
Describe the NuRD (nucleosome remodeling deacetylase) complex
A mediator of methylation induced gene silencing
It is a group of associated proteins with both ATP-dependent chromatin remodeling and histone deacetylase activities.
One associated protein is a CpG binding domain protein (MBD2 or MBD3).
What is MeCP2?
A mediator of methylation induced gene silencing
- KO is embryonic lethal
- Methyl CpG binding domain and transcriptional repression domain
- Interacts with the mSin3 co repressor complex which associates with HDAC to repress transcription
What is a proposed mechanism for stable repression of gene expression through only development with DNA methylation?
A methylated gene can be silenced by binding of methyl binding domain proteins (and other complexes, like Sin3a). But the MBP can be phosphorylated (or modified in other ways) to cause it to come off and allow active transcription, while the gene is STILL methylated
Transcription factors are transient while DNA methylation is not.
Give an indirect mechanism for crosstalk between DNA methylation and chromatin modification that results in transcriptional repression
- Methylation upstream of gene gives signal for MeCP of histone deacetylase complex (HDAC)
- HDAC deacetylases chromatin in CpG region, inactivating gene
