L5 : Epigenetics and Modifications Flashcards
What is epigenetics?
Inherited phenotypic changes caused by chromatin changes other than changes in DNA sequence (not mutations)
Modifications can occur at both DNA and chromatin level, changing interactions of transcription machinery
What are primary epigenetic mechanisms?
- DNA methylation
- Histone modifications
- Non coding RNA
How does DNA methylation occur and in which species?
Addition of methyl group at CpG sites on DNA
Forms 5-methyl-cytosine
Found in mammalian cells and plant DNA
Lack in model organism appears to be lineage specific loss (worms, flies, yeast)
Describe deamination of C and 5-me-C and consequences?
C deaminated to form uracil
5-me-C deaminated to form thymine
Replacement of 5-me-C with T can lead to mutation
Uracil not normally found in DNA
What are CpG islands?
Dinucleotide CpG underrepresented in vertebrate genome
Regions of >200 bases with GC content >50%
Higher frequency of CpG compared to rest of vertebrate genome
Found mainly near transcription start sites (promote regions)
What is the importance of DNA methylation in mammals?
- Regulates gene expression by repressing promoter activity
- Regulates alternative splicing
- Maintains genome stability
- Represses transcription of repeated seq
- Prevents relocation of transposable elemtns - Controls X-chr inactivation and expression of imprinted genes (key regulators of development)
What are CpG island shores?
Regions of lower CpG density that lie in close proximity (~2 kb) of CpG islands
Involved in tissue specific gene expression
How does DNA methylation regulate gene expression?
Hypermethylated promoter regions silence genes (transcriptionally inactive)
Hypomethylated promoters associated with active genes
Methylation within gene body associated with higher expression levels and alternative splicing
How does DNA methylation regulate transcription?
Promoter region bound by TFs and subsequently RNA pol
Active transcription of gene occurs where there is unmethylated CpG island
Gene body shows positive correlation between levels of methylation and transcription
CHECK
How much of the human genome exhibits alternative splicing?
90% human genes
22% alternative splicing regulated by DNA methylation
How does methylation status regulate alternative splicing?
Exons have higher levels DNA methylation, esp at splice site
Shown more by exons flanked by short introns rather than long introns
- Altering RNA Pol II transcription rate
- Mediated by CTCF (CCCTC-binding factor) and MeCP2 (Me-CpG binding protein 2) - Facilitating interactions with HP1 (heterochromatic protein 1) to recruit splicing factors
Only accunts for small proportion of alternative splicing
What are mechanisms for gene silencing?
- Unmethylated DNA adopts closed conformation
- Less accessible for non-histone proteins (eg. TFs) - Steric hindrance of methyl groups for TFs
- Physically impeding interaction - Me-CpG binding proteins (MBDs) preferentially bind methylated DNA and inhibit transcription
- Me CpGs recognised by MBPs can then interact with other effectors/chromatin remodellers
- May change density of chromatin and nucleosome clustering
What are two ways to methylate DNA?
Maintenance methylation:
Methylation of newly synthesised DNA strand following existing methylation patterns
De novo methylation:
Establishment of new methylation patterns by de novo methyltransferases
How does maintenance methylation work?
Before replication, DNA fully methylated at CpG dinucleotides
During replication, DNA strands synthesised without methyl groups so resulting DNA molecule has methylation on template strand but not newly synthesised strand (hemimethylated)
Methyl groups attract methyltransferase enzyme, which adds methyl groups to newly synthesised strand, forming fully methylated DNA
How does de novo methylation work?
De novo methyltransferases, which are directed to DNA by sequence specific DNA binding proteins
Occurs during early development, after DNA meythlation is removed following fertilisation
What enzymes mediate DNA methylation?
DNA methyltransferass (DNMTs)
Dnmt1 - maintenance methyltransferase (ubiquitination expressed in mammals)
Dnmt3a/b - de novo methyltransferase (developmentally regulated)
Dnmt2 - methylates cytosine on tRNA
What is evidence for epigenetic effects?
Importance of methyltransferases
Role of environmental factors (diet)
Inheritance of epigenetic traits
Experimental techniques
What evidence is there for phenotypic effects of Dnmt1 mutations?
Homozygous (-/-) mutants for Dnmt1 is recessive lethal phenotype
Die at 10.5 days gestation
Shows importance for maintenance of methylation by Dnmt1
What evidence is there for phenotypic effects of Dnmt 3a/3b?
Homozygous mutant 3a (-/-) survives through gestation but unable to sustain life
Homozygous mutant 3b (-/-) is embryonically lethal
Dnmt3a/3b (de novo) essential for mammalian development
Effect of high methyl diet for mothers for offspring coat colours?
Isogenic mice can have spectrum of colouring (same genome, phenotypically different)
Agouti driven by cryptic promoter within transposon upstream of gene which is not entirely repressed
Results in higher level agouti phenotypes
Supplemented mothers show offspring with more dispersed level of methylation across different CpG sites
CHECK
Explain how this supplemented diet causes different phenotypes?
In utero exposure to methyl donors increases number of pseudoagouti phenotypes
Representative bisulphite allelic sequencing progiles of invidiaul alleles from y and pa mice, allowing differentiation between methylated and non-methylated C
LTRs (long terminal repeat) flank transposon at 3’ and 5’ end
Would expect total methylation if only factor responsible for transcriptional inactivation, suggesting not only factor
CHECK
What is developmental origins of health and disease in humans?
Prenatal environment contributes to developmental programming
- Dutch famine offspring have lower brith weight, high risk of obesity/T2D/cardiovascular disease
Epigenetic patterning can be disrupted b y maternal adverse conditions such as undernutrition
Infants with low birth weight show strong correlation in later adulthood to develop hypertension/insulin resistance/T2D/cardiovascular disease
Other stressors can have similar outcomes (smoking/stress/trauma/maternal obesity)
What evidence is there for inheritance of epigenetic traits?
Maternal rat licking/grooming (LG) behaviour
Mothers showing high LG behaviour tend to have offspring that deal with stressors more effectively (less fearful and reduced hormonal responses to stress)
Molecular evidence:
Differential DNA methylation in glucocorticoid receptor gene (Nr3c1)
In low LG offspring. ERalpha1b promoter shows signifcantly elevated C methylation and reduced binding of stat5 TFs
