Week 7 Flashcards
(33 cards)
What is epigenetics?
Modifications in the genome that result in altered gene expression with no changes to the DNA sequence
During development, what does epigenetics do?
Creates a heritable memory of gene expression
Programmes associated with specific cells and tissues
What are the levels of epigenetic regulation?
DNA modifications
- Methylation, hydroxymethylation
Histone modifications
- Methylation, acetylation, phosphorylation, ubiquitination
Chromatin accessibility
- Open and closed chromatin
Higher orders of structure and nuclear organisation
Why is epigenetic regulation a dynamic process?
Hierarchical organisation of genetic material at multiple levels of hierarchy
What is canonical DNA methylation?
DNA methylation - addition of a methyl group to a cytozine in position C5 of the DNA - epigenetic modification
Essential for normal development, X Chr inactivations, genomic imprint establishment and gene expression
Cytosins in a CpG-context are underrepresented in the genome
- General hypermethylation of CpGs
High CpG-density in CpG islands (CGIs)
- General hypomethylation of CGIs
- Often associated with gene promoters
Histone modifications support DNA methylation pattern
- H3K4me3, corresponds with unmethylated, active promoters
- H3K36me3 overlaps with methylated gene bodies
What is DNA methylation?
The best studied epigenetic modification
Regulates gene expression
What does DNAme and histone mark?
Shape chromatin configuration
What are some epigenetic mechanisms?
Writers
Erasers
Readers
How can traits be passed onto the offspring?
Two major waves of DNA methylation
Germline and Early embryo
Complete DNA methylation reprogramming in the germline
- Setting of genomic imprints
- Preparing for fertilisation and embryonic genome activation
What happens in oocyte maturation?
GV is arrested at meiotic prophase I
Meiosis resumes upon induction of ovulation
MII stage: Ovulated oocyte, arrested at meiotic metaphase II, ready for fertilisation
What does transcription drive in oocytes?
De novo DNA methylation establishment
What is the epigenome of the sperm?
Mitotic period
(Spermatogoniogenesis)
- Conclusion of re-establishment of paternal-specific imprints
Meiotic period
(Spermatocytogenesis)
- Replacement of somatic histones by testis-specific histone variants
Haploid period
(Spermiogenesis)
- Hyperacetylaction of histones
- Transition proteins
- Nucleosome-protamine exchange
Spermatozoon
- Chromatin condensation
- Establishing of disulphide bridges between protamines
What is the unique epigenetic signature of Spermatozoa?
DNAmethylation
- Demethylation and de novo DNA methylation
DNA-associated proteins - protamines
- Somatic histones replaced by transition proteins and testis-specific variants
Nucleosome distribution
Post-translational histone modifications
- Histone to protamine transition
Stored RNA and non-histone and non-protamine proteins
- Storage of differnetially expressed RNAs
When are the two major waves of DNA methylation?
Germline
Early embryo
What does Ome mean?
Describes a collection of data related to a set of biological compounds, structures or organisms
What happens on the epigenome?
DNA modifications
- Methylation, hydroxymethylation
Histone modifications
- Methylation, acetylation, phosphorylation, ubiquitination
Chromatin accessibility
- Open and closed chromatin
Higher order structure
What does Royal Jelly do to the brain of worker bees?
Affects DNA methylation
What happens in ChiP-Seq and CUT&RUN?
Chromatin fragmentation
Immunoprecipitation
DNA purification
DNA analysis
Permeabilisation
Chromatin cleavage
pAG-MNase;
Add specific AB
DNA amplification
Sequencing
What is 3D genome organisation?
- Start with cell or tissue samples
- Crosslink chromatin
- Fragment chromatin
- Repair and biotinylate ends
- Ligate fragmented chromatin
- Shear DNA and pull down biotinylated DNA
- Paired-end Sequencing
- Bioinformatics
What is transcriptomics?
RNA sequencing
Post transcriptional modifications
How is gene expression looked at?
Extract total RNA from isolated cell or tissue population
Isolate specific RNA species
Poly-A selection
Ribo-depletion
Size selection
Convert to cDNA
Construct sequencing library
PCR amplification and sequencing
What is proteomics?
Protein profiling
Alternate splicing
Post-translational modifications
What happens in proteome analysis?
Sample of interest
Protein isolation
Band/spot excision
In-gel proteolytic digestion
Peptide mixture
Mass spectrometry
Database search
Protein identification
What is metabolome?
Metabolites - small-molecule chemicles within a biological sample
Metabolites are indirectly encoded by gene or act on genes or gene products
Endogenous vs exogenous
