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Summary lectures Epignomics in health and disease
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Complete summary of all lectures of the course 'Epigenomics in health and disease'
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Lecture 1 - Introduction lecturedinsdag 1 oktober 2019 08:26
Epigenetics: the study of heritable changes (mainly during cell division) in gene expression or cellular
phenotype caused by mechanisms other than changes in the underlying DNA sequence.
Most striking example epigenetics, is the amount of different cell types.
Every cell has its own epigenome. Epigenome provides a manual for how to read the genome.
Epigenetics is a major driver of embryonic development. A large part of studying embryonic
development is directed towards epigenetics.
Epigenomic building blocks
Different levels of packaging
DNA is wrapped around 4 pairs of proteins calles histones in a 'beads-on-string' fashion.
The 30 nm fiber stacks nucleosomes.
During cell division the fibers are wrapped around a protein structure to give maximal condensed
chromosomes.
Dynamic chromatin
N-terminal histone tails: are all modified with small chemical groups which can work as docking
stations for interaction with other molecules.
Histone modifications:
- Acetylation
- Methylation
- Phosphorylation
- Ubiquitylation
Open regions in the chromatin are binding sites for transcription factors.
DNA methylation: genomic material is directly modified, is a reversible event. --> methylates the C
when it is next to a G.
Chromatin structure; distant interactions and domains
Nowadays there is a lot of research on the folding of the DNA.
Transcription programs are instructed by epigenome in nucleus
Enzymes are being discovered that write, read or erase these marks
Profiling epigenetics
Chromatin/nuclear structure profiling using NGS
- Chromatin structure (interaction of chromosomes)
- Nucleosome occupancy
- Hypersensitive site (open sites)
○ DNAse-seq, FAIRE-seq, ATAC-seq
- Map proteins on DNA
○ ChIP-seq (histones, other factors)
- Direct reversible modification of nuceotiddes ((hydroxy)methylation)
○ Methyl-Cap, Methyl-Seq, RRBS, BS-Seq
Link between epigenetics and disease
~30% of all cancer driver genes are related to chromatin structure and function.
Epigenetics can also be used for profiling for biomarker discovery.
Epigenetics enzymes are very promising during targets, especially in cancer.
Lectures Pagina 1
,Lecture 2 - A reminder on sequencing and epigenomics
maandag 7 oktober 2019 08:26
Transcription regulation
RNA polymerase II dependent genes are regulated by DNA binding factors that can activate
and/or repress transcription.
However, DNA in eurkaryotic cells is not found as a naked helix. In stead, it is packaged in
chromatin.
Epigenomics studies the genome-wide distribution of epigenetic markers
Epigenetic modifications are reversible modifications on a cell's DNA or histones that affect gene
expression without altering the DNA sequence.
Epigenomics is the studiy of the complete set of epigenetic modifications on the genetic material
of a cell, known as the epigenome.
DNA seq by synthesis (Next generation sequencing) (Illumina sequencing)
For sequencing of random (or selected) fragments (200-300 bp) of dsDNA
1. dsDNA is fragmented (e.g. by sonication or restriction digestion)
2. 'Adaptor sequences' are ligated to the ends of the DNA
3. These adaptors are used to capture the DNA (single stranded) on a glass surface
4. These attached molecules are locally amplified in a 'solid phase PCR' to form 'clusters'
5. Like in Sanger sequencing, this procedure requires a primer, DNA polymerase and
fluorescent chain terminators to incorporate the complementary base
6. After each cycle, the glass surface is imaged (4 colors)
7. Reverse chain terminator (which cleaves of the fluorophore)
8. Initiate next cycle, etc….
Lectures Pagina 2
,These short read sequences are aligned to the reference genome
But how do we know which part of the genome that piece of DNA came from?
-> For this we make use of the results of the Human genome project: The reference genome
How many different sequences of 4 nucleotides are there?
-> 4 nucleotides, 4 positions = 4^4 =256 molecules
Thus, any stretch of 4 nucleotides repeats itself (on average) every 256 bases.
How many cycles would you need to perfom minimally to make sure that your sequence read
can be placed uniquely on the human genome?
-> minimally 20 cycles, in the lab we routinely do 43 cycles.
Different applications of NGS methods
Lectures Pagina 3
, --> Despite the differences in the goals of these applications, they are ll based on Next
Generation Sequencing of short fragments of DNA.
--> Therefore, the main trick to these applications is how you prepare your sample. The moment
you ahve turned your sample into short pieces of DNA, you can sequence them.
RNA-sequencing
In RNA-sequencing, one does not actually sequence the RNA. Rather, the RNA is first converted
to cDNA using Reverse Transcription (RT).
1. Isolate RNA
2. RT into cDNA
a. Start at polyA-tail (oligo dT primer)
b. Random hexamers
3. Produce second strand of cDNA
4. Fractionate DNA
5. Ligate adaptors
6. PCR amplify
7. Sequence
8. Map to the genome
--> mRNA does not contain introns, so it only shows exons since the introns are spliced out.
ChIP-sequencing
Is used to identify where a protein is contacting the chromatin.
Sonication: high frequency sound waves which can break (fragment) the DNA
--> Higher the signal, higher amount of reads found
--> Average of all cells in the population, that is why many reads are visible
Lectures Pagina 4
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