Transcript ppt - Chair of Computational Biology

Special-topic Lecture Biosciences:
Biological Sequence Analysis
Leistungspunkte/Credit points: 5 (V2/Ü1)
This course is taught in English language.
Lecture form: The students will be required to work actively at home and
during the tutorial in small groups to prepare half of the lecture content
themselves. The material (from books and original literature) will be
provided in the lecture. The lectures will then be a mixture of ex-cathedra
teaching, student presentations, and discussion.
Topics to be covered:
This course will enter into details of three selected topics in current genetics:
- Epigenetics
- Plant genomics
- Pharmacogenomics
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Aim of this lecture, „Lernziele“
The aim of this course is not to fully cover epigenetics, botany and
pharmacogenetics.
This course should improve your ability to compile the necessary biological
background that is relevant to your bioinformatics project from original literature.
During this course, you will have ample opportunity to explain biological details.
In this way, you practise presentation skills and to use simple language for
explaining difficult biology.
Also, you should practise your english discussion skills.
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Content (ca.)
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epigenetics: intro
epigenetics: CpG islands, DNA methylation, Human epigenome project
epigenetics: imprinting
epigenomics and cancer
test 1; plant genomes: Arabidopsis genome
plant genomes: biomarkers
plant genomes: genome rearrangement
plant genomes: gene expression
Zhang, X. et al. Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis.
Cell 126, 1189–1201 (2006). Zilberman, D. et al. Genome-wide analysis of Arabidopsis thaliana DNA methylation
uncovers an interdependence between methylation and transcription. Nature Genet. 39, 61–69 (2006).
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test 2; pharmacogenomics: X-ray structures of membrane transporters
pharmacogenomics
pharmacogenomics: SNP variations
pharmacogenomics: drug dosage response
test 3; pharmacogenomics wrap up
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Schein = successful written exam
The successful participation in the lecture course („Schein“) will be certified upon
successful completion of 3 written 30 minute tests. All tests have to be passed.
Each test covers the content of one lecture topic.
Dates: May 13, June 10, July 8 at the beginning of lectures V5, V9 and V13.
All students registered for the course may participate in the tests.
The final mark will be computed from the sum of the 3 test results.
The tests will cover the lecture material (slides on the lecture website) and the
required reading.
In case of illness please send E-mail to:
[email protected] and provide a medical certificate.
Those who miss or fail one test, will be given a second-chance oral exam.
If you fail or miss more than one test, you cannot get a Schein.
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tutorials
Barabara Hutter and Siti Azma Yusof – tutorials
Geb. C 7 1, room 1.09
[email protected]
Tutorial: one hour per week, to be announced
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What is epigenetics?
Epigenetics refers to alternate phenotypic states that are not based in
differences in genotype, and are potentially reversible, but are generally stably
maintained during cell division.
Examples: imprinting, twins, cancer vs. normal cells, differentiation, ...
The narrow interpretation of this concept is that of stable differential states of gene
expression.
A much more expanded view of epigenetics has recently emerged in which multiple
mechanisms interact to collectively establish
- alternate states of chromatin structure,
- histone modification,
- associated protein composition,
- transcriptional activity, and
- in mammals, cytosine-5 DNA methylation at CpG dinucleotides.
Laird, Hum Mol Gen 14, R65 (2005)
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Basic principles of epigenetics:
DNA methylation and histone modfications
The human genome contains
23 000 genes that must be
expressed in specific cells at
precise times.
Cells manage gene expression
by wrapping DNA around
clusters (octamers) of globular
histone proteins to form
nucleosomes.
These nucleosomes of DNA
and histones are organized into
chromatin, the building block of
a chromosome.
Rodenhiser, Mann,
CMAJ 174, 341 (2006)
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Bock, Lengauer, Bioinformatics 24, 1 (2008)
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Example: Monoallelic expression of odorant receptors
The nose recognizes chemical information in the environment and converts it into
meaningful neural signal, allowing the brain to discriminate among thousands of
odorants and giving the animal its sense of smell.
The mouse contains more than 1000 genes encoding olfactory receptors (ORs).
This makes them the largest mammalian gene family. They are putative GPCRs
and are located in clusters which are scattered throughout the genome.
The large number of receptors suggests that each odor elicits a unique signature,
defined by the interactions with a limited number of relatively specific olfactory
receptors.
From combinations of interactions, animals would then be able to sense more than
104–105 different odors.
Shykind, Hum Mol Gen 14, R33 (2005)
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Monoallelic expression of odorant receptors
Isolation of OR genes allowed studying the biology of olfaction.
RNA in situ hybridization studies revealed two fundamental characteristics of OR
expression.
(1) neurons expressing a given receptor are restricted to one of 4 broad zones
running across the olfactory epithelium.
(2) within a zone, individual receptors are expressed sparsely and without apparent
pattern.
Quantitative analysis of these in situ hybridization experiments led to the
suggestion that each neuron in the nose expresses only one or a few members
of the gene family.
Shykind, Hum Mol Gen 14, R33 (2005)
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Monoallelic expression of odorant receptors
Subsequent analyses of cDNAs synthesized from single olfactory neurons 
just a single OR species could be isolated from each cell.
This strengthened the ‘one neuron–one receptor’ hypothesis.
Additionally it was found that ORs are transcribed from just one allele.
Hypothesis by Buck and Axel in 1991:
the olfactory sensory neuron selects a single receptor from just one allele of a
spatially allowed subset of a widely dispersed gene family.
Shykind, Hum Mol Gen 14, R33 (2005)
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Axonal Wiring in the Mouse Olfactory System
The main olfactory epithelium of the mouse
is a mosaic of 2000 populations of olfactory
sensory neurons (OSNs).
Each population expresses one allele of
one of the 1000 intact odorant receptor
(OR) genes.
An OSN projects a single unbranched axon
to a single glomerulus, from an array of
1600–1800 glomeruli in the main olfactory
bulb.
Within a glomerulus the OSN axon
synapses with the dendrites of secondorder neurons and interneurons.
Axons of OSNs that express the same OR
project to the same glomeruli— typically
one glomerulus per half-bulb and thus four
glomeruli per mouse.
Mombaerts, Ann Rev Cell Biol 22, 713 (2006)
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Monoallelic expression of odorant receptors
The logic of the olfactory circuit rests upon this regulatory process as does the
formation of the sensory map, which is dependent on receptor protein to guide the
path-finding axon.
Aberrant expression of multiple ORs per neuron may disrupt olfactory axon
guidance and thus prevent accurate formation of the glomerular map.
Once a neuron establishes its synapse in the olfactory bulb, it must remain
committed to its OR.
Any change in receptor would change the ligand specificity of the cell and confound
the sensory map.
Shykind, Hum Mol Gen 14, R33 (2005)
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Visualisation of monoallelic expression:
Odorant receptor expression in axons
(A) Whole mount view of a compound heterozygous mouse, age P30, genetically modified to
express tau-lacZ and GFP from each allele of the P2 odorant receptor gene.
Neurons express P2 monoallelically (green or red cells) in the olfactory epithelium (oe), and
project their axons back into the olfactory bulb (ob) to form a glomerulus (gl, within white box).
Nuclei are counterstained by Toto-3 (blue).
(B) High power view of (boxed area in A) showing the convergence of P2 axons to a
glomerulus (red and green fibers). Neighboring glomeruli are indicated by asterisks.
How this mono-allelic expression works on a
molecular level is apparently still unknown.
Shykind, Hum Mol Gen 14, R33 (2005)
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Epigenetic modifications
Strands of DNA are wrapped around histone octamers, forming nucleosomes.
These nucleosomes are organized into chromatin, the building block of a
chromosome. Reversible and site-specific histone modifications occur at multiple
sites through acetylation, methylation and phosphorylation. DNA methylation
occurs at 5-position of cytosine residues within CpG pairs in a reaction catalyzed by
DNA methyltransferases (DNMTs). Together, these modifications provide a unique
epigenetic signature that regulates chromatin organization and gene expression.
Rodenhiser, Mann, CMAJ 174, 341 (2006)
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Cytosine methylation
3-6 % of all cytosines are methylated in human DNA.
How many cytosines are in „normal“ DNA?
How many CpG islands are in „normal“ DNA?
In mammalian genomes the CpG dinucleotide is depleted towards 20-25% of the frequency expected by the
G+C content. This is typically explained in the following way:
As most CpGs serve as targets of DNA methyltransferases, they are usually methylated.
5-Methylcytosine, whose occurrence is almost completely restricted to CpG dinucleotides, can easily
deaminate to thymine.
If this mutation is not repaired, the affected CpG is permanently converted to TpG (or CpA if the transition
occurs on the reverse DNA strand).
Hence, methylCpGs represent mutational hot spots in the genome. If such mutations occur in the germ line,
they become heritable.
A constant loss of CpGs over thousands of generations can explain the scarcity of this special dinucleotide
in the genomes of human and mouse.
Esteller, Nat. Rev. Gen. 8, 286 (2007)
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Cytosine methylation
Interestingly, repetitive genomic sequences are heavily methylated.
The maintenance of this DNA methylation could have a role in the protection of
chromosomal integrity, by preventing chromosomal instability, translocations
and gene disruption through the reactivation of endoparasitic sequences.
Esteller, Nat. Rev. Gen. 8, 286 (2007)
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effects in chromatin organization affect gene expression
Schematic of the reversible changes in chromatin organization that influence
gene expression:
genes are expressed (switched on) when the chromatin is open (active), and they
are inactivated (switched off) when the chromatin is condensed (silent).
White circles = unmethylated cytosines;
red circles = methylated cytosines.
Rodenhiser, Mann, CMAJ 174, 341 (2006)
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Basic principles of epigenetics:
DNA methylation and histone modfications
Changes to the structure of chromatin influence gene expression:
genes are inactivated (switched off) when the chromatin is condensed (silent),
and they are expressed (switched on) when chromatin is open (active).
These dynamic chromatin states are controlled by reversible epigenetic patterns of
DNA methylation and histone modifications.
Enzymes involved in this process include
- DNA methyltransferases (DNMTs),
- histone deacetylases (HDACs),
- histone acetylases,
- histone methyltransferases and the
- methyl-binding domain protein MECP2.
Rodenhiser, Mann, CMAJ 174, 341 (2006)
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DNA methylation
The loss of normal DNA methylation patterns is the best understood epigenetic
cause of disease.
Typically, unmethylated clusters of CpG pairs are located in tissuespecific genes
and in essential housekeeping genes, which are involved in routine maintenance
roles and are expressed in most tissues.
These clusters, or CpG islands, are targets for proteins that bind to unmethylated
CpGs and initiate gene transcription.
In contrast, methylated CpGs are generally associated with silent DNA, can block
methylation-sensitive proteins and can be easily mutated.
In animal experiments, the removal of genes that encode DNMTs is lethal; in
humans, overexpression of these enzymes has been linked to a variety of cancers.
Rodenhiser, Mann, CMAJ 174, 341 (2006)
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Uptake of methyl groups
DNA methylation patterns fluctuate in response to changes in diet, inherited
genetic polymorphisms and exposures to environmental chemicals.
Methyl groups are acquired through the diet and are donated to DNA through the
folate and methionine pathways.
Consequently, changes in DNA methylation may occur as a result of low dietary
levels of folate, methionine or selenium.
This can lead to diseases such as neural tube defects, cancer and atherosclerosis.
Imbalances in dietary nutrients can lead to hypomethylation (which contributes to
improper gene expression) and genetic instability (chromosome rearrangements).
E.g. hyperhomocysteinemia and global hypomethylation have been observed in
vitro in atherosclerosis models, which supports an emerging view that alterations in
global methylation patterns are characteristic of early stages of this disease.
In advanced stages of atherosclerosis, hyperproliferation may further contribute to
DNA hypomethylation and altered gene expression.
Rodenhiser, Mann, CMAJ 174, 341 (2006)
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Clinical consequences of epigentic errors
Epigenetic mechanisms regulate DNA
accessibility throughout a person’s lifetime.
Immediately following fertilization, the
paternal genome undergoes rapid DNA
demethylation and histone modifications.
The maternal genome is demethylated
gradually, and eventually a new wave of
embryonic methylation is initiated that
establishes the blueprint for the tissues of
the developing embryo.
As a result, each cell has its own epigenetic
pattern that must be carefully maintained to
regulate proper gene expression.
Rodenhiser, Mann, CMAJ 174, 341 (2006)
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Uptake of methyl groups
Rodenhiser, Mann, CMAJ 174, 341 (2006)
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Epigenetics and Assisted reproductive technology (ART)
Recent evidence suggests that the manipulation of embryos for the purposes of
assisted reproduction or cloning may impose inherent risks to normal development.
E.g. ARTs have been linked to an increased risk of intra-uterine growth retardation,
premature birth, low birth weight and prenatal death.
ART is apparently associated with Angelman syndrome and Beckwith–Wiedemann
syndrome.
Molecular analyses of patients with these 2 syndromes conceived by in vitro
fertilization or intracytoplasmic sperm injection revealed a loss of maternal-specific
DNA methylation at imprinting centres.
This indicates that the errors were epigenetic in nature.
Although individually rare, as a group, epigenetic errors may impose significant risk
for people conceived by ART.
Rodenhiser, Mann, CMAJ 174, 341 (2006)
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Esteller, Nat. Rev. Gen. 8, 286 (2007)
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Esteller, Nat. Rev. Gen. 8, 286 (2007)
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Epigenetic regulation during development
Surani, Hayashi, Hajkova, Cell 128, 747 (2007)
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Epigenetic regulation during development
Surani, Hayashi, Hajkova, Cell 128, 747 (2007)
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Epigenetic regulation during development
Surani, Hayashi, Hajkova, Cell 128, 747 (2007)
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Epigenetic signals in ES cells and in differentiated cells
Bernstein, Meissner, Lander, Cell 128, 669 (2007)
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Questions for next week
How can one detect methylation patterns experimentally?
- bisulfite treatment of DNA
- methylation-specific PCR
- Restriction landmark genomic scanning (RLGS)
- chromatin immunoprecipitation using the ChIP-on-chip approach
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