SS 2008 Biological Sequence Analysis 1 Special-topic Lecture Biosciences: Biological Sequence Analysis Leistungspunkte/Credit points: 5 (V2/Ü1) This course

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Biological Sequence Analysis1

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.)

1 epigenetics: intro

2 epigenetics: CpG islands, DNA methylation, Human epigenome project

3 epigenetics: imprinting

4 epigenomics and cancer

5 test 1; plant genomes: Arabidopsis genome

6 plant genomes: biomarkers

7 plant genomes: genome rearrangement

8 plant genomes: gene expressionZhang, 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).

9 test 2; pharmacogenomics: X-ray structures of membrane transporters

10 pharmacogenomics

11 pharmacogenomics: SNP variations

12 pharmacogenomics: drug dosage response

13 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.

mailto:[email protected]

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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)

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.


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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.


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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 second-

order 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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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.


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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 amolecular 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.


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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?

Esteller, Nat. Rev. Gen. 8, 286 (2007)

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.

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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.


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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.


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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.


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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.


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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.


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Uptake of methyl groups


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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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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

Documents

SS 2008 Biological Sequence Analysis 1 Special-topic Lecture Biosciences: Biological Sequence Analysis Leistungspunkte/Credit points: 5 (V2/Ü1) This course