1.

Name and describe a biological phenomenon requiring epigenetic Inheritance and name a chromatin
modification that is required. Describe the mechanical conseqs of this modification
Genomic imprinting: The Genex depends on which parent the allele comes from. The imprinting control
element (ICE, present on the chromosomes of both parents) is methylated or unmethylated. In the
unmethylated state, the binding of CTCF to the ICE blocks the interaction of an enhancer downstream
with genes upstream of the ICE, the enhancer calls for the expression of genes between ICE and
enhancer. If ICE is methylated, the enhancer can interact with the upstream seq of the ICE, but the
expression of genes between the enhancer and ICE is suppressed.
2. You want to determine the localization of an enhancer for gene y. a) which experimental approaches
(techniques) would allow you to collect information about the enhancer of Y in its genomic context? b)
Once you have identified a candidate enhancer in a), how could you confirm its function independently
of its genomic context?
Enhancers are usually distal upstream DNA region, which can be bound by transcription factors and by
help of mediator a DNA-loop can be formed which enhance the transcription rate. Enhancer regions
contains usually distinct histone modifications such as H3K4me1 und H3K4me2, H3.3, H2A.Z, CBP. With
CHIP- seq can be these modifications of histone in genome be localized.Steps:
1) DNA will be crosslinked by formaldehyde.; 2) DNA sonication→ it causes DNA- fragmentation into
small pieces 3) Immunoprecipitation: -Antibodies which recognize distinct modification will be added; for
instance an antibody recognizes onlyH3K4me1 or H3K4me2. Antibodies are coupled to heavy beads 4)
Centrifugation: separate DNA-Fragments coupled beads by centrifugation 5) Add Protease to degrade TFs
6) purify precipitated DNA fragments which contains probably enhancers 7) Sequencing
b) We can do structure function analysis. For this we need to make a co transfection reporter gene assay.
We can transform the suspected enhancer region into a plasmid region and express a reporter protein. If
the region is intact, we can express reporter protein. We can also do EMSA in order to detect whether
Transcription factors can bind this enhancer region.
3. Transcription factor x enhances transcription by increasing the transition from a stalled (pausing)
RNA pol II to an elongating RNA pol II. a) does this mean that an initiation complex is not needed.
Provide a brief explanation for your answer. b) what components of the transcription machinery might
x interact with? c) which modification of RNA pol II will be increased by the activity of x and how can
this be determined experimentally (keywords suffice)?
No, Translation initiation complex is always needed, First, the initiation complex must be formed at the
promoter region. For this event Transcription factors are required. After this, transcription can start but
RNA Pol stalls after tanscribing a few nucleotides. Because TFIIH (has CDK7) phosphorylates Ser 5 in CTD
of RNA-Pol II.Transcription machinery can interact with TF X : directly, through a mediator complex;
through interaction with CTD Domain (by allowing phosphorylation of Serines)
After Ser 5 in CTD of RNA-Pol has been phosphorylated (RNAP stalls), RNA-Pol must be phosphorylated
this time at Ser 2 by the activity of by CDK9 activity of P-TEFb. Transcription factor x can enhance the the
phosphorylation of RNAP by P-TEFb.
Experimentally this can detected by reporter gene assay (in vivo) two antibodies can be used which
recognize either the phosphorylated form of RNA pol or the unphosphorylated form. Antibody which
recognizes phosphorylated form was coupled to red fluorescence, the other Antibody which recognize
unphosphorylated form was coupled to green fluorescence
4. A computer algorithm detects a phosphorylation site in the C-terminus of a transcription factor.
Describe an exp set-up allowing you to determine whether this site is really phosphorylated in cells and
whether the phosphorylation is important for the activity of the transcription factor.
Posttranslationale Modification phosphorylation of TF → activates it
Structural function analysis: Need a reporter gene; Delete C-terminus of TF at DNA level → protein is
shortend → if it can’t activate reporter gene transcription → this modification at C terminus was
important ?end?
5. As a conseq of an infection an antimicrobial gene must be transcribed in epithelial cells. However, in
the genome of these cells the binding site for the relevant transcription factor is inaccessible due to
nucleosome packaging. Which events are necessary for transcription of this gene to occur? Which
changes in histone modification would you expect to result from transcriptional activation?
In order to make transcription of a gene possible, histones need to be removed/moved around to make
TF binding site accessable → way histones are orders contribute to cell specific gene expression
N-termini of histones protrude from nucleosome → easily acessable from outside and attach chemical
modification
Chromatin Remodellers (ATPases) → Outcomes: site exposure or altered composition
• Remodeller moves around histone, reposition histones via nucleosome sliding
• Remodeller eject nucleosome from DNA → free binding site
• unwrap DNA little bit → form bulge → through this “loop” binding site becomes acessable
• Histone exchange: exchange of canonical histone with histone variant or dimer ejection
The modifications of histones need to be interpreted. Histone code serves as purpose and proteins are
needed that convert this code into biological actions. Histone acetylation is always linked to increased
accessibility of nucleosomal DNA → because acetylation neutralize positive charge of lysin and tight
interaction is no longer possible Euchromatin is able to be transcribed.
6.Describe the CTD of the eukaryotic RNA Pol II. Which AA are phosphorylated during the initiation of
transcription? What is the importance of phosphorylation?
The C-terminal domain is made up of repeats of the aa seq YSPTSPS: Tyr-Ser-Pro-Threon-Ser-Pro-Ser. 1st,
Ser5 is phosphorylated by a cyclin-dependent kinase (CDK) contained by TFIIH. Then, capping enzyme
Paf1 can associate w/ the CTD. Finally, Ser2 is phosphorylated by a CDK in P-TEFb. This allows for
assembly of complexes on CTD that are required for elongation, splicing and processing of 3’ end of
mRNA.
Ser 5 phosphorylation → capping can occur.
Ser 2 phosphorylation → splicing and processing machinery can bind and at the end factors required for
3’ end processing.
7. Was machen die Elongationsfaktoren?
EF suppress RNA pol II pausing and render rate of transcription independent of transcribed seq. E.g. TFIIS:
Proofreading involves backtracking of pol on RNA and removing dio RNA that is mismatched w/ template
strand. For incorrectly transcribed RNA to be cleaved away, need TFIIS (analogous function in bacteria:
GreB). Some seq r difficult to elongate for RNA pol → DNA-RNA hybrid formed in transcr. bubble isn’t
stable, causing backtracking, cleavage & removing of wrong nt. TFIIS is EF because it helps RNA pol cleave
backtracked RNA & recommence elongation, it helps RNA pol transcribe difficult regions of DNA. Other EF
are P-TEFb & FACT.
8.) Erklären Sie die Begriffe pos und neg Regulation der Transkription‘ + Bsp.
Positive regulation → activators, e.g. STAT. Negative regulation → repressors, e.g. TCF-Smad4
Every gene has a certain ability to bind RNA pol bcs it contains a promoter, → every gene has a basal level
of transcription in the absence of any additional control mech. (-) control means that a protein called
repressor completely shuts off even that basal amount of transcription. The activator does the opposite:
It increases the level of basal transcription → (+) control. Different situations:
1) Glu +, Lac-: repressor active, no active CAP → no transcription
2) Glu +, Lac +: repressor inactive, but no active CAP → basal transcription
3) Glu-, Lac +: repressor inactive, active CAP → strong transcription
4) Glu-, Lac-: absolutely no transcription (although activator should be active)
- why? In the presence of the repressor, CAP helps with repression! How → Due to the DNA bending
caused by CAP, an O1 repressor dimer can interact with an O3 repressor dimer → the complex is super-
repressed.In the case of (+) reg, it is the other way round, i.e. the gene is not transcribed in the normal
state, if you want to switch it on, a transcriptional activator must be formed. e.g. CAP or transcription
factor MerR.
9. Skizzieren Sie den kompletten Initiationskomplex bei Transkription durch RNA Pol II. Welche dieser
Proteine assoziieren mit DNA-bindenden Faktoren. The initiation complex (IC) is formed by TFs.
- TFIIA stabilizes TFIID binding; TFIIB binds DNA directly. TFIIB directly contacts Pol II, TBP, DNA.
1. contact Pol II and helping bind (links TBP with PolII)
2. helping strand separation (linker)
3. helping position exactly/recognition at transcription start (reader) → Pol II cannot bind Promoter if
TFIIB is not there
- TFIID/TBP binds DNA directly. First step of initiation. Binds core promoter elements. (offer contact
surface for TF) - TFIIE/TFIIF enables docking of TFIIH - TFIIH phosphorylates CTD. Transcription initiation
and DNA excision repair - Med associated with CTD, PolII, TF (offer contact surface for TF). Bridges Pol
with the binding of various transcriptional → coactivaor of PolII - PolII catalyzes the transcription of DNA
to synthesize precursors of mRNA
10. Was sind die speziellen Funktionen von TFIIH Komplexen? 2 functions: transcription initiation & DNA
repair processes. How can TFIIH perform these 2 different functions? The core can associate to different
proteins to form 2 different holoenzymes. If it associates w/ CDK7, CycH & MAT1 it can drive promoter
clearance. If it associates w/ Rad & XPG it can function in DNA repair.
11. RNA pol II besitzt c-terminale domäne. a. Wie ist diese beschaffen? b. Welche Rolle spielen
Phosphorylierungsereignisse im Transkriptionszyklus? RNA Pol II possesses a carboxy-terminal domain
(CTD) comprising up to 52 heptapeptide repeats of amino acids which can be phosphorylated. The RNA
pol II transcription cycle is dependent on phosphorylation and dephosphorylation of the CTD, as its
phosphorylation by TFIIH and P-TEFb facilitates not only transcriptional elongation but also the
association of proteins that participate in mRNA processing. TFIIH phosphorylates Ser5 of CTD and IC
containing various TFs and RNA Pol II is formed at the core promotor, however promotor clearance is not
yet possible until a second phosphorylation event → Phosphorylation of CTD at Ser2 is conferred by the
elongation factor PTEFb → enables transcriptional initiation and lifts the elongation block
12. Welche Phasen lassen sich bei der Initiation der Transkription unterscheiden (in Stichworten)? -)
(pre)initiation → formation of a (pre-)initiation complex (PIC or IC) that brings RNA pol to the promoter -)
clearance → promoter opens & then clearance occurs, meaning RNA pol leaves promoter & transcribes a
few nt but then it stops again. -) elongation → mostly continuous mRNA-synthesis -) termination → RNA
pol leaves template, in eukaryotic cells termination has a lot to do w/ processing of mRNA at 3’ end.
13. Wie beeinflusst der σ Faktor die Assoziation der prokaryotischen RNA Pol mit Promotern? To
understand transcription initiation in bacteria, you need to understand what the σ factor does.
Transcription initiation starts off w/ the so-called holo enzyme. W/ the help of the σ factor, RNA pol
knows ‚that this is a promoter‘. Core enzyme → no σ factor, holo enzyme → σ factor. The σ factor has
domains that recognise consensus seqs. It allows RNA pol to discriminate between promoter seqs & any
DNA because it allows it to recognise the bipartite promoter regions. W/o σ factor, RNA pol would not be
able to distinguish
14. Ablauf von DNA microarray in Stichworten beschreiben. Was sind die Vorteile gegenüber anderen
Methoden? DNA microarrays = gene chips. Each spot contains a probe that will hybridise to mRNA from 1
of the genes present in yeast. So you isolate RNA from yeast, reverse transcribe it, & while doing that add
a nt tagged w/ a fluorescent dye → you get a cDNA that can be excited at a certain wavelength. The
hybridisation signals can be presented in a heat map. Genes are put together in clusters depending on
how strong the hybridisation signal is. The heat map is colour coded to reflect the expression levels of
genes. This enables a quick & easy interpretation of DNA microarray data. Advantage: Microarrays allow
simultaneous analysis of many genes (even all genes present in a genome, when you look at yeast f.ex.)
15. Nennen Sie 3 verschiedene Mechanismen der positiven Transkriptionskontrolle durch TF. -) Impact
on the initiation of transcription: contacts w/ TAFs or mediator, sometimes through coactivators -) Impact
on elongation (pausing pol II): pTEFb recruitment (through mediator or other mechanisms) -) Impact on
DNA structure: bending (e.g. TBP), DNA loops, chromatin hubs (CTCF) -) Impact on chromatin
modification & structure: recruitment of histone-modifying enzyme complexes (HAT, HDAC,
methyltransferases, demethylases etc.), association w/ remodelling complexes (e.g. SWI/SNF),
association w/ activator complexes (e.g. Trx), association w/ silencer complexes (e.g. PCR2)
16. Skizzieren Sie die Ereignisse die von der Bindung eines Wachstumsfaktors (z.B.: EGF) an seinen
Rezeptor zur Induktion von Genexpression führen. Benennen Sie Komponenten und relevante
Classical Phosphorylation Cascade. 1. Association of EGF with a receptor tyrosine kinase (RTK) first results
in autophosphorylation 2. Phosphorylation of the GTPase Ras → activation/phospho of kinase RAF. 3. RAF
activates MEK → activates MAPK 4. MAPK directly phosphorylates TF p90RISK → enter nucleus 5. MAPK
phosphorylates TCF, while SRF (serum response factor) phosphorylated by p90RISK
17. Wofür steht die Abkürzung ICR und welches Protein bindet an die ICR? Welche epigenetischen
Modifikationen reguliert die Bindung des Proteins an die ICR und welche Wirkung hat die Modifikation
auf seine Bindung? For genetic imprinting the main decisive point is wether CTCF binds to the imprinting
control region (ICR). CTCF frequently binds to insulator seqs. It part of a group of TFs that are sensitive to
methylation (won’t bind when methylation present). For the paternal allele, the ICR is methylated & for
the maternal it isn’t.
18. Womit interagieren folgende Proteinstrukturen? -) SH2-Domäne - phosphoryliertes Tyrosin -)
Chromodomäne - methylierte Histone -) Bromodomäne - acetylierte Histone -) Leucin Zipper - DNA
19. Posttranslationale Modifikationen an TF.
Cells react to environmental stimuli by changes in GeneEx, which are achieved either by de novo
synthesis or posttranslational modification of TF. Binding of a ligand such as heavy metals; protein
modifications such as phosphorylation; cleavage of a precursor molecule; dissociation of an inhibitory
molecule
-TF ACE1 is a sensor for excess Cu2+ and induces genes involved in heavy metal detoxification → at low
levels of Cu2+ ACE1 remains inactive. Cu2+ concentrations increase → ACE1 binds Cu2+ and associates
with its BS → transcription of Metallothionine → gene product binds intracellular metals like copper.
20. Welche Methoden um mRNA quantitativ nachzuweisen kennen Sie? Welche liefern exakte
quantitative Werte? Kann man damit Transkription nachweisen (Begründung)?
Northern Blots depict radioactively visualised band patterns which represent mRNA molecules separated
by their respective size. RT-PCR synthesises cDNA from mRNA isolates through reverse transcription with
help of primers specific for a certain gene. qPCR enables real-time observation of cDNA amplification as
fluorophores intercalate into dsDNA fragments. The intensity of fluorescence is proportional to the
amount of synthesised DNA, allowing determination and evaluation of the linear, quantitative phase of
PCR. Gene microarrays can only be applied if the whole genome of a target organism is known, as
complementary DNA seqs of each gene are fixed in defined positions onto a glass chip. NGS (next
generation sequencing) is a method for gene expression analysis whereby mRNA is reverse transcribed
into cDNA. It is based on automated massive parallel sequencing of cDNA (libraries) to render seq reads
which are complementary to mRNA isolates.
21. Nennen Sie 3 in vitro/vivo Methoden um Bindungssequenzen von TF zu verifizieren. Gibt es noch
andere Methoden außer TF-Bindungssequenzen nachzuweisen? In vitro: -) Electrophoretic mobility shift
assay (EMSA) - Radioactively label DNA fragment. Add cell extract, protein binds to the fragment resulting
in a complex that migrates slower in a gel than the DNA alone → mobility shift. Afterwards you put a film
on the gel that will blacken where radioactive stuff is. In vivo: -) Chromatin immunoprecipitation (ChIP) -
Covalently crosslink proteins w/ DNA, fragment by sonification. Purify protein-DNA complexes, use
antibodies against TF that are coupled to beads, precipitate via centrifugation. Purify DNA (w/ proteases)
→ all obtained fragments contain TF binding sites. -) Co-transfection reporter gene assay - Make plasmid
w/ reporter gene and upstream binding site for TF. 2nd plasmid that causes cell to make TF. When TF is
transcribed and binds to its binding seq, reporter gene is expressed.
22. Erklären Sie die Bedeutung von σ-Faktoren. Wie würden Sie nachweisen, dass ein Gen von σ70
reguliert wird? Depending on the presence or absence of a sigma factor, the RNA pol is defined as a
holoenzyme and a core enzyme, respectively. Different sigma factors confer particular RNA Pol
specificities and thereby allow transcription of various genes. The holoenzyme binds DNA nonspecifically
and performs a one-dimensional search for the promoter. After a specific promoter is found and RNA Pol
has associated, a closed complex is formed, followed by a conformational transition to an open complex
→ promoter clearance, dissociation of the sigma factor and transcriptional elongation by the core
enzyme.
DNA seq containing the gene of interest and RNA pol have to be isolated. Both are then incubated under
three different conditions: once with the target factor sigma 70, once with another sigma factor and once
without any sigma factor. If indeed sigma 70 is solely responsible for transcription of certain genes, no
reads (e.g. on a Northern Blot) should be visible under the other two conditions.
23. Abbildung des Chromatin-hubs, die man auch in seinen Folien etc. findet: Wie nennt man die
abgebildete Struktur? Was bedeutet in diesem Zusammenhang LCR und Insulator? Welche Chromatin-
Modifikationen würden Sie in Bereichen von active bzw. inactive genes erwarten?
Insulator (Ins) and locus control regions (LCR) separate distinct chromosomal regions. Insulator seqs:
insulate euchromatin from heterochromatin. → Insulator elements form boundaries between genomic
regions with different structural and functional properties. → Locus control determine by interaction with
the promoter or enhancer which genes within a chromosomal domain can be transcribed.
Chromatin hubs: There’s a loop that contains inactive and another that contains active genes. Only the
enhancers of the active genes are in vicinity of the LCR (locus control region). Depending on which TFs
appear, the LCR forms a loop-based complex w/ the individual promoters of the different globin genes.
Only those in the activated loop will be transcribed. Entire region isolated by CTCFs that bind to insulator
elements and cause loop formation. CTCF isn’t the only thing required for loop formation. It has been
shown that chromatin hubs require the interaction of CTCF w/ cohesins. They hold sister chromatids
together during meiosis, but they also hold other chromosomal regions together. How does DNA in the
nucleus know wether it should be hetero- or euchromatic? There are DNA seqs that play important roles
in determining these states of DNA. 2 important regions are the LCR or insulator regions (also called
boundary elements).
Chromatin regions which harbour actively transcribed genes are defined by acetylation, whereas
transcriptionally silenced regions are methylated.
24. GENE KÖNNEN DURCH EPIGENETISCHE MECHANISMEN REGULIERT WERDEN. a. Welches Kriterium
muss dabei erfüllt sein? b. Welche Rolle spielen Polycomb-Proteine (PRC1, PRC2 bei Säugern)?
Epigenetics describes mitotically heritable but reversible modifications to DNA or chromatin that lead to
changes in gene expression in the absence of genetic alterations. Epigenetic mechanisms are responsible
for different gene expression patterns from identical DNA in different cells and organs. Epigenetic gene
control results from the formation of distinct chromatin structures (eu-/heterochromatin) and DNA
methylation.
PRC1 binds H3K9Me + H3K27Me → interferes with transcription and makes sure that compact silence
chromatin is maintained through mitosis (stabilizing silencing and underlies cellular memory after cellular
differentiation)
PRC2 trimethylates H3K27Me3, corresponds with gen silencing → mark of transcriptionally silent
chromatin (initial targeting of genomic region to be silenced) (both required for long term epigenetic
silencing
25. POLYCOMB COMPLEXES ARE IMPORTANT FOR EPIGENETIC GENE REGULATION. a. What is their
effect on gene expression? b. How do they produce this effect? c. Which enzymatic activity of
polycomb complexes is important? Polycomb homologues found in mammals (PC) are responsible for
heterochromatin formation and propagation. Euchromatin is heterochromatised by polycomb complexes
through trimethylation of e.g. H3, resulting either in H3K9Me or H3K27Me. PC contains a histone methyl
transferase with ability to K9K27 modi → heterochromatin/ silence chromatin → leads to chromatin
structure that effects a whole DNA region → associated to eu/heterochrimatin formation in a way that is
inheritable. PRC 1 I PRC2 pitanje iznad
26.Warum muss DNA während der Transkription relaxiert werden? Nennen Sie die Namen der 2 dafür
relevanten Enzymgruppen und Unterschiede in ihren Mechanismen. Transcription of topologically
constrained DNA templates causes superhelical stress. If a DNA strand which has been fixated on either
end would be transcribed by a RNA polymerase unable to rotate, this would result in negative
supercoiling upstream of RNA Pol and positive supercoiling downstream of RNA Pol. Ultimately,
transcription would arrest. Topoisomerases are enzymes which relieve tension of topologically
constrained DNA: Topoisomerase I induces relaxation of neg. supercoils. No ATP is required as they only
nick one strand of DNA resulting in intermediate single-strand breaks through which the other strand is
passed. In contrast, Topoisomerase II facilitates ATP-dependent intermediate double-strand breaks
leading either to relaxation of DNA or establishment of neg. supercoils.
27.Beschreiben oder skizzieren Sie die Struktur eines Nukleosoms. Welche Komponenten des
Nukleosoms sind besonders gut für posttranslationale Modifikationen zugänglich? Nennen Sie drei
dieser Modifikationen. Können Sie in jedem Fall eine klare Aussage darüber machen, wie sich diese
Modifikationen auf die Transkription auswirken? Nucleosomes r composed of a histone core. DNA
wraps around it using 146 bp (wrapped twice around core). Between 2 histone cores is linker DNA of
variable length (20-60 bp). Histone core is formed of 4 diff histones: H2A/H2B dimers and a H3/H4
tetramer. Surface of histone core positively charged (high number of positive aa), DNA backbone
negatively charged due to phosphates.
H1 associates with linker DNA → compaction of nucleosomal DNA. H2 and H4 possess long N-terminal
unstructured regions (histone tails) protruding from nucleosome which are accessible for post-
translational modifications. Acetylation neutralise the positive charge of lysines → loosening of DNA-
nucleosome interaction and increasing accessibility of DNA for TFs. Deacetylation of lysines in histone
tails shows opposite effect. Methylation of arginine or lysine and phosphorylation of threonine or serine
→ functions of phosphorylation and methylation is not always definitive as the interplay with other
modifications of the histone code determine their effect
28. Skizzieren Sie die Struktur eines Nukleosoms einschließlich DNA. Benennen Sie die Komponenten.
Welchen Änderungen ist nukleosomale DNA im Zuge des chromatin remodelling unterworfen? Wie
bzw. durch welche Proteine wird die Energie für die chromatin remodelling erzeugt? Građa- iznad
If the transcription start or transcription factor binding sites are overlapped by nucleosomes, the
nucleosomes have to be moved in order for transcription to be possible →site exposure (Deposition,
repositioning, ejection, unwrapping) and altered composition (dimer exchange, dimer ejection)
Chromatin remodelling is achieved by ATP-dependent chromatin remodeller complexes such as SWI/SNF
or ISWI. Elongation factor FACT (facilitator of transcription) displaces the H2A/H2B dimer from the
nucleosome, allowing transcription, but also reinstalles the dimer by acting as a histone chaperone.
29.Beschreiben Sie kurz den Aufbau eines Nukleosoms (Skizze!). Beschreiben Sie die Aktivität des
chromatin remodelling complex. Was hat die (De-)Acetylierung der Histone zur Folge Chromatin
remodelling complexes (f.ex SWI/SNF in yeast) have certain characteristic features:
-) they all contain a protein w/ ATPase activity → for chromatin remodelling, the chemical energy of ATP
is converted to mechanical energy.
-) they must contain protein domains that are able to interact w/ histones. Often, histones contain
chemical modifications. Ex. for histone binding domains: bromodomain, SANT domain, PHD finger.
-) they all have the ability to slide histones, but not all of them can transfer (i.e. exchange for different
variant histones).
Acetyl groups, attached by histone-acetyl transferases (HATs) neutralise the positive charge of lysines.
This loosens the DNA interaction w/ the nucleosome & increases the accessibility of DNA for TF.
30. Bild von TFIID : Was wird hier gezeigt? Was ist seine Funktion? Beschreiben Sie den Nachweis von
seiner Bindung an DNA in vitro.
General TF TFIID (TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs)) → central role in
initiation of RNA pol II dependent transcription → initiating pre-initiation complex (PIC) assembly at the
core promoter. 1st binding of TFIID to the TATA box → TBP causes DNA to bend around initiation site →
GTFs recruited. Electrophoretic mobility shift assay (EMSA): allows determination of TF binding order by
incubating radioactively labelled DNA fragments carrying a promoter with various transcription factors.
Binding of the proteins to the promoter can be monitored by PAGE. With addition of each component a
larger complex was formed resulting in a shift on the gel.
31. Skizzieren sie den Signaltransduktionsweg mit G-coupled receptors und CREB. Wie können Sie in
vitro und in vivo nachweisen, dass CREB an die DNA bindet?
CREB is a conditionally active, signal-dependent, cell membrane receptor-dependent resident nuclear
factor. This means that CREB requires activation trough external factors which result in phosphorylation
of the transcription factor by second messenger signalling cascades. CREB resides in the nucleus
irrespective of its activation and possesses a basic region for DNA binding. The interaction of CBP with
phosphorylated CREB has been characterised in detail by using a fluorescence polarisation binding assay
and a genetic interaction assay in yeast. A central tenet of the CREB–CBP model is that CREB binds
constitutively to the CRE and that regulation occurs through the phosphorylation-dependent recruitment
of CBP. Immunoprecipitation assays show that CREB does not interact in vivo with CRE, or similar
elements in several other genes. Rather, CREB binding in vivo is regulated in a cell-specific manner, a
finding that was confirmed by using in vivo genomic footprinting assays.
32.NENNEN SIE 2 CHROMATIN-REMODELLIERENDE KOMPLEXE. a. Beschreiben Sie die biochemischen
Mechanismen von Chromatin Remodellers. b. Woraus beziehen sie die dafür nötige Energie? CDC are
not only able to bind to histones but also possess an ATPase domain which converts ATP to ADP (ATP-
dependent chromatin remodelling). The ATPase of SWI/SNF (human BAF complex) is bound to DNA and
moves in respect to the Bromodomain (translocation). This translocation induces stress which is released
through nucleosomal remodelling: stable products include nucleosomal sliding, DNA bulging, histone
exchange and nucleosomal transfer. ISWI facilitates nucleosomal sliding by binding to the H4 tail
protruding from the nucleosome and inducing the so-called HSS-out state (Hand-Sant-Slide domain;
primed state). ATP hydrolysis results in the HSS-in conformation, whereby HSS is moved in regard to the
ATPase and DNA is translocated 7 bp per hydrolysed ATP
33. Was bewirkt genomic imprinting von Genen? Welche Genmodifikation ist mit genomic imprinting
assoziiert? Die Bindung welches TFs wird durch diese Modifikation reguliert? Imprinting causes
differential expression of maternal and paternal imprinted genes through silencing, which is essential for
the development and homeostasis of mammalian organisms. There are three different models of
genomic imprinting. First, CTCF binds to the imprinting control region (ICR) on the maternal chromosome
enabling transcription of a downstream promoter while simultaneously repressing upstream gene
expression. Methylation of the paternal chromosome ICR prevents CTCF from binding. Thereby upstream
gene expression is now possible, while expression of downstream genes is repressed. Second, CTCF binds
to the non-methylated ICR on the maternal chromosome and recruits an associated protein which
induces formation of heterochromatin. Hence, CTCF insulates downstream active transcription sites from
the upstream heterochromatic sequences. On the paternal chromosome, ICR is methylated enabling
heterchromatisation of downstream sequences. Third, both maternal and paternal chromosome contain
a sequence for a long non-coding RNA (Air). On the maternal chromosome Air is not expressed, allowing
transcription of upstream promoters. However, on the paternal chromosome, Air is expressed which not
only leads to promoter occlusion on neighbouring promoters but also to inhibition of gene expression of
other upstream promoters by recruitment of HATs. Thereby, genetic imprinting ensures that only one
copy of each gene is active.
34. Nennen Sie reader, writer und eraser des histone codes. The histone code comprises covalent
modifications which are transduced by the histonemodifying writer enzymes and removed by the
antagonising activity of erasers. Writer: HAT (lysine), kinase (serine, threenine), PRMT (aragnine), HKMT
(lysine) Reader: bromo domain, unknown, Tudor, chromo domain; Eraser: HDAC, PPTase,
deiminase/citrulline, amine oxidase hydroxylase
35. Was ist der Unterschied zwischen de novo und maintenance Methylierung? Wie ist ein DNA
template für de novo/maintenance Methylierung beschaffen? DNA methylation is facilitated by DNA-
methyl-transferases (DNMTs). There are 2 kinds: -) De novo methylases are responsible for establishing
DNA methylation. They methylate a DNA strand even if there’s no methylation on the opposite strand. -)
Maintenance methylases make sure that methylations are passed on to daughter cells. They profit from
the fact that DNA replication is semi-conservative: 1 strand is already methylated, they then use this
template to methylate the newly formed strand. De novo means the DNA template is entirely
unmethylated. In all cells of our body we have a specific pattern of methylated Cs, so when cells replicate
the pattern of methylation also needs to be replicated, which is what maintenance methylases, Dnmt1,
do. This works because DNA replication if semi-conservative so 1 strand of the DNA (the parental strand)
is methylated and the other (daughter) isn’t.
36. Bild von genomic imprinting): Benennen Sie das abgebildete epigenetische Phänomen. Wofür steht
die Abkürzung ICR und welches Protein bindet an die ICR? Welche epigenetischen Modifikationen
reguliert die Bindung des Proteins an die ICR und welche Wirkung hat die Modifikation auf seine
Bindung?
Genomic imprinting depends on the so-called imprinting control region (ICR), a seq present on both
maternal and paternal chromosomes, which enables controlled, selective expression of upstream and
downstream promoters on each chromosome. CTCF can only bind to ICR if it is non-methylated which
results in inhibition of upstream gene expression while downstream promoters can be transcribed. When
ICR is methylated, CTCF cannot bind and upstream gene expression is ensured. Furthermore, binding of
CTCF regulates distribution and formation of heterochromatin: if CTCF is bound, upstream sequences will
heterochromatise while downstream gene expression active through insulation by CTCF. However, if ICR
is methylated and CTCF binding prevented, downstream seq will become heterochromatin, allowing
expression of upstream promoters.
37. Welche Base kann in eukaryotischen Organismen methyliert vorliegen? Skizzieren Sie die Struktur.
Welche biologische Funktion hat die Methylierung von DNA?
DNA methylation is mostly (but not exclusively) associated with transcriptional silencing. Most cytosine
bases within a CpG dinucleotide are methylated at the C5. Exception: CpG islands which are often found
at the 5’ end of house keeping genes. → epigenetic silencing (X Chromosome inactivation, imprinted
genes), stable silencing of endogenous retroviruses, retrotransposons and transgenes