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202 Cards in this Set

  • Front
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How do Illumia sequencing work?
1) Randomly framgent genetic DNA and ligate adapters on both ends to it. 2) Attach there pieces of DNA to a inner surface of flow cell channels.
3)Add unlabeled nucleotides and enzymes to initiate the solid phase bridge amplification. >>> Fragments become double stranded so they have 2 attached terminusen and 2 not attached.
4) denaturate the ds molecules
5) amplificate the clusters so you get like several millions of the finally.
6) determine the first base: add 4 reversible terminators, primers and DNA polymerase to the flow cell. Determine the sequence, with fluorescence imaging.
Which modification can undergo lysine?
*acetylation
*sumoylation
*ubiquitination
*biotinilatyion
Which modification can undergo arginine?
*citrullination
*methylation
Which modification can undergo glutamic acid?
ADP-ribosilation
Which modification can undergo proline?
cis-trans isomerization
Discribe the solid phase nanopore?
- A protein in a membrane has a hollow tube of a few nanometers in diameter. A potential is applied across the bilayer resulting in a current through the nanopore. Single molecules that enter the nanopore cause a disrupture in current. By measuring the disruption the molecule can be identified. DNA sequencing methods are exonuclease sequencing (not optical because not all nucleotides fall in the pore) and strand sequencing.
What is the goal of International cancer genome consortium ?
to obtain a comprehensive description of genomic, transcriptomic and epigenomic changes in 50 different tumour types and/or subtypes which are of clinical and societal importance across the globe.
How is the genome analyse within ISGC take place?
*whole genome shot gun analyse
*catalogues of somatic mutations
-sequencing of all exons
-analysis of paired read ends for reareggements
-copy number variation and breakpoint information
*expression analysis
-protein coding genes
-noncoding RNAs
-miRNAs
What is the goal of BLUEPRINT?
BLUEPRINT will generate epigenomes of >60 distinct types of
primary haematopoietic cells from healthy individuals and of >60 blood cancer subtypes
How do sanger sequencing works?
- DNA is denatured into single strands using heat. Next a primer is annealed to one of the template strands (comes next to the DNA sequence of interest). Then 4 tubes labelled G, A, T, C. Then reagents are added, four dNTPs, ddG/A/T/C(fluorescent) and DNA polymerase. When dideoxynucleotide is incorporated into the chain in place of a normal nucleotide it results in a chain-terminating event. Chains will terminate at all positions, and bands of all different lengths are produced. Then again denaturation and electrophoresis. The smallest band is the first nucleotide etc.
- Low error rate, reliable, long reads, low throughput, expensive, It is very slow, cloning step is essential.
What is the difference between the sanger sequencing and automated sanger sequencing?
- Same principal, but one single tube containing all four ddNTP’s, each labelled with a different colour dye. All run on the same lane. Then a laser reads the gel to determine the identity of each band according to the wavelengths at which it fluoresces. Then you have a diagram of coloured peaks that correspond to the nucleotide in that location in the sequence.
- Shotgun sequencing: sequence random pieces of DNA, computer figures out how it fits. Much faster but very expensive.
- Here one molecule is sequenced.
- Sequencing by blocking addition of nucleotides.
What is shotgun sequencing?
n shotgun sequencing,DNA is broken up randomly into numerous small segments, which are sequenced using the chain termination method to obtain reads. Multiple overlapping reads for the target DNA are obtained by performing several rounds of this fragmentation and sequencing. Computer programs then use the overlapping ends of different reads to assemble them into a continuous sequence.
What is next generation sequencing?
- For example illumine, roche/454 sequencer, solid sequencer.
- True high-throughput, in the same reaction you can sequence more than one molecule, sequencing by synthesis.
- Gene expression can be estimated. Sequence DNA, measure how many sequences (counting reads) you have of every gene of genome A & B and then you have a ratio.
- Massive parallel sequencing of complex samples, cheaper
- Early diagnosis become possible and looking at risks that are in your genome. Change lifestyle/medication. Disadvantage is that a selection of healthy people can take place
Describe the prokaryotic chromosomes.
- Usually circles of 10^6 a 10^7 basepairs
- Also horizontally transmission of genes, conjugation between bacteria.
- Chromosome condensation: electrostatic repulsion between the negatively charged phosphate groups in the DNA backbone can be neutralized by binding of positively charged molecules such as polyamines. The major polyamines are spermine and spermidine. Magnesium can do the same with its two positive charges. Factor 1000 condensation to fit inside the bacterium.
- DNA binding proteins condense DNA by wrapping DNA around themselves, for example H-NS.
- Supercoiling can effectively condense chromosome molecules, the take up less space when supercoiled.
- Gyrase, a type II topoisomerase, employs ATP to negatively supercoil bacterial genomes.
-supercoiled DNAs migrate fast on gel
Describe chromosomes in Eukaryotes.
Chromosomes in eukaryotes
- 10^8 basepairs
- Are in nucleus, so transcription and translation are separated spatially.
- Chromosome replication and segregation are also separated, temporally in eukaryotes taking place in S and M phase.
- Condensation with a factor ~1 million is needed.
- In the metaphase there is maximal condensation.
- Each chromosome tends to stay in its own territory. Their place differs from cell to cell and is dependent on their spindle position during mitosis.
- Looping is a very likely mechanism to constrain centimetre-sized chromosomes into micrometre-sized territories
- Rings are wound around the DNA to stop diffusions from their territory (cohesion-kleisin)
What is deoxyribonucleo-protein complex ?
Is a chromatin. It is bound to the equivalent mass of DNA.
What are the components of the chromatin?
*histones
*linker histones
*nucleosome
*core histone tails
What is a histone?
Basic proteins rich in lysines and arginines. The sequences are highly conserved in evolution in all eukaryotes. Form octamers and wrap 146 bp of DNA in a left handed fashion.
What is linker histone?
- H1 and H5 that are less well conserved in amino acid sequence. Cannot substitute for histone tails.
- The linker histone H1 binds the nucleosome and the entry and exit sites of the DNA, thus locking the DNA into place and allowing the formation of higher order structure.
What is the role of core-histone?
- The core histone tails are necessary for inter-nucleosome contacts
-Two of each of the core histones assemble to form one octameric nucleosome core particle, and 147 base pairs of DNA wrap around this core particle 1.65 times in a left-handed super-helical turn
What is C-value paradox?
the fact that the amount of DNA does not correlate with the complexity of that organism.
How is the complexety of DNA can be measured?
*by melting characteristics ( complex, longer temperature trajectory) , also A-T content
*DNA renaturation experiments: when a genome has a sigmoid curve on a % ssDNA versus C(0)t graph, it is less complex then a genome with not a sigmoid curve.
What do one octamer contains?
*One octamer consists of a H3(2)-H4(2) tetramer plus two H2A-H2B dimers
*Each nucleosome has ten histone “tails” that protrude from the nucleosome particle
Why do histone tails are necessary?
*for inter-nucleosome contacts
Which three types of DNA are there?
Fast (10-15%),
intermediate (25-40%)
slow (50-60%)


DNA of these three types was cloned by chromatographic separation over hydroxylapatite, a chemical that will bind dsDNA but not ssDNA (under specific ionic conditions).
Describe fast DNA?
Fast (10-15%)
-Simple, highly repeated 2-200 bp DNA sequences.
-‘Tandem repeat’ can be as long as 10^5 bp and are called satellite DNA
-Do not code for protein
-Some are found in the vicinity of telomeres and of centromeres where they may play a structural role.
-Alpha satellite: accommodates exactly one nucleosome
-Length of these repetitive DNAs varies amongst individuals because of unequal crossing over during meiosis (genetic fingerprinting)
-Can be amplified by replication slippage:
• Backward slippage, one daughter with one extra repeat and normal daughter.
Which two types of intermediate DNA there are?
intermediate (25-40%)
*mobile elements
*Tandem repeats of ribosomal RNA genes, transfer RNA genes and histone genes, multiple copies of these genes are present in the genome to satisfy the high demand for these gene products
Describe slow coding DNA.
-25%-50% of genes are only present once per genome
-50%-75% of the genes belong to gene families
-Gene families can be found in clusters or they can be distributed over multiple chromosomes
-A particular fraction of the slowly renaturing DNA sequences consists of pseudo-genes.
-Processed pseudogenes:
•Are the products of reverse transcription of mRNAs (cDNA) that are subsequently integrated into the genome. L1 LINE may play a role in their genesis in humans.
•Have not a promoter, are often 5’ truncated, are fully or partially spliced versions of the primary mRNA transcript from which they were made. Still have all their introns, since they are generated via a DNA duplication event rather than via a RNA intermediate that is subject to splicing.
Describe slow coding DNA.
Slow (50-60%) coding DNA,
- 25%-50% of genes are only present once per genome
- 50%-75% of the genes belong to gene families
- Gene families can be found in clusters or they can be distributed over multiple chromosomes
- A particular fraction of the slowly renaturing DNA sequences consists of pseudo-genes.
- Processed pseudogenes:
What are the processed pseudogenes?
• Are the products of reverse transcription of mRNAs (cDNA) that are subsequently integrated into the genome. L1 LINE may play a role in their genesis in humans.
• Have not a promoter, are often 5’ truncated, are fully or partially spliced versions of the primary mRNA transcript from which they were made. Still have all their introns, since they are generated via a DNA duplication event rather than via a RNA intermediate that is subject to splicing.
What are the SINEs?
Short INterspersed Elements are short DNA sequences (<500 bases) that represent reverse-transcribed RNA molecules originally transcribed by RNA polymerase III into tRNA, rRNA, and other small nuclear RNAs. SINEs do not encode a functional reverse transcriptase protein and rely on other mobile elements for transposition.
What is Alu sine?
*300 bp long and makes up 13% of the human genome
*The DNA sequence of Alu elements resemble the 7SL RNA and is very probably derived from it. 7SL RNA is part of the signal recognition particle (SRP), this transports the ribosome to the ER.
*There seems to be a positive correlation between local gene density and SINE density.
*The Alu elements are most likely transposed by the L1 line enzymes
*ALU can be used as probes for PCR reaction. SINEs of hamsters are not identical to the human ALU SINE.
Describe L1 line enzymes?
- L1 LINE is ~6 kb long, makes up no less than 21% of the human genome. Codes for a reverse transcriptase/integrase that reverse transcribes the L1 mRNA and subsequently integrates the L1 cDNA in the chromosome.
- Has 2 ORF, 1 see above and 2 codes for an RNA binding protein whose function is not yet known. Both ORFs products are found in most human cells and they are most likely responsible for SINE transposition as well as for the generation of “processed pseudogenes”.
What are the transposons?
- Are the cause of diversity (genetic change) on which selection can act.
- Can also spread genetic material ‘horizontally’ between individuals of 1 species and even across different species.



A transposable element (TE) is a DNA sequence that can change its relative position (self-transpose) within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's genome size.
Which two general classes of transposons by eukaryotes there are?
transposons and retroposons
Describe the eukaryotic transposons?
*Related to the prokaryotic IS elements
*Have 10-40 bp inverted repeats at both ends that serve as binding sites for the transposase
Describe the retroposons? Which types of retroposons there are?
*Are predecessors of retroviruses (or can also be other way around)
*Undergo transposition via an RNA intermediate and reverse transcriptase activity
*LTR
*Non-LTR retrotransposons
What are the LTR retroposons?
• Comparable with aids, but have no genes for the viral envelope
• They have 200-600 bp “long terminal repeats (LTRs) that are in the same orientation on the ends of the retroposon.
• Cause 3-6 bp duplication of the host DNA integration sequence wherein their integrase (cq transposase) integrated them.





The LTRs are partially transcribed into an RNA intermediate, followed by reverse transcription into complementary DNA (cDNA) and ultimately dsDNA (double-stranded DNA) with full LTRs. The LTRs then mediate integration of the retroviral DNA via an LTR specific integrase into another region of the host chromosome.
What are the non LTR transposons?
• One end of these elements has a polyadenine sequence
• Often miss the extreme 5’part, due to incomplete reverse transcription.
• SINE
• LINE
>>>major ones make up 45% of the human DNA
What are the LINEs?
Long INterspersed Elements are a group of genetic elements that are found in large numbers in eukaryotic genomes. They are transcribed (or are the evolutionary remains of what was once transcribed) to an RNA using an RNA polymerase II promoter that resides inside the LINE. LINEs code for the enzyme reverse transcriptase, and many LINEs also code for an endonuclease (e.g. RNase H). The reverse transcriptase has a higher specificity for the LINE RNA than other RNA, and makes a DNA copy of the RNA that can be integrated into the genome at a new site.
How do transposons play a role in the evolution of genes and genomes?
1) Integrative mutagenesis: Some transposons code for promoters/enhancers, splicing sites and polyadenylatie signals that can change local gene expression patterns
2) generation of duplicated genes or even chromosome translocations via “unequal cross over” between two copies of a transposons.
3) generation of “processed” pseudogenes: if these integrate close to a new promoter, this „third copy‟ of the transposed transcription unit can now display new expression patterns.
What are the differences between male and female recombinations?
- Female: not around centromere
- Male: around centromere, never breakage at centromere, so female recombination more.
What are the tandem dublications?
duplication of exons within one gene >25%, next to each other. First it is duplication, then small differences, then two different genes.
What are the large segmental dupliations?
segments of DNA with near-identical sequence, whole piece of chromosome is duplicated.
What is genetic fingerprinting?
-based on difference in the tandem repeat leghts.
-The length of these repetitive DNAs varies amongst individuals because of unequal crossing over during meioisis.
-after meiotic division the germ cells will get tandem repeats of different lenghts.
How do generation of microsatellite repeats by backward slippage of the nascent daughter strand during DNA replication works?
If during replication the nascent daughter strand "slips" backward relative to the template strand by one repeat, one new copy of the repeat is added to the daughter strand when DNA replication continues. THis new copy of repeat forms a single stranded loop in the daughter strand of the daughter duplex DNA molecule. If this single stranded loop is not removed by DNA repair proteins before the next round of DNA replication, the extra copy of the repeat is added to one of the double-stranded daughter DNA molecules, the other daughter molecule will be normal.
Which classes of somatic mutations in cancer there are?
1. Point mutations
2. Rearrangements
3. Copy number changes
Which two type of mutations there are?
*drivers - are the peaks and cause initiation
*Hills, results of gene shuffling, not present in every cell, only occurs in small subset. Provide no positive or negative selective advantage to the tumour, but are retained by chance during repeated rounds of cell division and clonal expansion.
What is IDH1 mutation?
-Result in a new ability of the enzyme to catalyse the NADPH-dependent reduction of a-ketoglutarate to 2-hydroxyglutarate (2HG). a-ketoglutarate is needed for the reduction methyl lysine to lysine. >>>> hypermethylation phenotype
what is CGH array?
DNA from a test sample and normal reference sample are labelled differentially, using different fluorophores, and hybridized to several thousand probes. The probes are derived from most of the known genes and non-coding regions of the genome, printed on a glass slide.

The fluorescence intensity of the test and of the reference DNA is then measured, to calculate the ratio between them and subsequently the copy number changes for a particular location in the genome
What is cloned paired end sequencing (shotgun sequencing)?
In shotgun sequencing, DNA is broken up randomly into numerous small segments, which are sequenced using the chain termination method to obtain reads. Multiple overlapping reads for the target DNA are obtained by performing several rounds of this fragmentation and sequencing. Computer programs then use the overlapping ends of different reads to assemble them into a continuous sequence

-They should contain enough sequence information to be uniquely mapped to the genome and thus represent the whole DNA fragment of interest.
-Look at the reference gene and patient genome with a certain sequence, to look if a deletion or insertion has occurred. Reference genome is always for example 3 kb. When patient genome is >3kb, it is a insertion, when it is <3kb It is a deletion.
what is chromothripsis?
catastrophic chromosome breakage, then rearrangement, loss of some pieces. This is a single event that causes cancer and not like multihit.
What is the 5 hit scenario of colorectal cancer?
Normal colon cells: APC mutation
Adenomateous polyp: one RAS mutation
Dysplastic polyp: two p53 mutations
Colon carcinoma: other events chromosomal abberations.
Metastatic carcinoma
Why people use chip seq?
to determine the epigenetic and transcription factor profiles
Describe which histones are there in the nucleosome? Describe the positions? ( zie tabel ) .
There are 2 copies of each histone per nucleosome. H2A,H2B,H3,H4.
What is constitutive heterochromatin?
-Constitutive heterochromatin is always there, enriched at centromeres and telomers
What is facultative heterochromatin ?
-Facultative heterochromatin is environmentally or developmentally regulated.
Describe Rb , E2F repression?
-H3K9 methylation, which recruits heterochromatin binding protein 1 (HP1)
-Also HDAC binds to Rb and E2F and deacetylates H3K9, then HP1 binds to H3K9 methylase , which can generate the new bindins sites for HP1, which itself also recruits methylases and it results in spreading of heterochromatin.
How do HDAC are recruited?
* by site specific repressors
*methyl-binding proteins (MBP) which bind to methylated CpG

The repressor proteins (rep) binds to tails in their deacetylated state and stabilizes nucleosome positioning, blocking of cis regulatory elements.
Describe H3K4me3.
-Deposited by a family of SET domain proteins: MLL’s and SET1A/B
-Are methylation complexes, methylate histones at promoter sites.
-MLL2: needed for development of the embryo, no methylation at promoters, but needed for neuronal development
-MLL1: at birth you know if it is a knock out, so it is needed at different times.
What are MLL and which MLLs are when needed?
-Are methylation complexes, methylate histones at promoter sites.
-MLL2: needed for development of the embryo, no methylation at promoters, but needed for neuronal development
-MLL1: at birth you know if it is a knock out, so it is needed at different times.
What does HAT?
HAT acetylates the histone tails and enables the binding of activators to enhancers and of general transcription factors such as TFIID and RNA polII to the promoter. The remodeler mobilizes the nucleosomes so that important cis sites are revealed.
Which modification are there in the euchromatin and heterochromatin?
*Heterochromatin: Low compaction, gene rich, mostly acetylated.
-H3K27me3
-H4K20me3
-H3K9me3

*Euchromatin : H3K4me3,
H3K9ac,
H4ac,
H2A-­‐Z
How do marks get onto promoter regions?
-Via recruitment of modifying enzymes, by DNA sequence specific transcription activators that bind to cognate response elements.
-Via RNA polymerase II
What happens upon desregulation of HAT complexes?
-MLL-CBP fusions
-MOZ, MORF and TIF2 fusions
What happens upon misregulation of HDAC complexes?
-PML-RAR fusions
-DNA methylation and HDAC recruitment
-Treatment is HDAC inhibitors
What does - Mono-ubiquitination?
Transcription, histone function, endocytosis and membrane trafficking
What does Lys48, Lys11 or Lys29 linked poly ubiquitination?
Target proteins to the proteasome
What does Lys63 linked poly ubiquitination?
Signalling, DNA repair, stress response, endocytosis and signal transduction
Whereto leads terminal ubiquination of H2A and H2B?
H2A: repression
H2B: activation
- no polyubiquination and degradation
-The methylation of H3K4 is dependent on mono-ubiquitination at H2B. The ubiquitin recruits SET1 and this can methylate H3 for example.
What is hallmark of cancer?
*Aberrant DNA methylation is a hallmark of cancer
* Mutations of chromatin regulator genes are prevalent
What is common for neuropsychiatric, metabolic, developmental disorders?
• Mutations in chromatin regulators (MeCP2), aberrant epigenetics
• Long-term consequences of early environmental exposures
How is H3K4me3 deposited?
H3K4me3 is deposited by a subfamily of SET domain proteins: MLL’s
and SET1A/B
How do DNA methylation occurs, which enzymes are involved?
The 5e base: 5-methylcytosine. DNA methyltransferase (DNMT) catalyses the reaction, S-adenosyl methionine, SAM, is converted to S-adenosyl-L-homocysteine, SAH.
Methylation due to de novo methylation, or due to maintenance of methylation.
How do demethylation of DNA occurs?
Demethylation due to active demethylation or due to DNA replication, that is passive demethylation.
What are the models of DNA demethylation?
- Glycosylation, abasic site, place cytosine back.
- Deamination causes the switch from methylated cytosine to thymine, then T-G mismatch glycosylase, abasic site, repair with cytosine.
- Oxidation causes the switch to 5-hydroxymethylcytosine then also via a unknown path a abasic site, repair with cytosine.
Where is TET enzyme involved and what is its function?
- Oxidation causes the switch to 5-hydroxymethylcytosine then also via a unknown path a abasic site, repair with cytosine.
-Here the enzyme TET is involved. The C terminal part is the enzymatic part of TET1.
-TET1 is sitting at the start site at CpG islands, here no 5mc or 5hmc is seen, so is it too fast or not there?
-TET can also make more variants of 5mc, 5hmc, 5fc, so this can be more epigenetic factors.
-The question is if 5-hydroxymethylcytosine is an in between product or an important regulator, epigenetic factor?
How do imprinting work in gametes?
-In gametes all the chromosomes will be either maternally imprinted (when female) or paternally imprinted (when male)
-Zygote: male methylation disappears due to oxidation by TET enzyme
-So “re”-methylation due to paternal imprinting
-- In female a protein is bound to DNA with methyl, so that TET cannot bind.
What is parental inmprinting?
genes rendered inactive depending upon parental source , appear hemizygous.
only paternal mouse Igf2 gene expressed ( maternal imprinted)

only maternal mouse H19 gene expressed ( paternally imprinted)
What happes by Prader Willy syndrome?
-Male: normally have an active SNRPN gene, but due to mutations the gene is inactivated
-Female: has a SNRPN gene that is inactivated by imprinting
o So both copies of SNRPN gene are inactive
What happens by Angelmann syndrome?
Angelman syndrome
-Female: normally have an active AS gene, but due to mutations the gene is inactivated
-Male: has AS gene that is inactivated by imprinting
*So bot copies of AS gene are inactive
How do methylation and cancer are related and which proteins are involved there?
-Promoter regions of CpGs are normally not methylated (Active)
-So when methylated it becomes inactive and causes cancer.
- Proteins involved are:
o HDACs
o DNA methyl transferases
o Methyl-DNA binding protein
o Proteins with chromatin remodelling activity
Which ways there are for global DNA methylation profiling?
-- Bisulphate conversion
--- Digestion with methylation sensitive restriction enzyme
- Capture of methylated DNA fragment
How do bisulphate conversion works?
Cytosine is converted to uracil by an intermediate bisulphate. When cytosine is methylated it will not be converted into uracil. So this is a way to detect methylated cytosine
How do digestion with methylation sensitive restriction enzyme works?
o MeDIP: methylated DNA immunoprecipitation, a chip type experiment.
o Only the pieces of DNA with methylation will be precipitated
o Single-gene analysis using qPCR, and global analysis using microarrays.
How do capture of methylated DNA fragment works?
o MethylCap
o Capture using methylated binding domain (MBD)-column. A higher salt concentration needed means that there is more methylated CpGs bound in the column.
Whereto IDH mutation leads?
- IDH mutations > increased 2HG > inhibits TET 2 enzyme > reduced hydroxylation of 5-methylation, so more methylated aminoacids.
How do X-inactivation works?
*Sequences at the X inactivation center (XIC), present on the X chromosome, control the silencing of the X chromosome. The hypothetical blocking factor is predicted to bind to sequences within the XIC.


-The XIC contains four non-translated RNA genesalso contains binding sites for both known and unknown regulatory proteins.

-The X-inactive specific transcript (Xist) gene encodes a large non-coding RNA that is responsible for mediating the specific silencing of the X chromosome from which it is transcribed.[12] The inactive X chromosome is coated by Xist RNA,[13] whereas the Xa is not (See Figure to the right). The Xist gene is the only gene which is expressed from the Xi but not from the Xa. X chromosomes which lack the Xist gene cannot be inactivated.[14] Artificially placing and expressing the Xist gene on another chromosome leads to silencing of that chromosome.[15][16]

Prior to inactivation, both X chromosomes weakly express Xist RNA from the Xist gene. During the inactivation process, the future Xa ceases to express Xist, whereas the future Xi dramatically increases Xist RNA production. On the future Xi, the Xist RNA progressively coats the chromosome, spreading out from the XIC;[15] the Xist RNA does not localize to the Xa. The silencing of genes along the Xi occurs soon after coating by Xist RNA.

Like Xist, the Tsix gene encodes a large RNA which is not believed to encode a protein. The Tsix RNA is transcribed antisense to Xist, meaning that the Tsix gene overlaps the Xist gene and is transcribed on the opposite strand of DNA from the Xist gene.[17] Tsix is a negative regulator of Xist; X chromosomes lacking Tsix expression (and thus having high levels of Xist transcription) are inactivated much more frequently than normal chromosomes.

Like Xist, prior to inactivation, both X chromosomes weakly express Tsix RNA from the Tsix gene. Upon the onset of X-inactivation, the future Xi ceases to express Tsix RNA (and increases Xist expression), whereas Xa continues to express Tsix for several days.
What are the hallmarks of heterochromatin?
histone H3K9 hypermethylation
global histone hypoacetylation
CPG DNA methylation
Late DNA replication
Which hallmarks are specific for inactive X?
histone H3K27 hypermethylation
Macro H2A incorporation
Where is histone phosphorylation involved?
- Mitosis, meiosis, chromatin compaction during late stages of gametogenesis
- DNA damage
-- Enzymes: kinases (H3: Aurora kinases), phosphatases
Where is histone methylation involved and which enzymes are there?
-H3K4: activation of transcription
-H3K9, K27: repression of transcription
-Enzymes: PRMTs, HMTs (SET domain), histone demethylases (LSD1 amine oxidase, JmjC domain hydroxylases)
What is poised RNA polymerase?
- A poised RNA polymerase is a polymerase present at an inactive promoter. They can initiate transcription but not elongate. H3K4 tri-methylation occurs at the transcription start site, but no H3K36 trimethylation has occurred so no elongation can take place and mRNA levels are low. Only short 5’RNA.
What are the bivalent marks on the chromatin domain?
- There are modifications for transcriptional activation ánd transcriptional repression present. So H3K4me3 for activation and H3K27me3 for repression. Since both are present, activation can rapidly take place.
What does methyl binding protein?
- When it is methylated it is a sterical hindrance for a transcription factor (tf). But a methyl binding protein (MBP) can bind to the methyl and recruit corepressor complexes.
-- MBP and meCP2 are readers of the modification
What does DNA methyl transferase?
when there is methylation, DNMT can bind on methyl and methylate DNA.HDAC, HMT(histone methyl transferase) and a tf are recruited by the DNMT.
What does DNMT1?
- DNMT1 makes the modification and is a maintenance DNA methyl transferase
-
What does DNMT3A/B?
- DNMT3A/B is a de novo DNA methyl transferase
What is differentially methylated region?
regulates imprinting of the region
-Is in the promoter of the gene (cytosine methylation)
-The methylated allele will not be expressed
-Complicated scenarios: DMR corresponds to insulator
Describe the imprinting in early development?
- In the early development are imprinted regions maintained, other methylations are removed. So then you can tell if it is from the mother or father.
Describe the imprinting in testes?
- In the testes all the maternal imprinting will be replaced by the father imprinting. So there you cannot tell which is from the mother/father even so in ovary. So there is resetting in the germline.

-- Evolutionary it is for the female more important that her offspring is small so see can fed all her offspring, the male wants a strong offspring and wants them to grow, so there is maternal imprinting for the growth factor IGF2.
How too see the distinct folding pattern of the chromosomes?
- A method called Hi-C is used. Cells are cross-linked with formaldehyde, resulting in covalent links between spatially adjacent chromatin segments. Then the chromatin is digested with a restriction enzyme and the resulting sticky ends are filled in with nucleotides, one of which is biotinylated. Then DNA is purified and sheared. The biotinylated junctions are isolated with streptavidin beads and identified by paired-end sequencing. Then you get interaction maps and corresponds to intrachromosomal interactions.
- The genome is spatially constrained within the nucleus, non-random long-range interactions.
What is embryonic patterning?
- the process of establishing positional information at the molecular level
-- causes patterned gene expression, it is established (is intermediate and outcome of patterning), axis formation and morphogenesis.
What is the function of Bicoid mRNA?
mRNA localized anterior, protein translated after fertilization (anterior) diffuses to posterior. Has a homeobox domain factor, a transcription factor, also inhibits translation of caudal (CAD). (homeobox = a DNA sequence found within genes that are involved in the regulation of patterns of anatomical development in animals, fungi and plants.
What is the function of Caudal mRNA?
- Caudal(CAD): mRNA uniform in embryo, translation is regulated by BCD, has a homeo domain factor.
What is hunchback mRNA?
maternal HB mRNA translation inhibited by Nanos (NOS), zinc finger transcription factor.
What are the pair rule genes?
account for the pattern of repeating stripes, are 8 genes. The transcription of these genes is controlled by transcription factors encoded by gap and maternal genes.
What is eve-skipped gene and how is it regulated?
- For example even-skipped (eve) gene in stripe 2 is a pair-rule gene and controlled by bicoid protein (maternal), hunchback, Krüppel and Giant (gap proteins). All four of these tf bind to a clustered set of regulatory sites, or enhancer, located upstream of the eve promoter.
What do Hunchback,bicoid,Giant and Krüppel ?
- Hunchback and bicoid are activators and Giant and Krüppel are repressors. Only in parasegment 3 the eve stripe 2 enhancer drives eve expression. This enhancer integrates multiple inputs.
How do enhancer works?
- Enhancer has a loop-back mechanism, activation from a distance, orientation-independent. More than one enhancer per promoter achieves versatility in regulation
How do you estimate regulators?
with chip
How do you study the looping?
by 3C/4C method and LCR

3C(one specific locus to another one)/4C(one-to-many, circular 3C):
How do 4c chromosome conformational capture works?
-Crosslink pieces of DNA in vivo
-For example promoter with down-looped enhancer
-Ligation
-De-crosslinking
-One primer on enhancer one on promoter , circles PCR
-The PCR product represents the genomic environment of the fragment of interest
-Characterization by microarray or by sequencing
What is LCR?
: locus control region: enhancer regulating several genes. In fetal it LCR interacts with alpha and gamma, in adult with delta and beta. It is a developmental regulation of mammalian globin expression. Also in fetal stage more histone acetylation at G and A and in adult stage more acetylation at delta and beta.
How do enhancers functions?
by forming a loop to promoters.
How do epigenetic regulation of enhancers work? And what is the consequence of it?
enhancers are marked with specific histones variants(H3.3 and H2A.Z, unstable nucleosome) and specific modifications (H3K4me1/2 = enhancer mark, H3K4me3 = promoter mark.)


o A consequence is that there is also RNA polymerase II at enhancers, making ‘eRNA’(enhancerRNA)

*o 88% of enhancers outside conserved regions.
Which promoters do enhancers prefere?
promoters with specific core promoter elements
The core promoters needs an activator. Which activators there are?
-Caudal, activates DPE but not TATA-BRE promoters. When BRE is not there it activates also TATA. So caudal discriminates between promoter types. Caudal is a regulator of the Hoc gene network, is a core promoter specific activator.
What does tethering element?
- It is not the core promoter but a tethering complex part of the promoter that binds to enhancers. The promoter closest to the tethering element is activated. So when the tethering element is moved, and it becomes closer to another promoter, that promoter will be activated.
By which three mechanism is binding of an enhancer to the promoter is regulated?
o Selective core promoter recognition/competition
o Tethering elements
o Insulators
What do insulators?
-Road blocks of the genome, cis acting elements, boundary elements, block enhancer function when in between enhancer and promoter
-Role in imprinting
-Block heterochromatin spread
-Barrier can also cause chain termination by tRNA genes that block Sir spreading in yeast. So no acetylation of histone tails.
-Neutralise “position-effects’ of transgenes in gene therapy
How can an insulator block heterochromatin spread?
An insulator with HAT can acetylate K9 that also can be methylated. So when acetylated no methylation can take place, so not all the gene is heterochromatic.
Which two types of insulators there are?
• Enhancer blocking
• And barrier for spreading of methylation/acetylation
• Many insulators show both activities
What is a - SINE B2 element?
o Considered as junk, and dangerous (mobile)
o But acts as an insulator for growth factor
o Depends on pol2 and pol3
o Has a tRNA type promoter
Which protein recognize insulators?
o CTCF: 11 zinc-finger protein
o Sometimes repressor, sometimes activator
o It binds insulators and blocks the enhancer.
Can promoters compete with enhancers?
- Competitiveness neighbouring promoters also determines effectiveness of insulator.
What are the enhancer -promoter preferences?
- How higher the dissociation constant (Kd) how smaller the affinity. So when K(2)<K(1) the enhancer will bind to K2.
- When the distance is smaller, the enhancer will act on this promoter, they will find each other easier. When d1 = d2 they will both bind.
- Also a part of the chromosome will bind to itself, because it is closest to it.
- it depends on where the insulator is and what the affinity is of the enhancer to an promoter.
What happens when there are two insulators present?
- When two insulators are present there can be no insulation.
- Insulators can bind to each other and form loops of their own, shortening the distance and not hindering the interaction.
- by binding to each other the insulators will separate promoter and enhancer in two distinct domains.
- binding of two insulators to each other can facilitate the interaction of a promoter and enhancer.
-enhancer blocking can be strenghtened by the interaction of two insulators ( forming a loop..).
Which insulator mechanisms there are?
#Insulator-insulator interactions
- Nuclear relocation and compartmentalization
- Distance effects
# Insulator-enhancer interactions
- Sequestration of enhancer availability
# Boundaries of active/inactive domains
- Spread of epigenetic marks
Describe the lambda switch?
o Has two repressors.
o Stable state 1, prophage state: lambda repressor is made and no ‘cro’ transcription occurs
o Stable state 2, lytic state: lambda cro protein repressor is made and no ‘cI’ transcription occurs. When stress in cell
o on/off switch

It determines on which ORFs RNA polymerase can bind.
What is morphogen molecule?
molecule that is present in different amounts, like Wnt8 and dorsal protein
What is dorsal protein and where is it expressed?
o Dorsal protein is highly expressed at ventral side, important for ventral development. It is named after the phenotype of mutant (dorsalized).
What is bicoid protein?
highly expressed at anterior side, when mutated no expression
What is HB protein?
o HB is a target of BCD. HB has more than one promoter, BCD works on one promoter. It has a very complex regulation. By every 2-fold increase in BCD there is an expansion of HB expression by 10% EL. (anterior, 100% EL, posterior, 0% EL)
Which two functions has dorsal?
o Dorsal acts as a repressor at dorsal patterning, and as an activator at ventral patterning, binds as homodimer
What is a downstream target of dorsal?
-Rhomboid is a downstream target of dorsal. It has a high affinity binding site
Which targets of dorsal has low affinity binding sites?
-Twist and snail have low-affinity binding sites
What is and what does snail?
-Snail is repressor, binds to thomboid locus and turns off rhomboid in high levels of dorsal. When there are low levels of dorsal, it binds only to the high affinity rhomboid.
What is the purpose of autoregulation?
: increased stability of gene expression, decreased biosynthetic cost of regulation, reduced response time to stimuli

the protein A which binds to promoter gives an output which regulates the amount of the protein A
What is an multicomponent loop?
loop closed by two or more factors, feedback control, bi-stable systems (ON/OFF switch) stable even after trigger signal is gone.

protein A binds on promoter A which output triggers protein B which binds to promoter B which output regulates protein A.
What is feedforward loop?
MCM1 proteins binds to SWI4 promoter which triggers >>> SWI4 protein which binds to CLB2 promoter. MCM1 induces ALSO CLB2 promoter.


- Feedforward loop: sensitive to sustained, not transient input, a stable input has a stronger effect, both simulations at same time on z. multistep ultrasensitivity (amplification of signal), activating/repressing feedforward.
What is coherent feed forward loop?
o Coherent feed forward loop:
-Can be AND-logic/OR-logic
-AND-logic: stimulation ON-delay
In Z expression
-OR-logic: stimulation OFF-delay
In Z reduction
What is incoherent feed-forward loop?
o Incoherent feed forward loop.
- When Y reaches a certain
Threshold, only then a decrease
of Z.
- It has a shorter Km than simple
Regulation.
Why we should use yeast for the experiments?
- It’s cheap and fast to culture
- Homologous recombination very efficiently, easy to engineer chromosomes using PCR products
- Homogenous cell population
- Propagated as haploid and as diploid.
How is Homologous recombination with transfected disruption constructs works?
- PCR is used to generate a disruption construct containing a selectable marker that subsequently is transfected into yeast. Homologous recombination takes place an and the homologous chromosomal site is exchanged efficiently. Then selection for G-418 resistance takes place, the non-transfected cells die. Then sporulation. If the disrupted gene is essential, the spores with disruption construct will be nonviable.
What do the activated pheromone receptors do by yeast?
The activated pheromone receptors halt the cell cycle and prepare the cells for cell fusion and the nuclei for nuclear fusion (karyogamy) by morphogenesis. > mating
What happens when there are no enough nutrients in yeast?
sporulation
Protein-protein interactions can be measured in yeast as transcriptional output by a transcription factor composed of two hybrid proteins. How does it work?
There is one protein: DNA binding domain + bait domain , second protein: activator domain + fish domain. When these two proteins will come togather on one DNA domain , there will be activation bait+fish.
How do you determine what the gene product does?
-Two dimensional projection of quantitative ‘protein-protein’ interactions
-Compare mass spectroscopy with predicted data
-The tandem affinity purification (TAP) strategy: isolation of native yeast protein complexes
-Often can be deduced by protein sequence homology (BLAST, automatic)
How do mass spectroscopy works?
o Mass spectrometry: cut your proteins with trypsin, an endoprotease that cleaves after Arg and Lys sample ionic source mass analyser detector(peptide fingerprint) > data analysis
How do tandem affinity purification works?
o Step 1: make strains with tagged genes, (spacer-CBP-TEV site-protein A) they will bind to the protein of interest. Break open the cells, bind the protein A moiety to sepharose beads coated with IgGs, wash the beads
o Step 2: cleave off the protein using a tomato etch virus protease
o Step 3: let calmodulin binding peptide (CBP) bind to calmodulin in a Ca2+ dependent fashion, wash the calmodulin beads
o Step 4: elute the complexes by chelating Ca2+ with excess EGTA (a molecule that binds Ca2+)
o Step 5: if protein of interest was in a complex, the complex is now pure
o Step 6: analyse the proteins with mass spectroscopy.
-When A and B bind, they have to be in the same part of the cell, with the same mRNA levels and same expression
describe how the matrix works?
- Off-diagonal spots indicate:
o Interaction points between cellular complexes
o Subunits shared by 2 complexes
- Chromatin conclusion:
o DNA replication, transcription and chromatin remodelling are performed by very large multisubunit protein complexes, not by isolated enzymes, This very probably reflects the need for a high level of signal input integration by these processes.
What does deletion consortium achivement?
o Mutant yeast strains whereby each of the deletion alleles is tagged with an UPTAG and a DOWNTAG that can be used like barcodes to quantify allele frequency in a mixed population
o Microarray-based quantitative detection of mutant yeast cells
o You can measure optical density or a fluorescent protein that is expressed in the knock-out strains. Then you can quantify cell numbers in each well.
What are the examples of assigning a biological process annotation to a gene.
o Sensitivity to nutrient source
o Cellular morphology of single gene deletion mutants
o Sensitivity to pharmacological agents
o Synthetic genetic interactions implicate genes in a biological process
o Chemical genetics
What is epistasis?
analyses of what happens in a double mutant, can indicate if A & B is in the same pathway. Can be positive or negative epistasis.

AxB=C
o No epistasis if the double mutant has a fitness of C
o Suppression when the double mutants fitness is >>C
o Enhancement (synthetic sickness/lethality) when the double mutants fitness is <<C
Where is the studying of a double mutant usefull in?
o Biosynthetic pathways in which a precursor material is converted via one or more intermediates to a final product
o Signalling pathways that regulate other processes and involve the flow of information rather than chemical intermediates.
- Two mutations must have opposite effects on the output of the same regulated pathway
o Suppressor mutations: a structural change in A will be suppressed by a structural change in B, allowing the mutant proteins to interact.
- Relatively rare
o Synthetic lethality 1: one heterodimeric protein is partially, but not completely, inactivated by mutations in either one of the nonidentical subunits. In double mutants there is a severe defect.
- More common than suppression, because more residues in protein B can be mutated to give this situation.
o Synthetic lethality 2: if either pathway alone is inactivated by a mutation, the other pathway will be able to supply the needed product. When both pathways are inactivated at the same time, the essential product cannot be synthesized and the double mutants will be nonviable.
-Result: cells can make product via more than one route.
Describe chemical genetics?
- If a drug poisons a process, then a mutation weakening that process will be hypersensitive to the drug.
- Instead of eliminating gene function at the DNA level on the chromosome, it is eliminated at the protein level through drug binding. Allele A is a genetic mutation and ‘allele’ B is a pharmacological inhibition of a protein.
- Overlaying epistatic interaction maps with chemical –genetic interaction maps reveals the target of compound X (poison)
- Any pharmacological agent can be tested for its mode of action, using the yeast deletion library as a cheap, fast and comprehensively described functional genomics substitute for human or other higher eukaryote.
What are the miRNAs?
18-24 nucleotides that base-pair with target mRNAs
What is PIWI domain and where is it found?
- Argonoute proteins interact with miRNA, is a core protein component of RISC. Contain a PIWI domain which structurally resembles RNase H and it provides the RISC endonuclease activity.
How Argounate complex recognizes miRNA?
- Mostly base pairing at the 5’ end of the miRNA
- Seed sequence: nucleotides 2-8, complementarity to this region is most important, although not always essential for target recognition.
What are the experimental strategies to study miRNA function?
-The use of antisense inhibitors: to block the targeted miRNA function
-Point mutations of miRNAs (or their targets) to study the direct interaction of miRNAs and their targeted genes
-Molecular, genetic and bioinformatics techniques can be used
-Finding miRNA targets in silico
-Microarray analysis shows that some microRNAs down regulate large numbers of target mRNAs.
How can you use antisense inhibitors to block miRNA function?
o An antisense RNA competes with cellular mRNAs to bind miRNAs, when pairing it inhibits the miRNA function.
o Antagomirs: cholesterol-conjugated RNAs with a partially modified phosphorothioate backbone and 2’-O-methyl oligoribonucleotide modifications
o ASOs: 2’O-methoxyethyl phosphorothioate-modified antisense oligonucleotides.
Where the point mutations must take place???
o 1 or 2 nucleotide changes in the “seed-region” will dramatically decrease the possibility of the miRNA binding to its targets  overexpression of the targets
How do microRNA analysis works?
o Discovered that there are over-represented motifs in the 3’UTRs of the down regulated genes
o Also there is an important role for miRNAs in establishing and/or maintaining tissue specific gene expression patterns
What miRNAs are abnormally expressed within cancers?
o BCL2: antiapoptotic gene, when miRNAs are reduced, abnormal survival of cells.
o RAS: low levels cause differentiation, high levels cause proliferation, so when miRNAs are reduced, regulation is gone and there will be more RAS, and more proliferation. (the same with CDK6 and CDC25)
o When there is overexpression of oncogenic miRNA, there is down regulation of suppressor protein coding targets PTEN and P27
Which oncogenic miRNAs there are?
o miR-21 interacts with tumour suppressor gene PTEN
o mir-221-222 interacts with the p27 tumour suppressor
Which miRNAs are abnormally expressed within the metastasis?
miR-10b is highly expressed in metastatic breast cancer cells. It positively regulates cell migration and invasion. Twist regulates miR-10b. then miRNA binds to HOXD10 mRNA and inhibits the translation. Reduced HOXD10 protein levels lead to an increased RHOC expression, leads to tumour cell motility > metastasis.
Which antisense miRNA and oligonucleotides exist to target miRNA in cancer treatment?
o antagomir and ASO
o also use of locked nucleic acid(LNA)-modified oligonucleotides
-these compete with cellular mRNAs leading to functional inhibition of the miRNA
How do genome wide RNAi screens in different organisms work?
- C elegans: multiple siRNAs, drawback: is u use worms you have to inject it in every worm
- Drosophila, need no additional techniques for transfecting, in wells
- Humans: better resemblance, drawback: transfection methods needed, dsDNA, 1 siRNA.
What is de novo sequencing?
In shotgun sequencing,DNA is broken up randomly into numerous small segments, which are sequenced using the chain termination method to obtain reads. Multiple overlapping reads for the target DNA are obtained by performing several rounds of this fragmentation and sequencing. Computer programs then use the overlapping ends of different reads to assemble them into a continuous sequence.[1]
What is the mechanism to constrain the centrimeter sized chromosomes into micrometer sized territories?
looping
Which two categories of GBM there are and by which mutations these are driven?
IDH1 mutations. Infrequent p16,EGFR, PTEN mutations.

IDH1 WT develops rapidly on late age, the prognosis is 1 year

IDH mutant, is on age 33, develops slowly, prognosis is 3.8 year.
Which mutation occurs in IDH1?
in the catalitische zijde, arginine-histidine mutatie.
Where to lead leukemic IDH1,2 mutations?
hypermethylation phenotype, TET2 impairment, impair hematopoeitic differentiation.
What are the small alterations?
insertions, deletions, inversions, duplication.
Does methylation of H3K4 is
dependent on mono-Ubiquitination at H2B?
yes
When do Glucocorticoid
Receptor appears?
Glucocor*coid Receptor binding
and appearance of in the presence
or absence and a chromatin remodeling complex SWI/SNF
Where is The Nuclear DNA Base 5-Hydroxymethylcytosine is Present?
Purkinje Neurons and the Brain
How is the imprinting in maternal/paternal DNA regulated?
maternal: there is an insulator which causes barrier in IgF2 activation. But H19 is activated

paternal: there is no insulator (CTCF) so IgF2 is activated. But H19 is methylated so these gene can not be activated. Insulator bindigs place is methylated as well so it can not bind an insulator.
What causes prader willy sindrome?
inactivation of SNRPN gene both on paternal as maternal chromosomes in zygote*
What do IDH mutations cause?
increased production of 2HG
Which Inhibits the TET2 enzyme
Reduced Hydroxylation of 5-Methylcytosine and thus increased methylation
What does DNMT3a/3b?
can place methyl on previously base to keep it methylated. DNMT3a/3b can be nove methylated the base which were not methylated before.
WHat does TET1 enzyme?
Tet1 can change methyl C to DH methyl c. TET1 binds to cPG island and rit over the transcriptions start site.
TET proteins help to lose the methylation pattern in the male and it will make hydroxy methylated.
What is bisulphide conversion?
Leads to formation of uracil from cytosine. All C will be U, so if C remains , it must be methylated. This reveal the presence of mCGH,mCHH,mCG.
What are MeDIP antobodyes?
reveal methylated DNA
What is methylCAP ?
capture using MBD-column. Methylated proteins will bind in the column.
Embryogenesis in Drosophila?
when Even-skipped is expressed during embryogenesis in drosophila in odd-numbered parasegments.
What do the coordinate genes?
they regulate the broad domain of expression of maternal genes.
What are the pair rule genes?
they have a complicated pattern of expression.
What are the gap genes and pair genes?
gap genes- have more complicated pattern of expression

pair-rule genes expressed in stripe patterns.
What are the homeotic genes?
determines which parts of the body form what body parts.
How do the A-P patterning looks like?
Coordinate genes > gap genes > pair rule genes > segment polarity genes > homeotic genes.
How do pair-rule gene regulation in drosophila works?
Multiple enhancers mediate stripe-specific gene expression

Eve stripe 2 enhancer drives eve expression in parasegment 3

Eve stripe 2 enhancer integrates multiple ‘inputs’
What are ther locus control regions?
Locus control regions (LCR) are defined by their ability to enhance the expression of linked genes to physiological levels in a tissue-specific and copy number-dependent manner at ectopic chromatin sites.
How do enhancers are marked?
Enhancers are marked with specific histones
variants (H3.3 and H2A.Z) and specific modifications (H3K4me1)
Enhancer functions by forming loop to promoters, what is the consequence of it?
RNA Polymerase II at enhancers, making ‘eRNA’ (enhancer RNA)
What do tRNA with SIR?
tRNA genes block Sir spreading in yeast

SIR binds aan hypoacetylated histone N terminal ends.
What does HAT to H3K9 methylation>?
it can block H3K9 methylation.
Where do SINE B2 element play a rol:?
Developmental activation of GH locus in pituitary
What is CTCF?
Nucleus: High expression
>11 zinc-finger protein
Transcription:
< Activation
< Repression
# Binds Insulators
< Enhancer blocking
What is the one advantages of yeast?
One of the advantages of yeast is that pair-wise combinations can be made through mating a strains to a strains, allowing cells with combinations to be generated
How do you use mass spectroscopy to obtain proteins?
*Cut your proteins with trypsin, an endoprotease that cleaves after Arg and Lys.

* Ionize the peptides, fragment them further ‘on-line’ and obtain a mass spectrum (peptide fingerprint).
* The mass spectra obtained are then compared by a computer algorythm to theoretically calculated spectra for the entire proteome, as deduced from genomic DNA sequence. Again, this relies on sequencing the genome !!!!