Intracellular processes Flashcards

1
Q

What is epigenetics?

A

Changes in phenotype, without changes in genotype -> by regulation of expression

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2
Q

Epigenetic changes can come about as a result of influences from the environment. When is the effect of influences from the environment greatest?

A

If they occur early in gestation

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3
Q

What is lyonization?

A

X-inactivation in females

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4
Q

What are housekeeping genes?

A

Genes needed by (nearly) all cells

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5
Q

What is a nucleosome?

A

DNA wrapped around histones

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6
Q

How many types of histone are there?

A

4

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7
Q

What is the effect of DNA being wrapped around nucleosomes?

A

It is less accessible to transcription factors and transcription machinery

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8
Q

What are the two key players in regulating gene transcription?

A
  1. Transcription factors
  2. Gene regulatory elements
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9
Q

Which epigenetic mechanisms does the body have to regulate gene transcription? (4)

A
  1. DNA methylation
  2. Nucleosome positioning
  3. Histone modifications
  4. 3D genome folding
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10
Q

In which way are epigenetic modifications related to immunological memory?

A

After immune cells have been activated, epigenetic changes remain that allow them to quicker upregulate immunological functions -> allows for quick response

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11
Q

What are gene regulatory elements?

A

Short stretches of DNA with certain motifs, to which transcription factors can bind

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12
Q

In which two groups can gene regulatory elements be divided, based on proximity to the gene they regulate?

A
  1. Close to the transcription start site (=promotor)
  2. Distal to the transcription start site
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13
Q

Which gene regulatory elements can be found distal to the transcription start site? (4)

A
  1. Enhancer
  2. Silencer
  3. Insulator
  4. Locus control regions
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14
Q

What is an enhancer?

A

Sequence of non-coding DNA that enhances formation of transcriptional loops

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15
Q

What is a silencer?

A

A repressor of transcription activation

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16
Q

What is an insulator?

A

A gene regulatory element that prevents transcriptional looping

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17
Q

What is a locus control region?

A

A group of enhancers that work together, often in a cluster of genes

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18
Q

What is an example of genes which are modulated by a locus control region?

A

Cytokine genes

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19
Q

How are transcription factors (often) activated?

A

Signalling cascades, often from receptors

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20
Q

How can distal gene regulatory elements influence DNA activity if DNA is linear?

A

DNA is not linear, but rather folded and curled, allowing for the formation of transcriptional loops

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21
Q

What is the function of transcription factor Pax5? What happens when it is absent?

A

Needed by B-cells to differentiate and maintain B-cell phenotype

In abcence of Pax5, B-cells will differentiate into different phenotypes depending on environmental signals

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22
Q

True or false: after a cell has differentiated into a specific phenotype, it no longer needs its cell-type specific transcription factors

A

False; cells need their transcription factors to maintain their phenotype

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23
Q

What are mechanisms and mutations in epigenetic regulation that can lead to disease? (4)

A
  1. Mutation of regulators can block their binding to gene regulatory elements
  2. Fusion of regulators can lead to dysfunctional binding to enhancers
  3. Mutations in GRE-sequences in DNA can block (complete) binding of regulators
  4. Mutations in DNA sequence of GREs can weaken/strengthen binding of regulators -> phenotypic variation of gene activity
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24
Q

True or false: mutations in the GREs often cause complete blockage of the binding of regulators to GREs

A

False; often there is only partially decreased activity of regulator and not complete failure

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25
What can be the effects of phenotypic variation of gene activity caused by mutations in DNA-motifs of GREs leading to stronger/weaker binding of regulators? (3)
1. Non-harmful phenotypic variations 2. Higher susceptibility to disease 3. Disease
26
What is DNA methylation? Which nucleotide is methylated?
Coupling of a methyl group to cytosine
27
Which enyzme methylates DNA?
DNA methyl transferase (DNMT)
28
Which enzyme demethylates DNA?
TET
29
What is the effect of DNA methylation? (2)
1. Prevention of TF binding by blocking enhancer/promotor, causing genes to be silenced 2. Recruitment of repressor molecules, causing genes to be silenced
30
What is the source of methyl groups of DNA methylation?
Folic acid
31
What is genomic imprinting?
An epigenetic regulatory mechanism in which only either the maternal or paternal gene is expressed, and the other one is shut down
32
How many genes use genomic imprinting?
<100
33
What is the effect of loss of genomic imprinting?
Often not compatible with life, and otherwise leading to severe syndromes
34
By which two ways can disrupted methylation lead to oncological processes?
1. Methylation of tumorsuppressor genes 2. Demethylation of oncogenes
35
How does nucleosome positioning dictate gene transcription?
Nucleosomes prevent transcription factors from binding -> positions of nucleosomes can make genes more/less accessible
36
How do chromatin remodelers change the accessibility of DNA? (2)
1. Sliding nucleosomes to create space for transcription factor binding 2. Assembling/disassembling nucleosomes
37
What are histone modifications?
Addition of groups to histone tails, influencing how tightly DNA is bound around nucleosomes
38
What is the most important kind of histone modification?
Acetylation
39
Closed chromatin has a [low/high] amount of acetyl groups as histone modifications, whereas open chromatin has a [low/high] amount of acetyl groups
Closed chromatin = low acetylation Open chromatin = high acetylation
40
Which enzyme adds acetyl groups to histones?
HAT
41
Which enzyme removes acetyl groups from histones?
HDAC
42
By which mechanism do histone modifications influence how tightly DNA is bound around nucleosomes and the accesibility of DNA for transcription factors? (2)
1. Histone modifications impact charge of DNA 2. Histone modifications can lead to steric hindrance
43
True or false: one histone tail can only have one modification
False; histone tails can have multiple modifications
44
True or false: silencing by DNA methylation is usually also associated by deacetylated histones
True
45
What is the chromatin configuration of the cytokine genes in memory T-cells?
Open configuration -> readily accessible for rapid respoonse
46
How can DNA methylation/modifications be identified?
Induce DNA breaks and incubate DNA with antibodies that recognize specific modifications to fish these fragments out. Fragments are then mapped back to the genome
47
What is Tn5? How can it be used to show DNA binding around nucleosomes?
An enzyme that wants to cut DNA at every possible site. Cannot cut histone-bound DNA -> these fragments can be fished out and mapped back to the genome
48
What is bisulfite sequencing? How can it be used to identify methylation patterns?
It causes unmethylated C to turn into U -> can be sequenced and mapped back to the genome
49
What is the most common method of DNA folding?
DNA looping
50
What is the approximate size of DNA loops? What is the area inside this loop called?
~200.000 base pairs; area inside this loop = contact domain
51
How are DNA loops created?
Created by various proteins that recognize DNA motifs
52
What is often a common characteristic of genes within the same contact domain created by DNA folding?
Often have the same epigenetic characteristics -> all turned on or off
53
What are nuclear subcompartments? What is their function?
Compartments within the nucleus that contain contact domains with comparable activity -> active and inactive regions grouped, allowing for efficient concentration of transcription machinery, nucleic acids, etc.
54
What is an important function of DNA function in VDJ-recombination?
It ensures that all V-genes are equally close to the recombination centre, giving all V-genes an equal chance to be incorporated
55
Which epigenetic change is most often used as a biomarker?
Methylation
56
What is an example of drugs that are used to modify epigenetics? What is their effect?
BET-inhibitors -> alter DNA methylation
57
What is the problem in using drugs that target epigenetic modifications?
Aspecific -> epigenetic profile of all cells altered, causing side effects
58
What is signal transduction (definition)?
Any process by which a cell converts one kind of signal/stimulus into another, involving ordered sequences of biochemical reactions inside the cell
59
What is the prototypic basic signalling cascade? (4)
1. Ligand activates receptor 2. Receptor activates protein at membrane 3. Protein at membrane activates protein in cytosol 4. Protein in scytosol = activated final target of pathway, leading to a response
60
What are two common locations of ligand binding receptors?
1. Cell surface 2. Intracellularly, such as in endosomes
61
Which groups of proteins are often involved in signal transduction? (4)
1. Linkers/adaptors 2. Kinases/phosphatases 3. Lipid-metabolizing enzymes 4. Guanine nucleotide exchange factors (GTP/GDP)
62
What are linkers/adaptors?
Building bocks that interact with other signaling molecules, without having enzymatic activity themselves
63
How do signaling proteins interact with one another?
Via modular protein domains
64
Where are the first intracellular signaling proteins in a signaling cascade often found?
Bound to the plasma membrane
65
What are ways in which signaling molecules can be recruited to the plasma membrane? (3)
1. Binding to phosphorylated sites on membrane-associated proteins 2. Being recruited by RAS-proteins 3. Binding to membrane lipids
66
How can phosphorylation lead to recruitment of signaling molecules?
By providing a docking site for these signaling molecules
67
What are RAS-proteins, and how are they activated?
Plasma-membrane bound proteins that are activated through confirmational changes when a linked receptor is activated -> provides docking sites for intracellular proteins
68
Which two types of linkers can be identified?
1. Scaffolds 2. Adaptors
69
What are scaffolds?
Proteins without enzymatic activity that can function as a docking site for other proteins
70
How are docking sites created on scaffolds?
Phosphorylation
71
What are adaptors?
Proteins that don't require phosphorylation and can directly connect to other signaling proteins
72
True or false: phosphorylation leads to activation, whereas dephosphorylation leads to deactivation
False; this is not always necessarily the case
73
How can signaling proteins be involved in multiple pathways?
Having multiple phosphorylation sites with different functions
74
What is a common method of intracellular signaling amplification?
Kinase cascades
75
What is a second messenger?
Soluble messenger inside the cell
76
What are common second messengers? (2)
1. Calcium 2. cAMP
77
What is the function of calcium and cAMP as seond messengers?
Rapidly diffuse throughout the cell and induce confirmational changes in calmodulin
78
Why are calcium and cAMP good second messengers?
They can be rapidly produced (cAMP) or released (calcium) upon activation
79
Why is intracellular signal amplification necessary in (most) signaling cascades?
Allows for a robust signal, even when little ligand is present
80
What are the three general outcomes of signaling pathways?
1. Alterations in gene transcription 2. Cytoskeleton changes, leading to migration 3. Alterations in cell metabolism
81
What are three common methods to switch off cellular signaling cascades? (3)
1. Altering phosphorylation 2. Ubiquitin-mediated degradation in proteasome 3. Ubiquitin-mediated degradation in lysosome
82
What is a characteristic that the BCR and TCR share when it comes to signaling?
They don't have an intracellular domain, requiring signal transducers
83
What are the signal transducers of the BCR?
Igα-Igβ
84
Which BCR is an exception to the rule that BCR's dont have a signaling domain? What is the effect of this?
IgG-receptor -> IgG can bypass signaling steps, allowing for a quicker response
85
What is the signal transducer of the TCR?
CD3
86
Why do drugs that interfere with the TCR often also have (some) effect on BCR signaling (and vice versa)?
They use a very similar signal transduction pathway
87
What is the shortest known signaling cascasde in immune signaling? What are its steps? (4)
Cytokine signaling through JAK-STAT 1. Binding of cytokine leads to receptor dimerization 2. Dimerization leads to JAK activation 3. Activated JAKs activate STAT through phosphorylation 4. STAT activates transcriptoin
88
Why do defects in (immune) signaling pathways (often) not immediately lead to disease?
Most signaling pathways have redundancy
89
How can targeting Notch signaling to combat T-cell leukaemia lead to skin problems?
While Notch signaling is important for T-cell proliferation, it is also important for protection against cancer in the skin
90
What are the effects of: 1. No BTK signaling 2. Permanent BTK activation 3. Increase of BTK protein
1. No signaling = no development of B-cells -> XLA 2. Permanent activation = constant B-cell stimulation -> CLL 3. Increased = higher survival of autoreactive B-cells -> spontaneous auto-immune phenotype
91
How can targeting of BTK kill CLL cells?
BTK inhibitors block CXCR4 chemokine signaling in B-cells -> CLL cells enter circulation, where they die
92
After some time of using BTK inhibitors, cells will lose their susceptibility to this class of drugs. Why?
Their pathways rewire in a way does not require BTK -> shows redundancy
93
How can phosphorylation of signaling proteins be measured? (2)
1. Western Blot -> bulk 2. Phosphoflow -> cell-specific