Health and Disease Dr Boyes Flashcards
(123 cards)
0
Q
What causes different genes to be expressed?
A
Transcription factors.
1
Q
Is DNA or genetic information lost during differentiation?
A
No
2
Q
What does tissue specific gene expression rely on?
A
A combination of the activity of specific transcription factors and chromatin modifications.
3
Q
What is Epigenetic regulation?
A
A change in state of expression of a gene which does not involve a mutation but is inherited, after cell division, in the absence of the signal, or event, that initiated the change.
4
Q
What determines which gene is activated?
A
The DNA binding domain on the transcription activators as they determine which promoter is activated.
5
Q
What determines rate of expression of a given gene?
A
Transcription factors and promoters, with the transcription initiation complex playing a role in the rate.
6
Q
Do promoters have multiple binding sites?
A
Yes, for various transcription factors.
7
Q
Describe how DNA is packaged?
A
In a nucleosome, wrapped around a histone octamer with a diad centre, then 10 nm fibre, then 30 nm fibre and the attached with loops to the protein scaffold.
8
Q
What are DNase 1 hypersensitive sites?
A
Found at promoters and enhancers of expressed gene, generated by the binding of transcription factors and the displacement of histone octamers. DNase 1 digests hypersensitive sites around 100x faster that other sites.
9
Q
How are DNase 1 sites mapped?
A
Cleave with DNase 1 in nuclei, prepare the DNA, cut with a specific restriction enzyme, electrophorese, Southern blot and probe with region next to the restriction site. The calculate the distance the hypersensitive site is from the restriction site.
10
Q
What has to occur before transcription?
A
Removal or relocation of the histone octamer at promoters by nucleosome remodelling complexes.
11
Q
What two mechanisms are there to remove or relocate the histone octamer at promoters?
A
Sliding histone octamers to new positions or displacement of the histone octamers.
12
Q
How does the nucleosome remodelling complex translocated the DNA around the nucleosome?
A
Using ATP taking 10 nm steps, coordinated by the DNA binding and translocation domains. The remodeller binds DNA in the linker and pushes it histone octamer to create a loop which passes through the translocation domain.
Push-me, pull-me idea.
13
Q
What are the other functions of the nucleosome remodelling complex?
A
Correct nucleosome spacing, DNA exposure, exchange of H2A/H2B dimers, CHD involved in repression.
14
Q
How can histone tails be modified?
A
Many sites, many acetylation via acetyltransferase co activators.
15
Q
What are changes in DNA methylation nearly always?
A
Inherited
16
Q
What are histone modifications?
A
Can be inherited and therefore Epigenetic. Eg. Polycomb H3K27me3 and Trithorax H3K9me3.
17
Q
What can Epigenetic changes cause?
A
The same transcription factor to activate different genes in different cell types.
18
Q
Where does CpG methylation occur?
A
On the cytokines of CpG dinucleotides and can occur at CNG in plants.
19
Q
What is required for the determination of genes being on or off?
A
Inherited Epigernetic mark and for it to do something for transcription.
20
Q
How are DNA methylation patterns inherited upon replication?
A
Newly synthesised daughter strands are hemi-methylated and converted to fully methylated state by Dntm1. The ‘perfect’ Epigenetic mark is maintained as somatic cells divide and the methylation is maintained by Dnmt1, required every time DNA is replicated, de novo methylation by Dnmt3 a and b.
21
Q
When does de novo methylation occur?
A
Only at high levels during embryonic development but can occur at low levels during different developmental stages.
22
Q
What does Dnmt stand for?
A
DNA methyl transferases.
23
Q
Describe Dnmt’s?
A
C-terminal highly conserved catalytic domain. Unconserved N-terminal domain.
24
What does Dmnt3 a and b associate with?
Regulatory factor Dmnt3L.
25
What are the two types of DNA demethylation?
Passive and active.
26
Describe passive DNA demethylation?
One strand fails to be converted to the fully methylated form at replication and then becomes inherited in daughter cells via exclusion of the maintenance methyl transferase.
27
Describe active DNA demethylation?
Methyl-cytosine is removed from DNA via enzymatic activity. This occurs much quicker and it's important during embryonic development to re-set Epigenetic marks.
28
What percentage of CpG dinucleotides are methylated?
70-80%
29
What percentage of the mammalian genome is CG?
40%
30
Why is methylation of CpG underestimated?
As 5-me-c is mutagenic and spontaneously deaminates. Methyl-cytosine deaminates to thymine, non-methyl-cytosine deaminates to uracil.
31
What accounts for 50% off point mutations in genetic disorders?
5'-methyl-cytosine mutations which are not efficiently repaired.
32
How are CpG dinucleotides distributed in a) invertebrates and b) vertebrates?
a) all of the genes are in unmethylated blocks.
b) methylation seems to have spread and there are small blocks without methylation which are found around gene promoters.
33
What are small blocks in CpG dinucleotides without methylation called?
CpG Islands.
34
Describe CpG Islands?
~1 kb long, GC rich, unmethylated in all (particularly germlines) tissues, at the promoter region of all housekeeping genes and 40% of tissue-specific genes, they span the promoter and into the first exon, some replication origins map to CpG Islands.
35
What are the orphan CpG Islands often?
Sites of transcriptional initiation.
36
What two methods are used to detect DNA methylation?
Restriction enzymes: use non-methyl specific to cut DNA when DNA sites are not methylated.
Bisulphite sequences: treat DNA with sodium sulphite which accelerates the deamination of un-methylated cytosine to uracil but it doesn't affect the methylated cytosine.
37
How does DNA methylation repress transcription directly?
Prevents transcription factors from binding.
38
How does DNA methylation repress transcription?
Methylated DNA binding proteins bind to methylated CpG and prevent transcription factors from binding to promoters. This is more common.
39
When can CpG Islands become methylated?
On the inactive X chromosome and in some cancers.
40
What do methylated CpG Islands do?
Strongly repress transcription.
41
What are the 3 classes of promoters?
CpG Islands, weak CpG islands, low CpG promoters.
42
How do the three types of promoters vary?
CpG Islands: one CpG every 10 bp
Weak CpG Islands: one CpG every 20-30 bp
Low CpG promoters: one CpG every 100 bp.
43
What is the methylated DNA binding protein MeCP1s activity equivalent to?
Kaiso and MBD2/HDAC
44
What does the histone code hypothesis predict?
That modifications on the histone provide binding sites for chromatin-associated proteins and modifications on the same or different histone tails may be interdependent and generate various combinations on any one nucleosome. And this leads to the generation of active or repressed regions of chromatin.
45
What does the histone code hypothesis imply?
That the chromatin structure of euchromatin or heterochromatic domains are dependent on the combination of differentials modified nucleosomes.
46
What are the four key examples of histone modifications that lead to the binding of activators or repressors?
Acetylation of histone tails (activators bind via their bromodomains), tri-methylation of H3K4 (binding of activators via their PHD fingers), tri-methylation of H3K9 (binding of a repressor HP1 via its chromodomain), tri-methylation of H3K27 (binding of a repressor complex, polycomb repressor complex 1, via the chromodomain)
47
During the formation of active or repressive chromatin what cooperates?
Chromatin marks and activator/repressor proteins to re-I force the activation/repression.
48
What are the promoters and enhancers of active gene characterised by?
The presence of DNase 1 hypersensitive sites.
49
What are DNase 1 hypersensitive sites?
Typically lack canonical nucleosome and are bound by a variety of transcription factors. Become activated from a repressed state via cooperation between nucleosome remodelling factors and histone acetyltransferases.
50
What do nucleosome remodelling factors do?
Use the energy from free ATP to remodel nucleosomes at the hypersensitive site.
51
What do histone acetyl transferases do?
Acetylate histone tails of nucleosomes in the vicinity of the promoter.
52
Why are nucleosome remodelling complexes required?
To remodel the nucleosomes to allow for the pre-initiation complex to bind, for transcription to initiate. Specific upstream activators recruit them.
53
What else is recruited to aid in activation?
Co-activators are recruited to facilitate activation from the factor to the pre-initiation complex.
54
What do a number of the co-activators have?
Acetyltransferase activity, leading to acetylation of the nucleosomes in the vicinity of the promoter.
55
What does the acetylation of the nucleosomes in the vicinity of the promoter provide?
Binding sites for proteins with bromodomains. This activating modification helps to recruit other chromatin activating proteins thereby reinforcing a it's activity of this region of chromatin.
56
What is a different activating chromatin mark?
The trimethylation of histone H3 at Lysine 4.
57
What is the other activating chromatin mark mediated by?
SET1 and other related proteins.
58
What recruits the mediator for the other activating chromatin mark?
The imitated form of the RNA pol 2, found only in the immediate vicinity of promoters (modification therefore found only on about 5 nucleosomes in the vicinity of the promoter).
59
What are H3K4me3 often bound by?
Proteins with a PHD (plant homeodomain) finger. The binding of these helps to recruit other chromatin activating proteins thereby reinforcing activity of this region of chromatin.
60
What is a common features of the two well characterised repressing chromatin marks?
These repressive marks are bound by repressors that then recruit enzymes that catalyse the addition of the repressive chromatin marks to adjacent nucleosomes. Thus, the presence of these modification helps to recruit other chromatin repressor proteins, thereby reinforcing repression of these regions of chromatin.
61
What does the trimethylation of H3K9me3 provide?
Binding sites for heterochromatin protein 1, which binds via its chromodomain and them recruits methytransferase Suv39h which methylated adjacent nucleosomes via its SET domain. Facilitates the spread of heterochromatin.
62
What does the trimethylation of H3K27me3 provide?
Binding sites for polycomb repressor complex 1, bonds via its chromodomain and recruits a second polycomb repressor complex 2 which includes Ezh2 which has methytransferase activity. Spread of the repressive mark through recruitment of the enzyme that catalyses addition of the histone mark.
63
How do transcription factors bind to DNA?
The outside of the DNA double helix can be read by proteins. Recognise helix-turn-helix motifs, zinc finger domains, leucine zippers, basic helix-loop-helix.
64
How do proteins bind DNA?
Bind in the major groove by hydrogen, electrostatic and hydrophobic interactions, typically 10-20 contacts. Contribute to strength and specificity. More information is present in major groove compared to minor and consequently more proteins bind here.
65
What is the helix-turn-helix motif?
N-terminal structural helix with a turn and a c-terminal recognition helix.
66
How do helix-turn-helix motifs bind DNA?
In the major groove, usually as dimers.
67
What are the two types of zinc fingers?
2 cysteine- 2 cysteine and 2 cysteine- 2 histidine
68
Describe 2 cysteine- 2 cysteine zinc fingers?
Bind as dimers with four zinc fingers, the two halves of the recognition site are separated by one turn of the DNA helix. The n-terminal finger is involved in DNA binding and the second fingers determines the optimal spacing between the two halves of the palindromic recognition site. Best example is DNA binding domain in nuclear hormone receptors.
69
Describe 2 cysteine- 2 histidine zinc fingers?
Has an antiparallel beta sheet leading into an alpha helix, the zinc coordinates four amino acids to hold one end of the alpha helix and one end of the beta sheet. Can have a number of zinc fingers which allows of almost continuous stretch of alpha helices that can form contacts.
70
Describe zinc fingers?
Used to coordinate and structure the DNA contacts, each recognises three bp.
71
Describe leucine zippers?
Bind as dimers with alpha helices contacting the DNA sequences in the major groove. Leucine is repeated every 7 amino acids and therefore allows dimerisation via hydrophobic interactions. Grips like a clothes peg.
72
What can heterodimerisation of leucine zippers do?
Can alter the DNA binding specificity.
73
Describe the basic helix-loop-helix?
Short helix mediates dimerisation and the loop and long helix mediates DNA binding. Flexibility of the loop allows odd the helices to pack against eachother.
74
How can basic helix-loop-helix be inactivated?
By dimerisation with partners that lack DNA binding domains as dimerisation regulates DNA binding.
75
Give the two types of designer transcription factors?
Zinc finger nuclease and TALE system.
76
What are zinc fingers used for and what are their disadvantages?
Can be hooked to Fok1 endonucleases to introduce breaks at specific sites in the genome.
Disadvantages: must bind palindromic DNA sequences as a dimer, each finger recognises a triplet of bp and not all are recognised specifically.
77
What are TALE systems?
Designer transcription factor newly developed for Xanthomonas. Can build up proteins that bind any sequence of choice. A 34 amino acid repeat where 12 and 13 recognise the specific base. They build-up seigneur DNA binding proteins by linking these individual repeats in the correct order.
78
How do transcription factors work?
They stimulate activity of the basal promoter complex. Can use multiple mechanisms to provide synergistic activation.
79
What are the mechanisms by which transcription factors work?
Using activation domains that contact different TAFs in the TF2D complex, activators can functions via TF2B to alter the configurations of the basal machinery to enable it to recruit components more efficiently or to enhance he rate of binding of TF2B, activators can interact with the mediator complex, activators can recruit co-activators/repressors to contact basal machinery or to open the chromatin.
80
How can genes be regulated?
By the presence or absence of a transcription factor or by converting a transcription factor between inactive and active states.
81
What are the advantages of regulation by transcription factors activations?
Allows a rapid response, allows regulation on response to external factors, frequently used by signalling pathways, allows fine tuning of transcription level.
82
How can transcription factors be activated?
By binding to a ligand, by protein modifications (phosphorylation, acetylation and ubiquitination), by cleavage of larger precursors, by dissociation of an inhibitor protein.
83
How does binding of ligand activate transcription factors?
Induces a conformational change in nuclear hormone receptors, triggering DNA binding and gene activation. Also permits interaction with co-activators such as CBP, SRC-1 and SRC-3.
84
Why is phosphorylation modification often used to activate transcription factors?
In response to cell surface receptor signalling.
85
What does activation of TF by phosphorylation do?
Allows cells to respond to the environment by increasing protein activation, interaction with other proteins and co-activators.
86
What is the most simple method of phosphorylation?
Direct phosphorylation by cell surface receptors. Cytokines activate JAK/STAT pathway.
Cell surface receptors transmit the signal to JAKs that phosphorylate STAT transcription factors. This causes dimerisation of STATs and translocation into the nucleus where they bind specific target genes.
87
What is a more complex method of activation of TF by phosphorylation?
Binding of ligands to receptor tyrosine kinases activates the Ras/raf/MAPK kinase cascade. Signalling cascade allows amplification of signal growth factors bind receptor tyrosine kinases. This triggers autophosphorylation and activation of Ras. Ras binds GTP activating it. This phosphorylates Raf which phosphorylates other kinases in the pathway.
88
What does phosphorylation of TF do?
Allows a transcriptional change based on growth/environmental signals.
89
What does acetylation do?
Increases TF binding to DNA, increases protein interactions and has positive or negative effects on protein stability.
90
How is acetylation typically added?
By histone acetyltransferases (HATs) co-activators bound to promoters.
91
What does acetylation of GATA-1 do?
Increased GATA-1/DNA binding.
Look into if you have time.
92
What are the properties of GATA-1 acetylation?
Stimulates GATA-1/DNA binding and GATA-1 dependent transcription, enhances GATA-1/protein interactions, is required for proper GATA-1 function during haematopoietic differentiation, modulates GATA-1 levels.
93
How does Ubiquitination TF activation generally occur?
Triggered by other protein modifications, usually phosphorylation.
94
What does poly-ubiquitination cause?
Protein degradation.
95
What is Ubiquitination?
Requires E1, 2 and 3 ubiquitin Ligases to add ubiquitin.
Look into more if you have time.
96
How is TF degradation and pol2 transcription linked?
The activator interacts with basal transcription machinery recruiting ubiquitin Ligases that modify the transcription factors. This Ubiquitination recruits the 26S proteasome simultaneously destroying the activator and promotes pol 2 elongation.
97
How does TF degradation linked to pol2 transcription limit uncontrolled transcription?
By destroying the activator each time the promoter fires and by ensuring that it's activator is at a promoter that are destroyed by the proteasome.
98
When is activation of TF by cleavage of larger precursors important?
During neuronal development.
99
Give the three long-range control elements?
Enhancers, LCRs and insulators.
100
Describe enhancers?
Long range, from 1 to 1000 kb from the gene controlled, positive control elements, may be up several kb from promoters and contain multiple transcription factor binding sites.
101
Describe LCRs?
Very strong enhancers, gives position-independent copy number dependent expression of the transgene, discovered as essential elements to generate transgenic mice, either 5' or 3' of the gene, often found as string DNase 1 hypersensitive sites, bound by many tissue-specific transcription activators.
102
Describe insulators?
Long range, prevent interference by regulatory elements in neighbouring genes, block enhancers from interacting with the promoter, second function is to block the spread of heterochromatin.
103
Why is it beneficial to block the spread of heterochromatin?
Barrier function: important to protect genes from silencing.
104
How was the barrier function assayed?
Using stable transfection reporter constructs.
105
What occurs in the absence of an insulator?
The reporter gene becomes repressed followed by heterochromatin formation.
106
What can reporter constructs be used for and why is this beneficial?
To assay the regulation of gene expression.
| Rapid, quantitative assay and can use the same assay to quantify expression of different constructs.
107
How can transfection into cells differ?
Can be transient or stable, can be into cell lines of type where genes are normally expressed, can be into heterologous cell lines together with co-transfection of an expression vector.
108
How are transgenic mice generated?
The transgene is injected into he male pronucleus of one cells embryo. Forster mothers are made pseudopregnant by mating with a vasectomised male.
109
How is the transgene inserted into the genome?
Randomly, head-to-tail manner. DNA is isolated from the vector backbone prior to injection of the transgene.
110
What vectors can be used to generate transgenic mice?
```
Plasmid- insert up to 20 kb
Cosmid- insert up to 45 kb
BAC- inserts 100-300 kb (look into more if you have time)
YAC- inserts up to 1 Mb
Initial analyses of LCRs-mini locus
```
111
Explain how promoters can be linked to reporter genes to more easily analyse expression?
Location of lacZ expression can be determined by fixing the embryos and then reacting them with X-gal. This enables the location of expression of the gene in the embryo to be readily determined. Promoter deletions and mutations can also be made to determine what regulates gene expression at distinct stages of development.
112
What are the advantages of analysing gene expression in transgenic mice?
In vivo: transgene is exposed to factors at correct stages of development. Reporter genes can be used to analyse when promoters are activated during development.
113
What are the disadvantages of analysing gene expression in transgenic mice?
Copy number is different from line to line, insertion site is random (insertional mutagenesis) position effects, need to analyse many transgenic lines to ensure that the effects are real.
114
How can you overcome some position effects?
Use LCRs and insulators.
115
How does generation of knock-in mice differ from he generation of knock-out mice?
Same but knock-out mice delete whole genes, regions of genes or gene regulatory elements and knock-in mice mutate regions of a gene of mutate specific regions of the gene regulatory elements. Substitution rather than deletions.
116
What are the disadvantages with knock-out mice?
15% are embryonic lethal, some knock-outs show no effects (compensated by closely related gene product), if the genes work in combination with other genes the effects are not always fully apparent in all organs.
117
How can conditional knock-out mic be made?
By flanking with LoxP sites, placing of the LoxP sites allows a number of different conditional rearrangements, look into more if you have more time.
118
What can Cre recombinant be used for?
To inactivate or activate gene expression, inducible cause changes in gene expression. Tissue-specific expression of Cre causes changes in the target heme only in those tissues, inducible Cre allows target gene inactivation at a controlled time.
119
Conclude how enchanters work?
Physically touch promoters, consistent with looping hypothesis. On come cases there is evidence that the enhancers reach promoters via facilitated tracking. However, it is currently unclear at how many promoters facilitated tracking will play a role.
120
What is essential for the enhancer blocking function of insulators?
CTCF an 11 zinc finger protein that can either activate or repress transcription.
121
What properties allow CTCF to have its enhancer-blocking function?
Interaction with cohesion and association with the nuclear lamina.
122
Conclude how insulators work?
Increasing evidence that insulators work by forming chromosome loops that prevent the enhancer from interacting with the wrong promoter. Insulators potentially also function by blocking tracking of the enhancer.
