Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

L3 Epigenetics > Chromatin structure and function > Flashcards

Chromatin structure and function Flashcards

1
Q

How is DNA organised in interphase nuclei? (2)

A
  • Discrete chromosome territories
  • Each chromosome is made of topologically associated domains with active chromatin and inactive chromatin domains
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What tools are required to examine chromosome structure and function? (3)

A
  • Biochemical methods (protein/nucleic acid purification)
  • Genetic techniques (model organisms)
  • Computational tools for analysing genomic/epigenomic data
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What methods are available for detecting methylated DNA sequences and histone modifications? (3)

A
  • Treatment of genomic DNA with restriction enzymes that selectively recognise and cleave DNA containing methylated OR unmethylated CpG dinucleotides
  • Chromatin immunoprecipitation with antibodies recognising specific covalent modifications
  • Bisulfite sequencing analysis of genomic DNA to map and quantify methylated CpG dinucleotides in biological samples
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What types of immunoprecipitation can be done to identify specific covalent modifications? (3)

A
  • MeDIP: immunoprecipitation of methylated chromatin with an anti-5-methylcytosine antibody
  • Chromatin immunoprecipitation with histone modification-specific antibody e.g. anti-acetyl-histone antibody
  • Deep sequencing of chromatin-immunoprecipitates to identify gene-protein interactions
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

How does mapping DNA methylation with restriction enzymes work? (8)

A
  • MspI restriction enzyme cleaves methylated and unmethylated CCGG equally
  • HpaII only cleaves unmethylated CCGG
  • Digest genomic DNA with MspI or HpaII
  • Electrophorese digests in agarose gel
  • Transfer DNA to nylon membrane for Southern blotting
  • Hybridise nylon membrane to radiolabelled DNA probe with sequence matching the gene of interest
  • Identify methylated and unmethylated CpGs within the genomic DNA fragment which matches the DNA probe
  • Can tell if a site is methylated/unmethylated based on fragment sizes produced
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

How can you monitor the plasticity of DNA methylation with restriction digests?

A
  • Treat cells with DNA methyltransferase inhibitor AzaC for a number of days then perform digests with MspI and HpaII
  • Observe changes in sensitivity to HpaII after AzaC treatment
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

How does MeDIP seq work? (7)

A
  • Sonicate genomic DNA to randomly fragment
  • Convert fragments into a library with primers for DNA sequencing
  • Incubate fragments with anti-5-methylcytosine antibody
  • Immunoprecipitate antibody-DNA complexes
  • Sequence each immunoprecipitated fragment and map to the genome to identify the location of methylated CpGs
  • Can monitor distribution across the genome and over time/in normal cells vs cancer
  • Relative numbers of copies sequenced tell you the methylation level across the sample
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

How does bisulfite sequencing work? (4)

A
  • Incubation of DNA with sodium bisulfite: cytosine deaminates and becomes uracil (pairs with A), 5-methylcytosine is protected (pairs with G)
  • Sequence the sample, compare to the reference genome
  • Can work out which cytosines are methylated and the extent of methylated
  • If a C is present in the final sample and matches with a C in the reference genome it means there is a degree of methylation at this location
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

How does Hoxd CpG methylation change in early zebrafish development? (4)

A
  • Initially egg has much more methylation than sperm
  • Oocyte methylation is preserved at first in embryonic cells
  • Erasure of methylation at sphere stage to look similar to sperm again
  • Evidence of dynamic process
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

How does ChIP work? (5)

A
  • Cross link DNA and chromatin proteins with formaldehyde
  • Fragment chromatin by sonication/shearing
  • Immunoprecipitation of chromatin fragments containing protein/histone modification of interest
  • Remove crosslinks, amplify fragments by PCR, library preparation and fragment sequencing
  • Map fragments to reference genome to identify the DNA sequences that are associated with the protein/histone modification of interest
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How is the distribution of histone modifications and specific chromatin proteins in chromatin studied?

A

Chromatin immunoprecipitation (ChIP)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What is special about H3K4 methylation? (3)

A
  • Correlated with H3K27 acetylation (active)
  • Marker of permissive activation (may not be activated now but prepared to be)
  • Histone methylation not always a silencing mechanism
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What is the impact of methylation and acetylation of lysines in histone H3? (2)

A
  • H3K4 methylation and acetylation are both associated with gene expression
  • H3K9 and H3K27 are competed for by histone methyltransferases and histone acetyltransferases for silencing/activating respectively
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What are polytene chromosomes?

A

Chromosomes in the salivary glands of drosophila that have been replicated many times without cell division so can be visualised by light microscopy

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What is the chromocenter in polytene chromosomes? (3)

A
  • Region of dense, transcriptionally silent heterochromatin where the chromosomes aggregate
  • H3K9me2 rich, H3K4me2 deficient: H3K9 is specific for transcriptionally silent heterochromatin in the chromocenter, H3K4 is specific for transcriptionally active euchromatin outside chromocenter
  • Sharp boundaries between the 2 regions
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What is an example of position effect variegation (PEV)? (4)

A
  • Drosophila eyes are usually red due to transcriptional activation of the white locus
  • The white locus is usually located in transcriptionally active euchromatin
  • Inversion mutation (wm4) repositions the white locus into heterochromatin in same chromosome, eye appears mostly white
  • Heterochromatin causes transcriptional silencing
15
Q

What is position effect variegation (PEV)?

A

When a gene normally in euchromatin is put into heterochromatin by rearrangement or transposition

16
Q

How was PEV of the white locus used to identify additional genes? (3)

A
  • Some mutants could override wm4 and eyes appear red (suppress PEV)
  • Some mutants enhanced wm4 PEV and eyes appear completely white
  • Su(var)3-9 KO rescues the wm4 silencing effect, prevents heterochromatin effect = Su(var)3-9 encodes a protein which is important for formation of heterochromatin
17
Q

What is encoded by Su(var)3-9? (2)

A
  • Histone H3K9 methyltransferase (the modification most abundant in the transcriptionally silent heterochromatin chromocenter)
  • Gene contains SET domain which encodes the catalytic domain of H3K9 methyltransferase
18
Q

What is heterochromatin protein 1 (HP1)?

A

Protein that specifically binds to H3K9 methylation mark made by Su(var)3-9

19
Q

How many methyl groups can be added to the lysine side chain? (2)

A
  • Up to 3 - mono, di or trimethylated
  • More methylation = stronger signal
20
Q

What is the mechanism of HP1alpha? (2)

A
  • HP1alpha has H3K9me binding domain and a domain to dimerise with another HP1alpha
  • Pulls adjacent H3K9 methylated nucleosomes to condense chromatin = heterochromatin
21
Q

Why is euchromatin transcriptionally active?

A

Open conformation is accessible to transcription factors which can then recruit RNA polymerase and start transcription

22
Q

How is euchromatin identified experimentally?

A

DNase I hypersensitive site-mapping

23
Q

How does DNase I hypersensitive site-mapping work? (6)

A
  • DNase I is a sequence non-specific endonuclease (cleaves any sequence within double stranded DNA that it can access)
  • DNase I degrades region of accessible DNA
  • Digest DNA with BamHI, absence of DNase I the fragment will be of known size and detected by DNA probe
  • Add DNase I, if DNA is accessible the fragment will be cleaved, section binding to the probe will be markedly smaller
  • Southern blot to hybridise with radiolabelled probe for BamHI fragment
  • DNase I hypersensitive = active euchromatin
24
Q

What is BamHI?

A

Restriction endonuclease with target sequence GGATCC

25
Q

What is the beta-globin locus control region? (4)

A
  • Series of DNase I hypersensitive sites separated by nucleosome-associated chromatin, upstream of adult β-globin gene
  • Contains sequences that are binding sites for transcription factors
  • Responsible for sequential activation of subsequent ε, Gγ, Aγ, δ and β globin genes
  • ε expressed very early in embryonic development then drops off, γ’s during pregnancy, β expressed after birth and into adult life
26
Q

How does the beta-globin locus control region work? (4)

A
  • Activity is constrained to the globin genes by boundary/insulator elements which stop the LCR activating other areas of the genome
  • LCR sequentially interacts with the downstream genes within a chromatin loop defined by the LCR HS5 and 3’HS1 sequences
  • LCR HS5 and 3’HS1 bind CTC factor
  • Loops correspond to topologically associating domains in chromatin which are defined by boundary elements like CTCF
27
Q

What are insulator elements?

A

Prevent heterochromatin spreading from adjacent chromatin whilst ensuring LCR can only act on the globin genes

28
Q

What are boundary elements?

A

Define the boundaries of a transcriptionally autonomous chromatin domain

29
Q

What is CTCF? (3)

A
  • CTC factor
  • DNA binding protein which contains zinc fingers which interact with a specific 34 base pair sequence that is present in the LCR HS5 and the 3’HS1
  • Restricts the activity of the beta-globin LCR to the genes within the loop by defining the boundaries
30
Q

What is ATAC-seq? (5)

A
  • Assay for transposase accessible chromatin-sequencing approach
  • Relies on the accessibility of euchromatin to Tn5 transposase
  • Introduce Tn5 transpososome (Tn5 plus transposable DNA material), finds open chromatin, cleaves and inserts transposon sequences without ligating again which tags the euchromatin and leaves the DNA fragmented
  • Sequence the Tn5 tagged fragments and map to reference genome to identify accessible euchromatin areas
  • Accessibility changes during development
31
Q

What are transposons? (3)

A
  • Genes that jump around the genome
  • Encode a transposase which mobilises the transposon
  • Some transposons move via cut and paste (like Tn5) or can encode a reverse transcriptase which makes a new copy of the transposon and inserts it somewhere else
32
Q

How does chromatin accessibility change during zebrafish early development? (3)

A
  • Chromatin accessibility increases during development
  • Correlates with erasure of methylation
  • Increases largely during zygotic gene activation (ZGA)
33
Q

What is zygotic gene activation (ZGA)?

A

When embryonic genes become active for the first time, before this point were transcriptionally silent

L3 Epigenetics (15 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions