Chromatin structure and function

Question 1

How is DNA organised in interphase nuclei? (2)

Answer 1

• Discrete chromosome territories
• Each chromosome is made of topologically associated domains with active chromatin and inactive chromatin domains

Question 2

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

Answer 2

• Biochemical methods (protein/nucleic acid purification)
• Genetic techniques (model organisms)
• Computational tools for analysing genomic/epigenomic data

Question 3

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

Answer 3

• 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

Question 4

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

Answer 4

• 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

Question 5

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

Answer 5

• 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

Question 6

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

Answer 6

• 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

Question 7

How does MeDIP seq work? (7)

Answer 7

• 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

Question 7

How does bisulfite sequencing work? (4)

Answer 7

• 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

Question 8

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

Answer 8

• 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

Question 8

How does ChIP work? (5)

Answer 8

• 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

Question 9

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

Answer 9

Chromatin immunoprecipitation (ChIP)

Question 10

What is special about H3K4 methylation? (3)

Answer 10

• 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

Question 11

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

Answer 11

• 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

Question 12

What are polytene chromosomes?

Answer 12

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

Question 13

What is the chromocenter in polytene chromosomes? (3)

Answer 13

• 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

Question 14

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

Answer 14

• 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

Question 15

What is position effect variegation (PEV)?

Answer 15

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

Question 16

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

Answer 16

• 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

Question 17

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

Answer 17

• 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

Question 18

What is heterochromatin protein 1 (HP1)?

Answer 18

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

Question 19

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

Answer 19

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

Question 20

What is the mechanism of HP1alpha? (2)

Answer 20

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

Question 21

Why is euchromatin transcriptionally active?

Answer 21

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

Question 22

How is euchromatin identified experimentally?

Answer 22

DNase I hypersensitive site-mapping

Question 23

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

Answer 23

- 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

Question 24

What is BamHI?

Answer 24

Restriction endonuclease with target sequence GGATCC

Question 25

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

Answer 25

- 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

Question 26

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

Answer 26

- 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

Question 27

What are insulator elements?

Answer 27

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

Question 28

What are boundary elements?

Answer 28

Define the boundaries of a transcriptionally autonomous chromatin domain

Question 29

What is CTCF? (3)

Answer 29

- 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

Question 30

What is ATAC-seq? (5)

Answer 30

- 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

Question 31

What are transposons? (3)

Answer 31

- 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

Question 32

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

Answer 32

- Chromatin accessibility increases during development - Correlates with erasure of methylation - Increases largely during zygotic gene activation (ZGA)

Question 33

What is zygotic gene activation (ZGA)?

Answer 33

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