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Flashcards in Chromatin and epigenetics Deck (55):

How is the DNA in chromatin usually bound

Hydrogen bond between the phosphodiester backbone and lysine/arginine residues of histones.


What is the structure of an individual histone molecule

Contains a structural domain - a 'histone fold'. This consists of 3 helices connected by 2 loops - causes a crescent shape.
Each molecule has N- and/or C- terminus made up of highly flexible tails which contain lots of basic residues (lysine/arginine)


What is the most basic histone/histone interaction

Histone dimerisation.
Specifically H3-H4 and H2A-H2B


How do histone dimers interact

In 4-helix bundles.
H3/H4 dimers interact to form a (H3-H4)2 tetramer.
H2A-H2B dimers bind to the H3-H4 tetramer.
Overall 2 H3-H4 dimers and 2 H2A-H2B dimers present in a histone octamer.


Why is nucleosome positioning important

It affects access to the DNA.
If the sequence is between nucleosome it is easier to transcribe.
Equally, DNA that is faced away from the octamer (facing out) is more accessible that the DNA facing inwards.


How often are the outer turns of DNA available for transcription

About 1 tenth of the time.
DNA association with the nucleosome is transient; in constant flux.


What is the importance of the histone tails

Not required for nucleosome assembly. Structure is unresolved.
Mutations result in defects in transcription (so important role in gene regulation)
Most are subject to lots of post translational modifications
One H4 tail is known to interact with the H2a-H2b dimer of an adjacent nucleosome.


Give examples of known variants of the H2A histone

H2A.Z; found in almost all eukaryotes (function unknown, but has a role in early development, chromosome stability and centromere function)
H2A.X (SQ C-terminal motif; becomes phosphorylated at sites of double-stranded DNA breaks)


Give examples of known variants of the H3 histone

H3.3 (replaces H3 at regions of active transcription. Involved in gene activation and heterochromatin formation)
CenH3 (centromeric H3 histone)(essential for assembly of proteinaceous kinetochore - in mitosis/meiosis)


What are linker histones

Histones that associate with ~ 50% of nucleosomes. Usually associated with less accessible chromatin (repressed regions).
Basically the 5th type of histone.


What happens to chromatin structure during cell division

Not well understood.
Cell morphology completely changes during this time. Chromosomes are organised into territories that are conserved throughout the cell cycle.


Where in the nucleus does transcription most commonly occur

The centre


Name two techniques used for studying gene regulation

Illuminated microarray
Highthroughput sequencing


What is involved in the technique Illuminated Microarray

Take the mRNA -> cDNA from cells in two different conditions (that you want to study). Hybridise them onto a microarray (plate). This is then scanned with a laser, and the absorbance is measured.
Used to map genome mutations and see where gene rearrangements occur.


What is involved in the technique High throughput sequencing

1 - Randomly fragment the DNA and ligate adaptors to both ends of each fragment
2 - Attach the fragments to the surface
3 - Add unlabelled nucleotides and enzymes. Initiate solid-phase bridge amplification. fragments become double stranded
4 - Denature double stranded fragments. Single-stranded templates remain.
5 - Amplification. Lots of each template is generated and can be analysed


Name some applications of genomics

Genome sequencing
RNA sequencing
Nucleosome mapping


How can nucleosomal DNA be isolated

Can be isolated based on resistance to nuclease digestion. DNA wrapped around a nucleosome is resistant to digestion.


Why is nucleosomal DNA isolation important

It provides a map of nucleosome positioning and occupancy. Tells us which regions are available for transcription and which regions are not.


What is Chromatin immunoprocipitation and why is it useful?

Can be used to identify DNA binding sites
Can measure chromosome conformation (and how a chromosome interacts with another chromosome)


Name some factors that can regulate DNA access

DNA methylation
ATP-dependent chromatin remodellers
Histone modifying enzymes
Histone chaperones


What does HAT stand for

Histone acetyl transferase


Name a cellular function of histone acetyl transferases

Co-activators of transcription


List modifications that can occur to histones

Lysine acetylation


What are the functions of the SAGA complex

Histone acetyltransferase
Histone deubiqutinase
It also plays important roles in transcription elongation, protein stability regulation, and telomere maintenance


What is the importance of Histone deacetylases

Referred to as HDACs. Act in opposition to HATs; they remove acetyl groups from histones
Some act as co-repressors (recruited to genes by repressor proteins) - act to reduce transcription.


How do histone modifications cause a functional change

A diverse class of protein motifs specifically recognise specific modifications.
Also, some modifications can directly alter the chromatin structure (mainly modifications to histone H3 and H4).


What type of histone modification has the potential to alter the structure of chromatin

Neutralisation of lysine residues (through acetylation)
This has a strong effect on chromatin condensation


What is the histone code hypothesis

The theory that multiple histone modifications in combination/sequence on one or more histone tails can specify unique downstream functions
Unlikely because the same combination of modifications can be found in other proteins.


Give an example of a positive feedback loop in histone modifications

H3 methylated at Lys9. This allows HP1 to bind. Causes heterochromatin formation (of SUV39H) which acts to methylate histones (and chromatin binding). It has the ability to spread - bad; a positive feedback loop can go on forever


What does PEV stand for

Position effect variegation


What is position effect variegation (PEV)

Variation caused by the inactivation of a gene in select cells due to its position in relation to heterochromatin (tightly packed chromatin).


Give an example of how PEV can affect gene expression

In drosophila, eye colour can be determined by the 'w' gene. When active causes the fly to have red eyes. When inactive it has white eyes. The 'w' gene is near to heterochromatin. If the sequence is reversed (and the 'w' gene is next to the heterochromatin) the gene is inactivated.


What does LCR stand for

Locus Control Region


What is the function of a locus control region (LCR)

Act as super-enhancers.
Are able to open chromatin (de-repress) and activate transcription. Able to overcome any repressive structure at the site of integration.


What is an insulator

A barrier to heterochromatin.
Can prevent heterochromatin from spreading. Also able to block enhancer action (when places between the enhancer and promoter)(does not affect the promoter)


How many major states of chromatin are there

6 (states of chromatin condenstation).


What are CpG islands

(sequence 5'-CG-3' )
They surround promoter of constitutively expressed genes (unmethylated).
When methylated, they prevent activation of the promoter they are near to (caused by proteins that can bind to methylated CpG islands)


Summarise the information currently known about DNA (de)methylation

Methylation patterns are erased during preimplantation and re-established through development (but are then stable)
It inhibits gene expression by affecting chromatin structure


What does SWI/SNF stand for

SWItching and Sucrose Non Fermentation
(Present in yeast, part of the SNF2 family)


What family of enzymes does SNF come from

Nucleic acid translocases
Activity enabled by helicase motifs present.


What are the main proteins related to SWI/SNF (in yeast)

ISW 1a/b


What effect on chromatin structure does ISW1 and CHD1 have

Able to more evenly space the nucleosomes. Organised over coding regions.
Can change gene expression because DNA bound factors can favour regular phasing (of nucleosomes)


What effect on chromatin structure does SNF2 have

Takes organised nucleosomes and disorganises them (ruins regular phasing).
Might make a binding site more available.
Also when nucleosomes touch each other, DNA dissociates and histone octamers can be lost. Therefore SNF2 can act to remove nucleosomes.


What effect on chromatin structure does SWR1 have

Can take a normal histone tetramer/octamer and replace certain histones with modified ones (H2A.X/Z H3.3 etc)


What is the main role of histone chaperones

To assist histones movement to and from chromatin. (Histones and DNA are not very stable when apart).
Enables constant dis/re-association of histones.


Give some examples of histone chaperones



What is the closest homologue of the SAGA complex in humans

The beta-interferon enhanceosome


What role does the beta-interferon enhanceosome play in chromatin modifications

Binding causes DNA distortions. Modifications could change the DNA arrangement.
Recruits specific complexes (such as GCN5) which modify histones. The enables polymerases to bind and transcribe.


Define epigenetics

The study of heritable changes in gene expression not controlled by changes in DNA sequence


Give some examples of epigenetic effects

Different cell types in the body
Differences between identical twins
Stem cells


Explain X inactivation as an epigenetic effect

In females one X chromosome is selected for inactivation at random (dosage compensation). Early steps are directed by Xist RNA
Change is stable.
Inactive X chromosome have slightly acetylated histones and lots of macroH2A histone variant.
Different X chromosomes can be inactivated in different cell types (e.g. cats that have a multicoloured coat)


What are imprinted alleles

Rare. One allele is expressed from a specific parent (maternal/paternal) only. dominance/recessive doesn't matter. Involves chromatin changes and DNA methylation.


Give examples of transgenerational epeigenetic inheritance

Metabolism changes in mice - changes detected in grandchildren.
Overkalix study: Observed food intake during pre-adolescent slow growth period. Grandfathers nutrition affects grandsons (not granddaughters)
Dutch famine of 1944: Woman suffering in the famine had grandchildren that grew up smaller despite being well fed.


Aside from chromatin, where else can epigenetic changes occur

In prokaryotes, Prions are heritable protein filaments. Can terminate/not terminate transcription at a certain point.


Why is epigenetics of interest to medical research

Histone modifications common in cancers - chromatin pathways are commonly mutated in cancer. There are many links to chromatin regulators.