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What does the term epigenetic refer to?

The concept that the function of a section of DNA is determined by factors more than just its nucleotide sequence


What are considered epigenetic changes that can affect gene expression?

Alteration of methylation status of DNA or alteration of underlying chromatin organisation (e.g the degree of compaction) it is possible to regulate DNA accessibility for activities such as recombinant and gene transcription


What does the term 'chromatin' refer to?

The complex of DNA that makes up the chromosomes


What does chromosomal organisation mediate?

Compaction of the genetic material
Efficient transmission to daughter cells upon division
Protection of DNA from damage
Regulation of DNA accessibility
Regulation of gene transcription


Various organisms have different compaction mechanisms. What do prokaryotic circular chromosomes need to overcome during replication? What do linear chromosomes need to ensure during this process?

Circular chromosomes need to find a way to separate the DNA strands of the supercoils whereas linear chromosomes need to ensure copying of the very ends


Which techniques helped in our understanding of chromosome structure?

Electron microscopy, nuclease and salt treatment but the detailed molecular understanding comes from the X-ray that revealed the crystal structure


The structure of the chromosomes change throughout the cell cycle. What is it like in metaphase (M phase)? What is it like in the G1-S-G2 interphase?

During metaphase/ M phase- the chromsomes are compacted (cells are just about to divide)
During interphase- decondensed chromosomes. In G1, the cells synthesise all components required for the S-phase (DNA synthesis) and during G2 phase, the cell prepares to divide


During interphase, what name is given to the parts of the nucleus that individual chromosomes occupy?



Centromeres are the centre parts of the chromosome. Centromeres contain highly repetitive DNA that extends over megabases. This is compacted into what?



What are kinetochores?

Large protein complexes that bind to centromeres and they also attach to microtubules (pull separated chromosomes to opposite poles during metaphase)


When are inner and outer kinetochores present?

Inner= all the time
Outer= only just before cell division


Where are telomeres found?

At the end off linear chromosomes


What are the functions of telomeres?

Prevent fusion of chromosomes to one another and from recombination and degradation
Also provide a mechanism for replication of the ends


What can you tell from telomeres on linear chromosomes?

Distinguish between natural ends and artificial breaks by X-ray


Are telomeric repeats well conserved between vertebrates?

yes! Found in both double stranded regions and single stranded over hang regions at the end.


Where did the first evidence of 'bead-on-a-string' model come from?

Electron microscopy studies of chromatin extracted at different salt concentrations


Extraction of chromatin using lower salt concentrations produce a 10 nm fiber that is the classic 'bead-on-a-string' depiction. What is the fiber showing?

nucleosomal beads joined together by linker DNA


What does the nucleosome comprise of?

A histone octamer core protein around which DNA is wrapped twice. The core DNA is highly conserved whereas the linker DNA is much more variable


What does micrococcal nuclease do?

Cleaves linker DNA. Reactions with lower levels of this will not cleave at all linker regions so a ladder will be created on gel


What core histones are found in each nucleosome?

Two copies of:

H2A, H2B, H3 and H4


What are histones?

Small proteins of which around 20-25% is made from lysine and arginine. They are highly conserved. All core histones are important


What are the two important parts of a histone?

N-terminal (histone) tail and histone-fold domain


What does the histone-fold domain consist of and what shape does it form? What is its main role?

3 alpha helices which form a Z-shape (long central part flanked by two shorter outer ones). It mediates interactions with other core histones through formation of H2A-H2B and H3-H4 heterodimers


The nucleosome assembles in a highly organised manner when individual core histones are mixed with DNA. What happens?

H3-H4 dimers assemble to form tetramers then bind to DNA. H2A-H2B dimers then join this complex (these do not form tetramers, only form dimers that interact independently at the edge)


Histone tails stick out of the nucleosome and may be cleaved off by protease treatment. Does this affect the structure of the nucleosome core?



Is it the histone core fold or the tails that interact with the DNA wrapped around?

The core fold! H3-H4 binds in the middle and ends of the DNA and H2A-H2B bins on either side


What interactions are involved between the histone fold domain and DNA?

Hydrophobic interactions with the faces of sugars in DNA, interactions of polypeptide amide groups with phosphates on DNA, and interactions of histone arginines with the minor groove. There are no sequence specific interactions with the histones and bases of the DNA


Most nucleosomes are positioned at random on the DNA however where is the minor groove on the DNA found?

Compressed on the inside of the nucleosome- AT-rich DNA easier to compress so there is sometimes a tendency of this to be on the inside


What does histone H1 do? What is its structure?

Binds to linker DNA and pulls fiber together to form solenoids. H1 has a central globular domain and basic N- and C-terminal tails that interact with the linker DNA. H1 protects the nucleosomal DNA from micrococcal nuclease digestion


Is the chromatin further compacted than just wrapping around the histone core particles?

Yes, the chromatin is further compacted into loops within the chromosomes (slide 20 first lecture)


When are the core histones normally made during the cell cycle?

In the synthesis phase (S) when DNA is synthesised. They are assembled into nucleosomes on the newly replicated DNA near the DNA replication fork


There are such things called histone variants (Literally just variants of the standard histones e.g H2AX). When are these made in the cell cycle?

Histone variants are made in interphase and are inserted into chromatin that has already formed by a histone-exchange mechanism to replace a core histone with the variant. This requires a chromatin remodelling complex to facilitate this energy dependent process


Each histone variant has a specialised has specialised function. Where are the following ones found and what do they do?


H2AX- present throughout chromosome, and becomes phosphorylated when DNA is damaged- this is recognised by DNA-repair enzymes
CENP- found at centromeres- has an extended N-terminal tail that attaches it to kinetochores and facilitate separation of sister chromatids during mitosis
H3.3 and H2AZ form less stable nucleosomes than core histones enabling looser structure that is associated with transcriptional activity


What is the difference in structure of euchromatin and heterochromatin?

euchromatin is looser and associated with transcriptional activity
heterochromatin contains compacted chromatin in which access to DNA is restricted and is transcriptionally inactive


What state is constitutive heterochromatin and what state is falcultative heterochromatin in?

Constitutative heterochromatin is always heterchromatic
falcultative heterochromatin contains regions of chromosomes that are not heterochromatic under all circumstances e.g. XX, one is active and one isn't


Genetic control of gene expression is based on nucleotide sequence and is controlled by what?

How can a gene be switched off knowing this?

cis-acting regulatory sequences (gene promoters, enhances and RNA splicing regulatory sequences) that interact with trans-acting regulators (transcription factors and splicing factors). A gene can be switched off if an element has a mutation in- these are ingerited by daughter cells and are in the germ cell line. It can also be switched off if it acquires a 'repressive' chromatin change (e.g happens in cancer)- these are often reset in gametes but are inherited by daughter cells (epigenetic inheritance usually reset!)


In epigenetic regulation, the chromatin environment may regulate the accessibility of a gene to a specific transcription factor. Even if the transcription factor is present in the cell, the gene will not be activated due to its repressive state. Use the example of how oestrogen has a different effect on chicken liver and chicken oviducts due to their different states of repression.

Oestrogen binds to the receptor in both organs then moves to the nucleus to activate gene transcription.
In the oviduct, the vitellogenenin gene is in a repressive state whereas in the liver the ovalbumin gene is in a repressive state, hence the oestrogen activates the ovalbumin gene in the oviduct and the vitellogenin in the liver


Gene activation often requires an 'opening' of chromatin structure and presence of appropriate transcriptional activators. Use the example of fibroblasts differentiating into carilage-producing cells, explain what this means. In addition, can they re-differentiate back? If so, why?

This differentiation requires the opening of chromatin structure of specific genes required for the phenotype (these cells secrete an extracellular matrix) and this is triggered by being put in a certain environment. They can re-differentiate back as cells have a 'memory' of what they were like before, as long as the structure is still open. Fibroblasts that have never been induced to differentiate have repressed chromatin and so remain that way


DNA binding sites need to be exposed so that transcriptional factors can access the DNA. Which parts of the DNA is rarely exposed but important that it is? Is ATP needed for chromatin remodelling?

The DNA near the centre of the nucleosome. ATP is needed to rearrange the nucleosomes to allow for this to happen.
Remodelling occurs either by sliding nucleosomes along the DNA, ejecting whole histone octamer cores or by replacing core histones with variants


How are the amino acid residues marked?

Numbered from the N-terminus of the proteins


Acetylation and methylation are modifications of histone tails and of DNA. Do both these processes occur on all core histone tails?

Acetylation occurs on all 4 core histone tails
Methylation occurs on H3 and H4 only


What are the enzymes which carry out the modifications called?

Writers (e.g histone acetyl transferase)


What are the proteins that recognise and interact with he specific histone tails modifications called?

Readers (e.g bromodomain and chromodomain-containing proteins)


What is acetylation?

Acetylation of the histone tails neutralises their basic charge, loosening chromatin structure- this is associated with transcriptional activation


Does methylation affect charge of the histone tails? Is it associated with thranscriptional activation or repression?

No effect on charge and is associated with both activation and repression


What is acetylation carried out by? What is happening during acetylation?

Acetylation is carried out by histone acetyle transferase activities (HATs) that transfer the acetyl group from acetyl Co-A to the terminal amino group of the lysine side chain. Many transcriptional activators interact with co-activators that recruit HAT activity


What is the correct nomenclature for acetylation activity? Use H3K9 as an example.

H3K9= the lysine residue (K) at position 9 on histone H3


What is the opposing reaction, histone deacetylation, carried out by?

Histone deacetylases (HDA or HDACs) whic interact with co-repressors that have HDA activity


As mentioned previously, acetylation of histone tails leads to transcriptional activation by neutralising the charge on the nucleosome thereby loosening interactions. Acetylation specifically of H4K16 has been shown to inhibit what?

Acetylation of H4k16 has been shown to inhibit formation of compact chromatin fiber,


Specific proteins that contain bromodomains recognise what?

Acetylated lysine residues on the histone tails- these are readers of the acetylation code.


What three classes of proteins contain these acetylated lysine recognising bromodomains?

Components of basal transcriptional apparatus
transcriptional coactivators
chromatin remodelling activities


Basal transcriptional apparatus is a complex that interacts with genes at their start site. What sequence is often upstream of a gene?

TATA box and the part of the basal apparatus that binds to it is called the TFIID complex (TBP is the sequence within this which recognises the TATA box)


What is meant by a cascade of histone tail acetylation?

When proteins that contain bromodomains also have acetyl transferase activity themselves so they are attracted to acetylated lysine and then catalyse more acteylation events which in turn attract more bromodomain proteins


Myc is a transcriptional factor that causes a cascade in histone acetylation. How?

Myc recognises CACGTG and recruite a co-activate that has HAT activity. The HAT activity leads to the acetylation of histone tails around the TATA box. The acetylated histones then attract bromodomain protein TAFII250, which is a component of the basal transcription apparatus (TFIID complex) that binds to the TATA box. The TBP component binds to the TATA sequence, TAFII20 itself has HAT activity leading to further acetylationand yet more recruitment of TFIID complex


The transcriptional repressor, Mad, also recognises CACGTG just like Myc. What does it lead to instead of a cascade of histone acetylation?

Mad recruits corepressor mSin3 that in turn recruits histone deactylase activity, This leads to the removal of acetyle groups and to compaction of chromatin


Myc and Mad antagonise eachother's activity during cell proliferation and differentiation. When Myc levels are high and Mad levels, what does this induce? How about when Myc levels are low and Mad levels are high?

High Myc levels and low Mad levels= histone tail acetylation so gene activation thus driving cell proliferation (many cancers are due to over expression off Myc levels)
Low Myc levels and low Mad levels= Deacetylation of histone tails and gene repression, blocking proliferating but inducing differentiation


Moving onto methylation which only takes place on H3 and H4, how many methyl groups can be added to the terminal amino group of the lysine side chain?

1 (monomethylation, 2 (dimethylation) or 3 (trimethylation)


Which enzyme catalyses methylation of histone tails?

Specific histone methyl transferase


What is H3K4me3 and H3K4me3 associated with? Where are high levels of H3K4 trimethylation seen? What does this sort of methylation lead to? How does it lead to an acetylation cascade?

Side note (me3 just means this has been trimethylated/ same as saying the methylation of H3K4 etc.)

Transcriptional activation. H3K4 methylation is seen in high levels at the transcriptonal start site and is carried out by a methylase (SET1) that is recruited by the initiating RNA polymerase.Once transcription intiation has taken place, RNA polymerase changes into an 'elongating' form that transcribes the body of the gene. This elongating form recruits a different type of methylase (SET2) that in turn methylates the H3 tail at position36 (also trimethylation). This then provides a binding sitr for proteins that contain a PHD domain finger (e.g TAF3 subunit of the TFIID) which sets of an acetylation cascade


What is the methylation of H3K9 and H3K27 associated to?
The methylation of both provide binding sites for what?

Transcriptional repression. It is found in the constitutive heterochromatin. This modification provides a binding site for the chromodomain protein heterchromatin protein 1 (HP1). In contrast, H3K27me3 provides a binding site for the chromodomain if the polycomb complex and is associated with falcultative heterochromatin


Bromodomains recognise acetylated lysines and chromodomains recognise methylated lysines. What is the difference between the specificities?

Bromodomain recognises non specific acetylated lysine whilst chromodomains recognise specific methylated lysines


How does HP1 lead to a spread of heterochromatin/ cascade of methylation?

contains a chromodomain that interacts with H3K9me3, It self asociated and also interacts with the histone methyle transferase SUV39H1 that methylates H3K9


Where does methylation of DNA in mammals take place?

This sort of methylation is different from methylation of histone proteins!

Exclusively at the 5-position of the base cytosine- the methyl groups lies in the major groove of the dsDNA and does not interfere with base pairings. It also only takes place when the C is immediately followed by a G-this sequence is known as CpG dinucleotide (p stands for the phosphate on the DNA backbone)


Are CpGs common and what state are they usually in?

No they appear 4-5 times less than would be expected but when they are present, they are usually methylated, hence 5meC accounts for around 1% of our DNA


What happens to unmethylated CpG dinucleotides? What about hemi-methylated DNA?

Unmethylated CpG dinucleotide has a de novo methylase added to give a hemi-methylated product that has meC on one strand only whereas hemi-methylated DNA is recognised by maintenance methylase which then methylates the unmethylated C of the palindromic site to result in a CpG dinucleotide that is methylated on both strands


What are three important consequences of CpG methylation in mammalian DNA?

CpG methylation is a hotspot for mutation- often seen in cancer and genetic disease
It is involved in stabalising heterochromatin into a compact and repressive state
It plays a critical role in gene regulation


What can deamination of cytosine, adenine or guanine lead to the formation of? What does this lead to and can this be corrected?

Uracil, hypoxanthine and xanthine respectively- leads to a mismatch of base pairs but is often picked up by the base excision pathway


What about deamination of 5me-C? What does this form and is it corrected?

Forms thymine which is naturally occuring. The resulting TG mismatch is converted to a TA base pair. Because 5me-C is frequently deaminated and not repaired, CpG sequences are gradually lost from the genomic DNA (except from CpG islands which are critically unmethylated!). Most point mutations found in genetic diseases and cancer are Cto T transitions that resulted from deamination of 5me-C at CpG sequences (50% genetic diseases cause by this)


Are males or females more likely to be affected by these C to T transitions?

Males since their sperm is heavily methylated


Where are CpG islands often found? Are they methylated or unmethylated?

Clusters of unmethylated CpG residues


Name some highly methylated sequences found in the genome?

Satellite DNAs, repetitive elements, non repetitive intergenic DNA, exons of genes


What do studies show about the association between methylation levels and gene expression levels?

Inverse correlation


Explain the relationship between methylation levels and gene expression levels using the human beta-globin genes.

In early life, genes are expressed are unmethylated at this stage. They do become methylated however in the adult and this us when they are repressed. Conversely, the adult-like genes are unmethylated in adult blood cells but are methylated when they are in the embryo and fetus (transcriptionally silent)


How does CpG methylation sometimes lead to inhibitiom of transcriptional activity??

By interfering with transcriptional factor binding
By attracting histone deacetylases (e.g MeCP2) to lead to compaction
By attracting histone methyl transferase (MBD1) to again contribute to compaction of chromatin structure


Which genetic diseases can arise from mutations and problems in methylation?

ICF syndrome, Rett syndrome, cancers