Epigenetics - Appunti di lezione

Epigenetics for the exam - lezione 1

Meaning of epigenetics

Epigenetics is the branch of biology that can answer to the question: why and how multicellular organism’s cells that have the same genetic material can use it in different ways? Starting from the zygote, the cells progressively differentiate into adult cells (passing through the blastocyst state), like neurons or pancreatic cells. How can this variety be possible? →

The original definition by Conrad Has Waddington. The father of epigenetics is Conrad Has Waddington, embryologist, palaeontologist, geneticist and philosopher, who introduced the term in 1942. He became interested in biology and problems of biology development and in how the gene function can be modified to produce a complex body shaping.

Epigenetics: genetics + epigenesis = development of organisms as plants, fungi, animals from a seed, spore or egg cell through a sequence of steps in which cells differentiate and organs form → the original meaning of epigenetics was the branch of biology that studies the causal interactions between genes and their products, which bring the phenotype into being. Epigenetics referred to all molecular pathways modulating the expression of a genotype into a particular phenotype, so how the gene function is regulated and modified during development to produce a complex body. → Epi = above, beyond + genetics field of biology that studies something that genetics alone cannot explain.

The epigenetic landscape by Weddington: a small red ball represents a cell at the beginning of differentiation process, then it goes down through the valley taking one particular branch, becoming an epithelial cell for example, or a neuron for example. There are hills that separate the branches of the valley, they represent the epigenetic mechanisms that make the cells stay in their state. For a neuron is not possible to become an epithelial cell, so these hills are the epigenetic mechanisms that maintain the difference in differentiation for each type of cells. →

The modern definition at that time, principles of genetics as Mendel and population genetics was known, as the new Darwinian synthesis = evolution is a slow substitution by selection of a fitter version of a gene set. Anyway, the DNA structure wasn’t already known (discovered in 1953 thanks to Watson and Creek). So, after the discovery of DNA structure, a new definition: studies of changes in gene function (thanks to gene regulation) that are meiotically and/or mitotically heritable (with long term regulation, the changes are transmitted from the mother to the daughter cells) and that don’t lead to changes in DNA sequence (it is a reversible process).

So, epigenetics is a long term, but reversible regulation of genes that involves:

• Differentiation of cells and development of multicellular organisms
• The effect of the environment, diet and lifestyle on the phenotype (they can have an impact on our health status)
• Epigenetics mechanisms are involved in many most common human diseases, as many age-related diseases and cancers

An example of gene expression regulation that is not an epigenetic mechanism: glucose regulation of insulin gene expression in pancreatic Beta cells, by glucose levels in blood. 3 transcription factors activate insulin gene expression in a co-ordinated and synergistic manner in response to increasing glucose levels. It has been shown that changes in glucose concentrations modulate the function of these β-cell transcription factors at multiple levels. These include changes in expression levels, subcellular localization, DNA-binding activity, transactivation capability and interaction with other proteins. It is a short-term regulation: as soon the levels of glucose decrease, this mechanism of regulation is reversed. → →

Difference in epigenetics mechanism long term and not epigenetics modification short term and cannot be transmitted.

Chromatin structure

Epigenetic regulation usually involves changes in chromatin composition, structure and function.

The chromatin is a complex of DNA and proteins (with up to twice as much protein as DNA) which condenses to form a chromosome during cell division. The primary functions of the chromatin are:

• To package DNA into a smaller volume to fit in the nucleus, *
• To strengthen the DNA to allow mitosis,
• To prevent DNA damage,
• To control gene expression and DNA replication.

* Considering the length of naked DNA in eukaryotic cells (2.17 metres) and the dimensions of the nucleus Commentato [MCB1]: The diploid human genome contains approximately a total of 6 billion base pairs of DNA (few micrometres of diameter), DNA must be packed by proteins in a smaller volume to enter within per cell. Because each base pair is around 0.34 nanometres nucleus. For this reason, there is no cell with native DNA, but it is always associated with proteins to form long, each diploid cell contains about 2 meters of DNA [(0.34 chromatin. 9 × 10^-9) × (6 × 10)].

Structure

Structure. The basic repeat element of the chromatin is the nucleosome. Nucleosome is formed by an octamer of 8 basic proteins called histones that form an interior core, and DNA that lies on its surface. They are small, basic, very well conserved proteins in eukaryote even between distant species. The octamer is formed by 8 histone proteins: 2 copies of H2A H2B H3 and H4 for each nucleosome, they are called the core of the histone. One octamer is formed by H3-H4 tetramer associated to 2 H2A-H2B dimers.

DNA (blue) enters in contact with the octamer (yellow) and do a little bit less than 2 turns around the octamer, then exits from the nucleosomes. The length of DNA that takes contact with each histone octamer is not variable, 146 bp, the length of the DNA that lies between one and the following nucleosome, DNA linker, can vary from 8 to 114 bp per nucleosome). →

Crystal structure of the top and side view of nucleosome. In white H3-H4 tetramer, in → blue H2A-H2B dimers (only one visible in the top view). In the top view (the first) the double helices of DNA take contact with the histones octamer, you can see that there are some bases that more strictly take contact with the histones of the octamer, while those that are in the external part do not take contact directly with histones proteins. →

Histone variants

Histone variants all core histones except H4 also have histones variant, differences in sequence from canonical histone that can be found in particular nucleosomes: →

• Centromeric H3 substitute normal H3 near the centromeres. →
• H2AX present in 10-15% of nucleosomes, very important because it is a sensor of DNA damage: it is the target for ATM/ATR kinases: when it is phosphorylated, it is referred to as γ-H2AX, this modification is required to stabilise the binding of repairing factors and the cell will not divide until the DNA damage is repaired. →
• H2AZ important to allow gene expression activation, in nucleosomes which surround the promoters of active genes, a part of the promoter which is free is surrounded by a part in which nucleosomes have the variant that is evicted from the nucleosomes and allow the binding of transcription factors and RNA polymerase.
• Many other histone variants are tissue specific: the variants confer specific functions to the chromatin for the gene regulation.

Chromatin hierarchical organization

Chromatin hierarchical organization. There are different levels of organization, starting from the naked DNA double helix structure: →

• 1st level: Chromatin in nucleosomes DNA about 6 folds more compact than naked one, resulting in a 10-11 nm of diameter fibre.
• 2nd level: 30 nm fibre by coiling the 10 nm into helical array, which allows the compaction of 40 folds,
• 3rd level: 30 nanometres fibre can be compacted to form interphase chromatin (1000-fold) or mitotic chromosomes (10000-fold compaction).

Histone H1 is important to form the higher-order chromatin structures: it’s not in the octamer and it can be present or removed without affecting the nucleosome structure.

Histone modifications

They can change the function of histones and chromatin in general. Histones, like all proteins, are subjected to many covalent modifications by PTM that change the functional properties of the octamers. These reversible modifications are dynamically added and removed to regulate transcription, but also replication and DNA repair.

Considering the octamer, each of the core histones has a histone fold domain that contributes to the globular core of the nucleosome, and a flexible N-terminal tail (H2A and H2B have C-terminal tails as well) that exits from the core, which contain sites for covalent modifications that can occur also in the globular region (but mostly in the tails).

There are dots in different colours that represent different modifications that can happen. The main kinds of modifications occurring at the histone tails that can affect gene expression are: →

• Histone acetylation acetyl group is added to the amino group of a lysine residue, removing the positive charge of the lysine: this leads to a lower grade of interaction between histone tails and DNA that has a negative charge thanks to the phosphor group. This PTM is associated with regions of active gene expression, involving nucleosomes surrounding a gene promoter: after acetylation, histone tails interact less tightly with the DNA and the nucleosome can be easily removed to allow the binding of transcription factors to the promoter. Different lysine acetyltransferases KAT or histone acetyltransferase HATs acetylate different histone targets (by transferring the acetyl group), mainly H3 and H4 histone tails. The number and the position of acetylated lysins can determine of how much an acetylated nucleosome can be removed. Acetyl groups are removed by histone deacetylases (HDAC). →
• Histone methylation the main residues that are the targets of this modification are: ✓ → Lysine in the tail or core of H3 or in the tail of H4; a lysine can be mono- or di- or tri- methylated.

✓ → Arginine can occur in each of the 4 histones of the octamer, the methylation can be:

• Symmetric => 2 methyl group (di-methylation) added to different nitrogen of the guanidinium group of the Arg, this is associated to transcriptional repression;
• Asymmetric => 2 methyl groups added to a single nitrogen, linked to transcriptional activation.

Histone methylations are associated to activation or repression of gene expression depending on the specific sites that are methylated. This occurs because each specific histone modification is then recognised by specific proteins responsible of one of the 2 consequences. For example, symmetric and asymmetric methylation sites are recognised by different proteins, so they have different effects. The epigenetics marks that are present on the chromatin, in fact, are read by other proteins that mediate the changes in gene expression. In particular:

• Di or tri-methylation of lysine in H3K4 (histone 3 lysine 4), H3 or H4 asymmetric arginine di-methylation are associated with transcriptional activation, in particular if modifications are found in the chromatin that surrounds the promoter.
• Methylation of H3K9, H3K27 and also H4R3 me2s (= H4 arginin3 di-methylation symmetric) are associated to transcriptional repression.

HMT (histone methyltransferases) are the enzymes, specific for Arg or Lys, that transfer the methyl groups; histones can be demethylated instead by histone demethylases, including families of proteins. →

• Histone phosphorylation occurs mainly on serine or threonine residues.
• During mitosis → very important ser10H3 phosphorylation that correlates with mitotic progression and chromosome condensation. →
• During interphase Ser10H3 phosphorylation is linked to chromatin relaxation and gene expression. So, even in this case different reader proteins during these 2 steps of the cell cycle (mitosis and interphase) can recognise serine phosphorylation with different effects: chromosome condensation or chromatin relaxation. →
• H1 phosphorylation elimination of H1 with effects on promoting higher order structures; it seems able to favour both cell cycle progression and gene expression. →
• During DNA repair phosphorylation of the H2AX histone variant is an important signal of DNA damage with recruitment of DNA repair proteins. →
• Histone ubiquitination addition of ubiquitin groups to histones. They are small peptides of 76 amino acids, highly conserved in all eukaryotic cells. The modification occurs mainly with the bond between C terminal glycine of Ub and the N group of Lys residue on the acceptor molecule. It occurs at specific lysine residues of C-terminal tail of H2A and H2B. It is important as a signal for protein degradation, but in case of histone, there is a role in transcriptional regulation: →
• In H2A repressive effect →
• In H2B activating effect on gene expression. →
• Histone sumoylation conjugation of a SUMO protein with a histone protein. SUMO = small ubiquitin-related modifier, member of ubiquitin-like proteins involved in PTM. Histone sumoylation involves the N terminal tails of H4. It is a signal for gene silencing through recruitment of histone deacetylases and heterochromatin protein 1. →

Epigenetic memory during DNA replication

Epigenetic memory during DNA replication. Histone modifications are epigenetic modifications Figure 1 - SUMO protein because the cells have memory: there are mechanisms that maintain the histone modifications even after the DNA replication. When the cell divides, the daughter cells maintain the same histone modification. During replication, to allow the replication machinery with DNA polymerase to produce new strands of DNA, the nucleosomes are displaced into H3-H4 tetramers and H2A-H2B dimers. Then they are attached again to the new double strand DNA after the passage of replication machinery, but they are not sufficient: a double number of nucleosomes is required to maintain the same density of nucleosome after the synthesis of DNA. So, newly synthesised histones (orange) are assembled in H3-H4 tetramers and H2A-H2B dimers, but they don’t have any histone modifications. Many different enzymes that catalyse histone modifications can recognise a modification pattern on the old histones and copy the modification to the new ones.

Lezione 2 – 9 / 03

Histone code hypothesis

→ Histone code hypothesis different regions of the chromatin of the same or of a different nucleosome are characterised by different histone modifications that specify the functional status of chromatin, representing a sort of code. The presence of multiple modifications at particular sites defines the function of these chromatin domains. This can be seen as an exception of the general rule of central dogma of biology (1956) => biological info goes from DNA to m RNA to proteins. Histone code is an exception to this rule: histone modifications determine the local function of the gene contained in that region of the chromatin. Histone code has an important rule: activating modifications are mutually exclusive with inactivating events: you do not find in the same gene promoter silencing together with activating modifications, with the only exception of stem cells where this rule is not true.

Representation of a gene and histone modifications

Representation of a gene and histone modifications that occur in different gene regions affecting its expression.

Upper line => representation of a gene with gene promoter (left) and gene sequence (right) and the transcriptional starting site (arrow).

2nd line => distribution of histones: denser in the central portion of the gene because if the gene is transcriptionally active, nucleosomes are removed from the promoter; but anyway, they can be present in the internal downstream part of the gene even if it is transcribed.

3rd line => H2AZ thicker in the promoter indicates gene expression, because it is a histone variant present at the level of active promoters.

Lower lines => distribution of histone modifications with respect to the structure of the gene to show which modifications are associated to the promoter or to the gene sequence. Histone modifications have, in fact, different gene distribution and different effect depending on their position on the gene. Some examples:

H3K9 methylation => it is associated with chromatin condensation and gene silencing, when it is present in the promoter (green) the gene is inactive, when it is present in the gene sequence (orange) it is active. The same kind of modification can be associated to gene expression or silencing: why? In addition to the main transcriptional starting site, the internal part of the gene (usually introns) can contain other cryptic promoters, cryptic gene transcriptional starting sites, that can be oriented in the opposite direction. Internal transcriptional starting sites must be silenced to allow the normal transcriptional start site work: when the modification occurs in the internal part of the gene, it suppresses the transcriptional initiation of the cryptic promoters of the gene. This histone modification favours the expression when it occurs in the internal part of the gene, silence instead the gene when it occurs in the main gene promoter. So, the position associated to the modifications is important.

A similar phenomenon is observed also for DNA methylation => associated with gene repression if the methylation occurs in gene promoters, but it is not true if it occurs in the gene body.

Another feature which we should know about histone modification is that in most kind of cells activating histone modifications are mutually exclusive with inactivating modification in the same chromatin region. For example, H3K9 methylation at the level of promoter (repressive) cannot be found at the same time in the same region in the same cell together with histone acetylation (activating).

H3K36 di-methylation and tri-methylation => Histone modification to repress the expression of the transcription of the internal cryptic promoter thanks to chromatin condensation that helps the expression of the gene in the main promoter.

Figure: typical active gene.

Promoter => nucleosomes containing H2AZ variant, that allows an easy displacement of the nucleosomes, and acetylated histone tails. Histone variant H2A.Z (yellow) is preferentially found in nucleosomes that flank a