Chromatin Remodelling

The overall chromatin remodelling is not only carried out by histone modifications, but is also affected by ATP-dependent chromatin remodelling complexes (Dinant, Houtsmuller et al., 2008). The importance of these chromatin remodelling complexes is apparent, considering that 80% of the nuclear DNA is packaged by nucleosomes. For all cell processes involving DNA, like transcription, recombination, replication and repair mechanisms, the chromatin needs to be dynamic in order to give access to the genes. Chromatin remodelling therefore plays a crucial role in regulation of gene expression and misregulations.

 DNA interacts with the histones in the nucleosome by forming hydrogen bonds and salt links. ATP-dependent chromatin remodelling complexes are able to either break or reform these interactions (Kundu, Dasgupta, 2007). These complexes use the energy from ATP-hydrolysis to change either the structure or the position of the nucleosomes. These ATP-dependent changes in the chromatin structure, can help transcription factors and other regulatory proteins, which normally would be occluded by the histone proteins, to gain access to DNA sequences (Allis, 2007). The alterations of chromatin by remodelling complexes can be done in four different ways, see Figure.

Fig. Mechanisms for ATP-dependent remodelling. The change in position or composition of the nucleosomes is relative to the DNA wrapped around it. a) Nucleosome sliding to expose a region that was previously hidden. b) Histone exchange where a histone variant is transferred in to the octamer, instead of a standard histone. c) Nucleosome eviction exposes an even larger region of the DNA, with the removal of an octamer. d) Altered nucleosome structure where the path of DNA is creating a loop on the surface on the nucleosome. Modified after (Allis 2007).

                                    

The first one is sliding of the nucleosome (a), meaning that they are moved along the DNA strand (Kundu, Dasgupta, 2007). The second way is the exchange of histones within the histone octamer (b), so the nucleosome is changed to another variant. The third way, which is called nucleosome eviction (c) (Allis, 2007), is the transfer of an octamer from one part of the DNA to another part (Kundu, Dasgupta, 2007). The fourth one alters the path of how the DNA is wrapped around the nucleosome, leading to a gap between the surface of the nuclesome and the DNA strand (d) (Allis, 2007).

DNA methylation 

DNA methylation changes the interactions between proteins and DNA, which can lead to alterations in chromatin structure and either a decrease or an increase in the rate of transcription (Jones, Takai 2001). All healthy cells contain DNA, which, to some degree, is methylated.

Hypermethylation is an increase in the number of methyl groups on the DNA strand and is often linked to gene mutations and gene silencing. Hypomethylation is a decrease in the amount of the methylated DNA and is linked to DNA instability and activation of genes that are normally silenced, such as oncogenes in cancer cells (Clark and Melki, 2002). Methylation is a regulatory mechanism involved in both initiation of transcription and silencing of genes, depending on the type of methylation and the gene that is methylated (Tost, 2008).

Fig. The methylation takes place on the 5-carbon in cytosine (Laboratory 2007).

Repression caused by DNA methylation can happen directly or elaborately. The direct way is when the methyl groups inhibit the transcription factors from binding to the promoter region. The elaborate way represses DNA expression with the use of other chromatin modifying factors, which bind to methylated CpGs (Bogdanovic and Veenstra, 2009). CpG is an abbreviation for cytosine and guanine separated by a phophate and is derived from the way they are connected in the DNA strands (Allis, 2007).

DNA Demethylation

The demethylation takes place in several steps. First the methylated cytosine residue has an oxidative deamination, meaning that the amine of the 4-position carbon is changed to oxygen, and thereby the methylated cytosine becomes a thymine.(Ooi, Bestor 2008).

The deamination gives a T/G mismatch basepair, which can be restored by the DNA repair system by removal of the thymine. Then the base excision repair system inserts an unmethylated cytosine, thus ending with an unmethylated C/G basepair (Ooi, Bestor, 2008). The demethylation process is, surprisingly, thought to be initiated by the DNMT3a and DNMT3b, which are normally associated with the DNA methylation (Gehring, Reik et al., 2009). This suggests that DNMT3a and DNMT3b are involved in demethylation and methylation, which both are important mechanisms during gene transcription. Absence of the methyl donor SAM seems to favour the conversion of methylated cytosine to thymine (Ooi and Bestor, 2008).

How Epigenetics Changes Gene Function(Cont.)

Histone acetylation

Histone modifications by acetylation and deacetylation have been associated with transcriptional activity, regulated by changes in the nucleosome assembly and higher-order chromatin structure (Grant 2001). Histone acetylation involves the attachment of an acetyl group from acetyl-CoA to the α-amino group of the specific lysine (K) side chains (Tollefsbol, 2009) and is carried out by the enzyme histone acetyltransferase (HAT) (Chung 2002).  In some cases acetylation can also occur at serine (S) or arginine (R) residues. The attachment changes the positively charged residue on lysine into a negatively charged residue, which results in the histones having a decreased affinity for DNA. The consequence is that the chromatin structure opens and the DNA is thereby more accessible for transcription (Tollefsbol, 2009). 

Fig. Acetylation of the histone tails is catalyzed by histone acetyltransferase (HAT). Acetylation activates gene expression by making the chromatin structure less dense. Deacetylation is carried out by the enzyme histone deacetylase (HDAC) and results in a denser chromatin structure, and therefore no gene expression. Modified after (Yoshida, 2008).

The reverse, deacetylation, catalyzed by histone deacetylases (HDAC) (Chung, 2002), removes the acetyl groups, which results in increased affinity for the DNA. The chromatin become more condense and is thereby less accessible for transcription, see Figure (Tollefsbol, 2009). The modification can either be global, involving large parts of the chromatin, or promoter specific. The global histone acetylation concerns the general transcriptional activity, while the promoter specific acetylation is important for specific gene activity (Vaissiere, Sawan et al., 2008).

Histone Phosphorylation

Another form of histone modification is phosphorylation, which influences processes such as transcription, DNA repair, apoptosis and chromatin condensation (Grant, 2001). In mitosis the phosphorylation has an essential role, because it correlates with chromosome condensation (Hsu, Sun et al., 2000). The negatively charged phosphate groups are thought to neutralize the charge of the histone tails, resulting in reduced affinity towards the DNA. Studies furthermore indicate that phosphorylation of H3S10 induces HAT activity, leading to an additional increase in transcription activity, caused by acetylation (Grant, 2001).

Histone methylation

Transcriptional activity is also regulated by histone methylation (Grant, 2001), but this form of histone modification is more complex than the others, since it can occur on both lysine and arginine (Allis, 2007). The methylation is catalyzed by the histone methyltransferases (HMTs), which transfer a methyl group from the methyl donor S-adenosyl-L-methionine (SAM) to the residues.  Depending on the residue getting methylated, histone methylation can either enhance or repress transcriptional expression. There are at least 24 identified sites of lysine and arginine methylation on the core histones. These residues have several methylated states, which add another level of complexity (Allis, 2007). Arginine can be either mono- or dimethylated, while lysines can be mono-, di- and trimethylated (Völkela and Angrand, 2006). This gives numerous combination possibilities, which are applicable in tightly regulated processes, such as transcription (Allis, 2007).

There are six well characterized lysine methylation sites: H3K4, H3K9, H3K27, H3K36, H3K79 and H4K20. Methylation on H3K4, H3K27 and H3K79 is associated with activation of transcription, while the others have been linked to repression (Allis et al., 2007).

Histone ubiquitylation

Ubiquitylation (Ub) is different from the other types of histone alterations, mainly because of its size. Ub is a large polypeptide which increases the size, of the histone by approximately two-thirds. Ub can, as histone methylation, be either repressive or activating, depending on which histone it binds to. H2B monoubiquitylation on K123 is activating the DNA transcription and leads to H3K4 methylation, whereas H2A monoubiquitylation on K119 is repressing the transcription (Allis, 2007).