Epigenetics is a term that was first defined by Doctor C.H. Waddington in 1942 to explain the interactions between “genes and their products, which bring the phenotype into being.” (20). Nowadays the term ‘epigenetics’ is used to coin all the genome modifications that cause differential gene expression, independent from changes in the DNA sequence (21). Some of the mechanisms included in epigenetics cause regulation of gene expression through changes in the level of chromatin condensation, changes in the mRNA resulting from transcription or changes in the DNA itself - DNA methylation. Since the DNA contains the genetic information that codes for chromatin condensation and mRNA synthesis, DNA methylation can be linked to modifications in the chromatin and RNA therefore resulting in cell-specific gene expression (22).
The epigenetic mechanisms that allow changes in the degree of relaxation of chromatin are commonly known as chromatin remodeling and can directly influence DNA transcription. The nucleosomes are composed of 147 bp of DNA wrapped around histones. If the wrapping is tight, the DNA is less likely to be available for transcription (19). The tightening of the chromatin fiber can be promoted by the presence of different modifications on the histone tails, which are mostly composed of residues of arginine and lysine (5). The specific modifications on the histone tails occur post-translationally and can be methylation, acetylation, phosphorylation, propionylation, butyrylation,
tails is known as ‘histone marks’ and a combination of several histone marks leading to a gene regulation event is known as ‘histone code’ (23).
One of the most studied examples of histone modification is the methylation of lysine (K) residues in tails of histone H3 that can either cause gene repression when tri-methylation occurs on lysine 9 of histone 3 (H3K9me3) or gene activation for H3K4me3 (24). Another type of histone modifications intimately related to gene expression consists on the presence of histone variants. Some histones contain differences in few amino acid residues when compared to the canonical histones. For example, in mammals there is a known variant of the histone H2 called H2A.X. When H2A.X is phosphorylated, it marks a double strand DNA break. Another variant known as H2A.Z, which is widely present at transcription start sites, shows a negative correlation with the existence of DNA
methylation. Even though information regarding the mechanism of action of these variants is limited, the existence of dedicated mechanisms responsible for the removal and addition of these histone variants into the nucleosome further reinforces the importance of these histones in gene expression (22).
Gene expression can also be regulated at posttranscriptional levels through RNA. Two main mechanisms are known to date: one is related to the RNA secondary structure and the other related to the binding of small regulatory RNAs to the mRNA.
Regulation through the RNA secondary structure occurs when a specific factor binds to certain mRNA transcripts preventing them from unfolding thus preventing translation. For example, in eukaryotes several proteins such as ferritin mediate the harmful accumulation of iron. Ferritin is an iron-storage protein mainly found in the liver and kidneys. The ferritin mRNA can only be translated when the 5’-end stem-loop is free
from another protein called IRP. Since iron binds to IRP strongly, in high concentrations of iron, the IRP preferentially binds to iron releasing the ferritin mRNA and promoting translation for higher protein availability (5).
Another type of gene expression at the RNA level can occur when specific mRNA transcripts are deleted. The deletion of mRNA after it has been transcribed occurs when certain small RNA – called micro RNAs (miRNA) – are transcribed from specific genes. After being synthesized the miRNAs bind specific proteins from the Argonaute family, which have the capacity to degrade mRNA transcripts complementary to the miRNA sequence. Since each miRNA sequence can be complementary to several mRNA transcripts, several types of mRNAs can be degraded in this manner (5). Recent discoveries show that this form of gene silencing mediated by miRNA can establish a repressed chromatin state through back-signaling to DNA. Such chromatin state
represents a more stable gene silencing mechanisms since it can be propagated through several cell divisions (22). Additionally, small nuclear RNAs can also operate through transcriptional gene silencing (TGS, see Figure 1.2) pathways. It has been proposed that in order to defend the organism from harmful DNA elements introduced by viruses, small RNAs can interact with effectors of histone lysine methylation pathway to silence the extraneous DNA (22).
The third type of epigenetic modification, the DNA methylation, has been widely studied. In humans methylation occurs primarily in the 5’ carbon of cytosines followed by guanines (CpGs) and because of the large effect of that methyl group in gene
expression, methylated cytosines have been referred by some as the 5th nucleotide (25). In fact, DNA methylation plays important roles in several genetic processes such as
repression of transposons and repetitive elements, genomic imprinting and X-
chromosome inactivation, where one of the X chromosomes is silenced in every somatic cell of female mammals (21, 26).
Figure 1.2 – Schematic representation showing all the genomic and epigenomic factors and mechanisms involved in gene regulation. The center of the figure represents 4 nucleosomes and the main mechanisms for epigenetic regulation such as DNA methylation (red circle with Me), histone modifications (Mod), histone variants (yellow nucleosome) and non-coding RNA (wavy blue lines). The brown boxes display genomic functions that affect epigenome modifications and help in its regulation. The main factors needed for genomic functions are shown near the boxes. Adapted from (22)
The close physical relationship between DNA and histones in the nucleosomes precludes possible correlations between the different states of chromatin and DNA methylation. Ongoing research aims to further draw a mechanism of how such
interactions affect gene expression. In 2010 direct evidence was found that connect the methylation status of CpGs to recruitment of proteins able to mediate modifications in the
histone tails. One example is the specific recognition and binding of protein CXXC-type zinc finger protein 1 to unmethylated CpGs which promotes the recruitment of enzymes called histone lysine methyltransferases (KMTs). Methylation of histone tails on their lysine residue by KMTs seems therefore to be influenced by unmethylated cytosines on associated DNA regions (27).