Structural Biochemistry/Epigenomics

Introduction
Derived from the Greek, epigenome means "above" the genome. The epigenome consists of chemical compounds that modify, or mark, the genome in a way that tells it what to do, where to do it and when to do it. The marks, which are not part of the DNA itself, can be passed on from cell to cell as cells divide, and from one generation to the next.

Effects of Epigenome
The protein-coding parts of your genome, called genes, do not make proteins all of the time in all of your cells. Instead, different sets of genes are turned on or off in various kinds of cells at different points in time. Differences in the types and amounts of proteins produced determine how cells look, grow and act. The epigenome influences which genes are active — and which proteins are produced — in a particular cell. So, the epigenome is what tells your skin cells to behave like skin cells, heart cells like heart cells and so on.

Epigenetic Modification
Epigenetics involves heritable gene functions that are not attributed to changes in the cell’s DNA sequence. Some known examples of epigenetic modifications include DNA methylation and histone modification.

DNA Methylation


DNA methylation is the process of adding a methyl group to a DNA sequence. This reaction is catalyzed by an enzyme known as DNA methyltransferase (DNMTs). There are two types of DNMTs: De novo DNMTs and Maintenance DNMTs. De novo DNA methyltransferase enzymes initially attaches the methyl group to a DNA strand. Maintenance DNMTs copy the methylation from an existing DNA strand onto the complimentary strand after replication. DNMTs recognize regions where a cytosine nucleotide is placed next to a guanine nucleotide along the base sequence. This region is known as CpG, where a phosphate group binds a cytosine to an adjacent guanine.

DNA methylation processing is used to repress gene expression. The methylation blocks the promoter site at which transcription factors bind to, thus stopping the gene expression. Though DNA methylation is very stable, the reaction can be reversed by a different group of enzymes.

De Novo Methylation in Cancer
Alterations to DNA methylation patterns play a major role in the onset of cancer. In fact, all tumors that have been studied exhibit changes in DNA methylation. Early studies seem to suggest that widespread demethylation is characteristic of cancer. One type of methylation pattern is called de novo methylation. In this process, the polycomb complex (PRC2) targets genes that, under normal conditions, have unmethylated CpG island promoters. These genes are repressed due to the methylation of H3K27. Methylation of H3K27 is mediated by the histone methylase EZH2. The methylated H3K27 then binds to the chromodomain-containing complex (PRC1), thereby giving rise to a type of heterochromatin. During normal development, targeted de novo methylation can take place at certain sites on DNA. Similarly, it can occur abnormally in cancer. The explanation offered for this phenomenon suggests that the histone methylase EZH2 recruits Dnmt3a and Dnmt3b. Some studies show that the polycomb complex PRC2 may be released from the methylated islands. This event results in a more stabilized form of methylation-mediated repression, which occurs instead of the flexible polycomb repression mechanism.

Many believe that de novo methylation is a completely random process. This theory helps to explain the presence of methylated tumor suppressor genes in some cancer types. However, more recent research reveal DNA methylation in cancer to involve hundreds of CpG islands that are methylated in a more pre-determined way. Under the older theory, it is believed that DNA methylation causes genes to become inactivated. In contrast, the newer theory revealed that the genes targeted for methylation have already been inactive. Many of these targeted genes have a polycomb complex nearby, which may explain how the methylases are recruited to carry out the gene modification. Thus, the newer theory based on programmed methylation suggests that de novo methylation in cancer occurs on gene sites that were targeted regardless of whether the genes are active or inactive. The target sites are also independent of whether the genes affect tumorigenesis. Nonetheless, studies on human colon cancer reveal the possibility that DNA methylation can cause a more permanent repression mechanism than that of the normally flexible polycomb repression mechanism; this observation links DNA methylation to tumorigenesis.

Fragile X Syndrome
Fragile X Syndrome is a developmental disease that eventually causes mental retardation. This disease occurs when abnormal de novo methylation causes the repression of the FMR1 gene early in development. Abnormal de novo methylation is believed to be the cause of Fragile X syndrome due to the fact that inactivation of the FMR1 gene is observed to occur alongside a process known as H3K9me3 heterochromatinization. From these studies, scientists believe that the programming of all abnormal modifications may proceed through a single mechanism in which histone methylases mediate random DNA methylation and repression.

The Demethylation Pathway
Active demethylation occurs in a step-wise manner that results in a modification of 5-methylcytidine (5mC). This modification allows the molecule to be recognized for removal and replacement by repair; the modified 5-methylcytidine is replaced with unmethylated cytosine. The following steps depict the pathway suggested for active demethylation:
 * 1) Hydroxylation: An enzyme known as ten-eleven-translocation (Tet) catalyzes the hydroxylation of the methyl group on 5-methylcytidine, thereby forming 5-hydroxymethylcytidine (5hmC).
 * 2) Deamination: Activation-induced deaminase (Aid) or Apobec family proteins replaces the amine group of 5-hydroxymethylcytidine with a doubly bonded oxygen atom. This new molecule is called 5-hydroxymethyluridine (5hmU).
 * 3) Glycosylation: A glycosylase (Mbd4 or Tdg) essentially removes the 5-hydroxymethyluridine structure from the sugar-phosphate backbone. What is left is an apyrimidinic acid residue.
 * 4) Repair: The apyrimidinic acid residue is replaced with cytosine (C) through a repair process such as nucleotide excision repair (BER or NER).

Histone Code
The hypothesis of the histone code, a recent postulation by Strahl and Allis, suggests that it is a number of histone modifications at a particular locus that is involved in determining what genes are expressed or suppressed. This expression is a heritable epigenetic factor that allows regulatory proteins to have an effect on gene expression. By changing the charge of histones or the addition of structural proteins, genes can be essentially turned on or off. But the question of whether it is the combination of histone modifications working in concert or a cumulative effect of numerous modifications is a topic of current debate. Histone modifiers consist of residue specific proteins and reader proteins allow for these epigenetic changes to be transmitted post-translationally.

Transcriptional Histone Modifications
Acetylation of histones has been associated with increased transcription of specific genes. Several acetyltranserfase compounds have been documented that allow acetylation of histone complexes and increase genetic transcription. Using genome mapping techniques, it has been shown that the most acetylated regions are responsible for the highest rates of transcription. However, the acetylation is relatively dynamic, often only lasting for several minutes. This suggests that it is the frequent acetylation that has a role in the promotion of a genetic region rather than simply the presence of acetylated histones.

Lysine methylation in histones that leads to decreased transcriptional activity is much longer lived in relation to acetylation. Mono, di, and tri-methylation is part of an indexing system that is involved in epigenetic control and each manner of methylation has a different longevity. Additionally, methylation has a longer turnover rate than phosphorylation.

Serine, Threonine, and Tyrosine can be phosphorylated as another impermanent epigenetic control factor that occurs on histone tails. During mitosis, core histone tails are occasionally phosphorylated in a process that is initiated then spreads throughout the genome. Phosphorylation has been linked to acetylation and other post transcriptional modifications.

Histone assembly and deposition
As chromatin is assembled, histones are constructed in the cytoplasm and transported through the nuclear membrane to be deposited on DNA. Generally, histone assembly and placement is concurrent with S-phase progression. Histones are initially modified on their journey into the nucleus and placed at the replication fork of the chromatin then further modifications are made within the first few minutes of placement. However, further modifications like the methylation of lysine occur after longer periods of time, indicating the importance of epigenetic changes.

Inheritance of the epigenetic code
For histone modifications and the epigenetic code to be heritable, the cell must be able to initiate modification, alter modifications, and transmit modifications to future copies of the genetic code. As mentioned previously, acetylation of histones has a rapid turnover, which does not strongly suggest a lasting or heritable epigenetic control. Even in situations where acetylation is observed for longer durations, it is usually seen to attract proteins responsible for increasing levels of acetylation, and is not likely responsible for the heritability of epigenetic codes. Methylation, however has been observed to have a prolonged binding and increased stability of reaction and could be responsible for a transmittable epigenetic code. Although it is still to be determined if there is a “histone code” that carries epigenetic changes as DNA is copied, a code based on methylation of histones is the most likely type of histone modification to be involved.

Epigenome and cancer
Cancers are caused by a combination of changes to the genome and the epigenome.

Adding or removing methyl groups can switch genes involved in cell growth off or on. If such changes occur at the wrong time or in the wrong cell, they can wreak havoc, converting normal cells into cancer cells that grow wildly out of control.

For example, in a type of brain tumor called glioblastoma, doctors have had some success in treating patients with a drug, called temozolomide, that kills cancer cells by adding methyl groups to DNA. But that's only part of a very complex picture. Cells also contain a gene, called MGMT, that produces a protein that subtracts methyl groups — an action that counteracts the effects of temozolomide. In some glioblastomas, however, the switch for the MGMT gene has itself been turned off by methylation, which blocks production of the protein that counteracts temozolomide. Consequently, glioblastoma patients whose tumors have methylated MGMT genes are far more likely to respond to temozolomide than those with unmethylated MGMT genes.

Changes in the epigenome also activate growth-promoting genes in stomach cancer, colon cancer and the most common type of kidney cancer. In other cancers, changes in the epigenome silence genes that normally serve to keep cell growth in check.

To come up with a complete list of all the possible changes that can lead to cancer, the National Institutes of Health (NIH) has started a project called The Cancer Genome Atlas. Beginning with glioblastoma, these researchers are comparing the genomes and epigenomes of normal cells to those of cancer cells. They are looking for any changes in the DNA sequence, called mutations; changes in the number and structure of chromosomes; changes in the amounts of proteins produced by genes; and changes in the number of methyl groups on the DNA.