Dna Methylation Changes In Development

Published Date: 02 Nov 2017

Abstract

DNA Methylation, while poorly understood, is becoming more and more important in our studies of genetics and epigenetics. We are now beginning to realise that various DNA methylation events are much more important than was previously thought, playing roles in development for both the better and for worse.

With this in mind, this paper was written in an attempt to highlight what we currently know about the process (particularly in embryonic development) and to show the mechanisms behind the action of the methylation events.

Introduction

DNA Methylation is an epigenetic process by which CH3 (i.e. methyl groups) can be added onto a cytosine or adenine nucleotide in DNA. This addition can cause changes in the cellular phenotype and can affect gene expression.

This can happen during various stages of embryogenesis, and is hence a vital process for the correct development of an embryo. Methylation is involved in many key developmental events, including Genomic Imprinting, Gene Regulation, X-Chromosome Inactivation, and even some more questionable events such as Angiogenesis.

The presence or absence of certain methylation patterns during development have even been linked to a variety of diseases and abnormalities, including Fragile-X syndrome, facioscapulohumeral muscular dystrophy (FSHD), and various other disorders.

By investigating some of the patterns of methylation chronologically, some insight can be gained regarding how all of the above processes can occur and what effects they may have.

DNA Methylation Process

DNA Methylation is a vital process in embryogenesis. It can also occur in adult cells, but it is generally associated more frequently with the correct development of an embryo. Certain patterns of Methylation have been observed, and their effects noted, though before the various patterns of DNA Methylation are discussed, it is important to be aware of how the process happens.

A group of enzymes known as "DNA Methyltransferases" catalyse the addition of methyl groups to DNA by taking a methyl group from a molecule called S-adenosyl methionine (SAM for short) and attaching it to a specific nitrogenous base at a specific site in the DNA.

These Methyltransferases are a broad group of enzymes, and can be further classified by the specifics of the reactions they catalyse. For example, the "m5C Methyltransferase" enzyme attaches the methyl group to the fifth atom in the six atom ring of a cytosine, resulting in the formation of 5-Methylcytosine. It is important to note that the methylation process does not alter the sequence of nitrogenous bases, but it can affect gene expression. The methylation of CpG Island regions in the genome is a classic example of how methylation can affect gene expression.

CpG Island Methylation - Effects

CpG Islands are regions of the genome that contain significant amounts of Guanine and Cytosine dinucleotides (~60% of a given region). These genomic regions are important with respect to DNA Methylation because the soon-to-be mentioned methylation events and patterns usually occur at either side of these regions in close clusters, though not often in the regions themselves. This implies that if a gene is to be expressed, the CpG regions generally remain unmethylated. Hence, CpG methylation is thought to inhibit the expression of a given gene.

As you’ll later see, various mass-methylation events occur during development. So, to allow certain genes to be expressed, these CpG islands will need to be protected during these events, otherwise they will become methylated and inhibit gene expression. The fact that almost all unmethylated CpG sites are regions initiating the start of gene transcription further indicates that the unmethylated regions are vital in gene expression.

The exact mechanism of the CpG protection is unknown, though experiments have been performed using embryonic stem cells, which have demonstrated their ability to somehow recognise and protect these CpG islands. Further study is needed to fully clarify how and exactly why this happens.

http://missinglink.ucsf.edu/lm/genes_and_genomes/images/methylation1.png

Figure 1: The above diagram clearly shows how gene expression can be affected by various DNA Methylation events. The CpG islands, when methylated, repress gene expression, and vice versa.

Erasure - Methylation Event in Development I

One of the first methylation events to occur in embryonic development is the process of erasure. Methyl groups are inherited from parental gametes, but are mostly erased during the morula / blastula stages of development, possibly as a result of restriction enzymes acting at the DNA sites. From an epigenetic perspective, this is a topic of particular interest as the methyl groups are inherited, yet removed.

Erasure is seemingly a two-step process. The first step, which occurs when the organism is still a zygote, is characterised by the mass demethylation of the paternal genome.

The second step occurs slightly later in development and sees further demethylation taking place throughout the organism during early successive replication phases of the embryo, possibly as a side-effect of the DNA-(cytosine-5)-Methyltransferase-1 enzyme (encoded by the DNMT1 gene) migrating from the nucleus of a cell into the cytoplasm.

The exact mechanism of this mass demethylation has yet to be conclusively accepted by scientists. As mentioned above, there seems to be a link between the migration of the DNAC5MT1 enzyme and the demethylation occurring, though more research is needed.

Generation of a Bimodal Pattern – Methylation Event in Development II

Shortly after erasure, around the time when the embryo becomes implanted into the wall of the uterus, the organisms’ genome becomes mass-methylated. Notably, the methylation pattern here is observed as the addition of methyl groups to new positions on a DNA molecule, implying that this is the first independent, non-inherited methylation event for the organism in question. Two enzymes called DNA-(cytosine-5)-methyltransferase-3 α and ß (encoded by the DNMT3 α and ß genes) are present in abundance during this phase of development and hence are suspected to be related to the process. The enzymes are particularly concentrated at the anterior pole of the embryo.

Most sequences become highly methylated (to over 80%) during this event. A bimodal (double-peaked) distribution of methylated DNA regions can be observed when this is graphed. The reason for the two peaks is apparently due to CpG Islands not being methylated (which is related to gene expression – see the appropriate above sections) which causes a dip in the middle of the graph.

It is assumed that this pattern is preserved throughout multiple cell divisions, as the pattern can be observed in mouse and human embryonic stem cells, which have been harvested and monitored directly during this developmental phase. A proposed reason for this is because the cells in question are derived from the earlier, inner cells which were present at the time of implantation.

Genomic Imprinting – Methylation Event in Development III

Genomic imprinting is a process that occurs during development which aims to achieve the expression of only one allele in a given allele pair. Imprinted genes in allele pairs are silenced (i.e. inactivated), leaving the other non-imprinted gene to function. Having zero or two active copies of an imprinted gene can cause complications, so it is a necessity.

Most imprinted genes have important roles in embryonic growth, such as in the development of the placenta, though many perform their roles earlier in embryogenesis. They are also related to gene expression, regulation and have been linked to certain diseases if there are abnormalities as a result of imprinting.

In order for imprinting to occur, it is thought that the DNA must be methylated in or near the previously mentioned CpG Islands. This is because the majority of imprinted genes test positive for high levels of methylation. However, certain imprinted genes (such as the KIP2 human gene) do not exhibit many signs of methylation. Nonetheless, due to the fact that most imprinted genes are highly methylated, DNA methylation events probably do have effects on gene imprinting, even if it’s in a more subtle manner than was originally thought.

X-Chromosome Inactivation – Methylation Event in Development IV

X-Inactivation is a female specific, random and (usually) permanent process by which the paternally donated copy of the X chromosome becomes inactivated during development, resulting in only the maternal copy being functional and producing gene products. This chromosomal inactivation occurs in an imprinted manner. It is an important process as it prevents females from producing twice as many X-chromosomal products as males.

During the inactivation of the chromosome, the cells chromatin gets converted into a substance known as heterochromatin, which is when Histone H3 at position Lysine 9 becomes methylated. This heterochromatin performs several key roles, including the regulation of genes, which probably helps to inhibit the X-Chromosome via methylation.

The cells methylate the paternally donated X-Chromosomes when the organism is a blastocyst with the intention of inhibition. Methylated CpG Island regions have been observed on the inactive chromosome, which is thought to prevent the DNA from being transcribed into RNA, as this is one of only very few differences between the two chromosomes. Plus, CpG Island Methylation has already been linked with inhibiting the expression of genes, so it seems a very plausible explanation.

Tissue Specific Methylation Patterns

Experiments have been performed detailing the methylation levels on human chromosome 1 in various organs in the body. Tissue samples from male and female donors were cross examined and comparatively analysed. One particular test in the experiments performed by Trask was a comparison between spleen tissue and numerous other organ tissues to evaluate their relative methylation levels. The tissue comparisons were all from the same male donor, with the exception of the ovarian tissue sample.

The results obtained (methylation profiles) show that the methylation of chromosome 1 does in fact vary from organ to organ. It is also hypothesised that the DNA Methyltransferase enzymes receive signals from cells during embryonic development. These signals supposedly notify the enzyme of the future cell-fate, and hence affect its action based on the signal. If this is correct, it would probably account for the varying levels of methylation in different tissues.

Another interesting result found that the methylation profiles of various brain lobes from the one donor were very similar. A correlation was observed between the four brain tissue samples. These results indicate that there is a difference between the levels of chromosome 1 methylation in different tissues. The spleen comparisons show that the overall methylation levels differ, whereas the brain comparisons show consistencies with a given organ (Trask 2009).

From these tests (along with others performed as part of the same experiment), it was also deduced that an outlier in the methylation ratio data always corresponded to a gene with an outlying expression ratio. These tests also proved that genes in less methylated regions often had much higher levels of expression.

Postimplantation Methylation Changes

Once the embryo is implanted, other methylation events can occur. However, most of the mass-methylation is over by now, and all of the methylation patterns from this point on are either tissue or gene specific.

Genes maintaining pluripotency need to be silenced by methylation, as undifferentiated embryonic stem cells will need to differentiate shortly after implantation. One such gene that needs to be silenced is NANOG, which is an activator for the Rex-1 promoter sequence, as Rex-1 is a pluripotency marker. Hence, the NANOG gene is inactivated by a gene-specific methylation event shortly after implantation.

Another gene which must be silenced by methylation is the POU5F1 gene, which encodes for the Oct-4 protein. This protein also plays a role in the regulation of Rex-1. It has been noted that both over and under expression of this can cause embryonic stem cells to differentiate. Hence, it must be stringently regulated.

Fragile-X Syndrome – DNA Methylation Events in Human Disease I

Now that we understand the importance of methylation in development, it is important to know the consequences of errors in the processes. Fragile-X syndrome is an x-linked disorder occurring on the Xq27.3 gene, resulting in severely hindered mental development. The disease is understood to be linked to a mutation in the 5’ untranslated region of the FMR1 protein, which is essential in the cognitive development of an embryo.

The mutation occurs at a particular CGG repeat in the above-mentioned protein sequence. Normally, there are between 6 and 52 copies of the CGG repeat in cells, though this number increases to over 200 copies in affected individuals, and slightly more or less in individuals suffering from other diseases. This increased expression of the trinucleotide can hinder cognitive development.

Most of these CGG sequences become methylated in affected individuals, which interestingly also results in the gene coding for the FMR1 protein to be silenced. The methylation causes the protein to become inactivated, and hence allows room for errors and room for developmental abnormalities to occur. Specific methylations of these trinucleotides can also result in different abnormalities.

Figure 2: The above diagram shows the CGG repeat sequence at the specified location being copied numerous times. Notice that the DNMT1 (Methyltransferase enzyme) is present, which of course implies methylation did occur and is a cause.

FSHD – DNA Methylation Events in Human Disease II

Another disease caused by errors in the methylation process is facioscapulohumeral muscular dystrophy (FSHD), which is an autosomal dominant disease causing muscular weakness, particularly around the face. The disease corresponds to the 4q35 genomic position, whereby repeats in the D4Z4 region appear to undergo deletion.

It is supposedly caused by a hypomethylation event, whereby the transcription of one or more genes either side of the repeat are either disrupted or overexpressed as a result. This is thought to result in the possible contraction or deletion of the region, which in turn can lead to FSHD.

The SLC25A4 gene (in the D4Z4 region) can cause cells to undergo apoptosis when it’s overexpressed – a common molecular symptom of FSHD. The exact genetic mechanism of the disease is not 100% clear, though a hypomethylation event would cause gene regulation to degenerate and allow the overexpression of certain genes. Hence, this seems like a probable cause of the disease.

http://ars.els-cdn.com/content/image/1-s2.0-S0959437X04000565-gr4.jpg

Figure 3: The diagram shows the deletion of the D4Z4 region. As mentioned above, it is thought that a hypomethylation event is responsible for this, as well as FSHD development.

Methylation Events in Mammalian Cloning

Mammalian cloning has been attempted numerous times with varying degrees of success. Natural DNA Methylation patterns are one (of many) barriers when attempting to successfully clone a mammal. This is particularly true for the method of "Somatic Cell Nuclear Transfer" (SCNT), whereby a nucleus from an adult somatic cell is transplanted into a donated female gamete with the nucleus removed. This cell then undergoes cell division and is implanted into a uterine wall.

However, there is a high mortality rate post-implantation, as a number of things can go wrong. Errors in the methylation process are (among a wide variety of other factors) one such cause of death. The process of erasure, where the entire genome becomes demethylated in naturally fertilised embryos, occurs irregularly in SCNT cells. This usually results in erasure happening too early.

As a result, later methylation events happen too early, and certain layers of the embryo will not have enough time to undergo the demethylation they need. This hypermethylation can cause numerous problems for the developing embryo. Hence, working out ways to combat this irregular methylation scheme will be an essential step in making any future SCNT tests more successful.

Summary

The process of DNA Methylation is vital to the development of an organism. The presence and absence of certain methylation patters is closely monitored by cells, as any minor changes in these patterns can result in abnormalities. These in turn could be detrimental to the fate of the organism.

Genes can be inactivated by DNA Methylation, which can be used to the advantage of a developing cell. However, if there are errors in the methylation process, an important gene could become inactivated, resulting in the underproduction of a possibly vital protein. Of course, this can be fatal and is hence strictly controlled.

The exact mechanism of certain methylation processes is still unclear. We can see this evidently in the numerous attempts at mammalian cloning, which were not always successful due to these processes not being fully understood.

Conclusion

In conclusion, the methylation events discussed are crucial in embryonic development. While there is still a lot to understand about the processes, it cannot be argued that they play a number of important roles which form the basis for the developmental process to succeed.