Fertilization is the fusion of sperm and egg gametes to form a zygote. In monozygotic twins (MZ) it is known that 1 fertilization event occurs producing a pair of zygotes. It was found that epigenetic modification was the main reason for the genetic differences of MZ twins with consistent genetic materials (Kaminsky et al, 2009). There is no known pathway by which this process occurs. “In mammals, methylation occurs almost exclusively at cytosine-guanine (CpG) dinucleotides which are found in high frequency at short regions knows as CpG islands” (Auclair et al 2014). Methylation of cytosine is carried out by DNA methyl transferases (DNMTs). DNMTs are necessary for many developmental processes, after two days of implantation the embryonic DNA begins to methylate. For example, DNA methylation regulates HERVWE1, a gene fundamental to the structure of the placenta (Gao et al 2012). “DNMT1 copies methylation on the new DNA strand at hemimethylated CpG sites after DNA replication, which mediates epigenetic inheritance in dividing cells” (Auclair et al.) Considering DNA methylation is one of the reasons for epigenetic differences in twins and that DNA methylation plays a role in development itself, it must play a role in the “splitting” of the blastocyst that produces two offspring.
Aim 1: “Several genes in the Wnt/ B-Catenin pathway gain exon CGI methylation” (Auclair et al 2014). Wnt and B-Catenin are signals for gastrulation and vegetal plate specification. Which genes in this pathway are methylated? Using DNMT1 as the indicator of methylation, an assay can be used to find where the protein is located in the early gastrulation stage. After determination, the genes that are methylated can be traced backwards finding a start point. For a control, antibody labeling for a set of housekeeping genes in early development can be compared to see expression differences.
Aim 2: Methylation occurs before any type of gene activation. To test this remove DNMT1 from the gene. Using HERVWE1, de-methylate it and observe what happens to the structure of the placenta. A positive control would be to allow HERVWE1 to be methylated at normal. Negative control would be to de-methylate a non-lethal gene and observe what happens.
Aim 3: Twins are supposed to have the exact same genes but expression can be different. Methylation must be independent of the normal processes of gene expression. Twins share a growth environment and exact DNA but despite this still may have phenotypic differences. Chromatin structure is blocked depending how methylated, which can block gene transcription. Looking for methylation using DNMTs, use in situ hybridization in order to locate where methylation of the gene for chromatin formation. Once located move to a different part of the body and observe functionality. Positive control is just observing normal chromatin in the brain.
Introduction
Monozygotic Twins
Sperm and egg gametes fuse in order to produce offspring known as zygote. From this one fertilization event those gametes produced two identical set of offspring. A monochorionic pregnancy is one in which twins share a placenta and diamonitic means they both have their own amniotic sac. “Approximately 80% of MZ twins originate from monochorionic/diamniotic pregnancies” (Noli et al 2015). Twins not sharing a placenta are known as dichorionic/diamniotic accounting for the other 20% of twin births. The inner cell mass splits early on in development, near the 4-6 day mark. Twins begin forming after collapsing and expanding in the blastocyst stage. There is still no known reason for this occurrence, but they share identical genes and growth environments. Despite having, the same genetic makeup differences still occur phenotypically.[image: ] Fig 1. Different types of twin births revealing the types of placenta and amniotic sac that can develop for all types of Twins. (https://eatinfor3.wordpress.com/)
DNA Methylation
DNA methylation is the addition of a methyl group to DNA normally inhibiting transcription of it due to condensing the chromatin. Identical twins are meant to share identical genes, however how much or how little these genes are expressed ultimately drives the differences in appearance. There is a difference for twins on the epigenetic level. They have been seen to have differences in X chromosome inactivation, histone acetylation and DNA methylation all of which change as age increases (Fraga et al 2005). “MZ twins have been used to demonstrate the role of environmental factors in determining complex diseases and phenotypes, but the true nature of the phenotypic discordance nevertheless remains extremely poorly understood” (Fraga et al 2005). Methylation acts to repress CpG islands of the promoter in order to block transcription. For twins with identical DNA, these processes occur independently of that fact leading to the question of why this is the case and how early on does this occur. Several DNMTs are responsible for methylation of genes. DNMT1 is responsible for methylation in early genetic inheritance and the dividing of those cells moving forward. DNMT3A and DMNT3B are “de novo” for setting up methylation early on in development. DNMT3 acts by targeting un-methylated CpG sites and creating new genes (Auclair et al. 2014). DNA Methylation is revamped during embryonic development and sperm and egg methylation sequences are nearly destroyed at the blastocyst stage. Once implantation occurs DNA methylation returns to normal levels in epiblast cells (Auclair et al. 2014). The removal of methylation, DNMT1 and DNMT3B, were lethal in mice during the mid-gestation period, while removal of DNMT3A was lethal after birth (Auclair et al 2014) which leads us to believe that methylation is necessary for early development.
“Methylation levels at different CpG sites through embryonic development. (B) Distribution on CpG methylation in RefSeq genes and 10 kb of flanking sequences throughout embryonic development. For each protein-coding RefSeq gene (excluding the X and Y chromosomes), we calculated methylation in 20 equal-sized windows within the gene and 10 1-kb windows of flanking sequences. (C)Violin plots showing the acquisition of CpG methylation in post-implantation embryos compared with blastocysts in various genome elements. TEs, transposable elements; HCPs, high CpG promoters; ICPs, intermediate CpG promoters; LCPs, low CpG promoters. (D)Pairwise comparison of CpG methylation (measured in 400 bp tiles) in E3.5 blastocysts and E8.5 post-implantation embryos. The density of points increases from blue to dark red. (E) Number of 400 bp tiles that gain or lose more than 20% CpG methylation at each developmental transition. (F) Kinetics of de novo DNA methylation in development. We selected all the genomic tiles (400 bp) that gain methylation in post-implantation embryos (defined as 50% methylation in E8.5 embryos) and then plotted their methylation as a function of the developmental stage (black line). The red line shows the methylation for 17 imprinted germline DMRs. The lines represent the median methylation and the error bars represent the 25th and 75th percentiles. (G)Kinetics of de novo DNA methylation in exons, introns and transposable elements (TEs). (H)Kinetics of de novo DNA methylation in classes of transposable elements. LINE, long interspersed nuclear element; SINE, short interspersed nuclear element. In (G,H), the lines depict the median methylation measured at each developmental stage” (Auclair et al 2014).
Nature and Nurture
Identical twins have the same DNA so it is important to look at growth environment as it effects genes. Sometimes sharing a common intrauterine factor, the placenta is important for a growing fetus to get nutrients from the mother. The syncytium is the main route for maternal and fetal interactions (Gao et al 2012). The gene HERVWE1 is important in maintaining the structure of the syncytium. HERVWE1 creates a protein known as syncytin-1, which is responsible for fusion, proliferation and immune suppression (Gao et al 2012). “Syncytin-1 promotes the merging of cytotrophoblast cells to form the syncytiotrophoblast cell, which secretes several hormones that promoting fetal growth, such as human chorionic gonadotrophin and human chorionic somatomammotropin” (Gao et al 2012). One of the most important regulations of HERVWE1 is DNA methylation. Hypermethylation diminishes HERVWE1 expression, while hypomethylation, a loss of the methyl group, increased transcription in placenta.
Experimental Method and Design
Human development is difficult to study because it cannot be directly seen and it is unethical to perform certain experiments, therefore, mice will have to act as the model system for this. Mice are similar in that they are mammals and also derive the placenta in a similar manner. Studies on DNA methylation in mice would help us to understand the role they play in embryonic development and guide us toward a mechanism for epigenetic differences.
Hypothesis A: DNA Methylation is responsible for specific pathways of embryonic development.
Certain genes in the Wnt signaling pathway gain CGI exon methylation (Auclair et al. 2014). Ransick and Davidson revealed that micromeres are responsible for vegetal plate specification and these cells through the Wnt pathway allow B-Catenin to enter the nucleus and begin transcription. Endo16 expression was the marker for vegetal plate specification that Ransick and Davidson used. To locate methylation on the genes that play a role in these early processes perform antibody staining for DNMT1. To make sure you get only the DMNT1 of the genes performing this early embryonic development, an in situ hybridization of Endo16 can help mediate so it is known that this specific methylation was present in that early gastrulation stage. A control experiment would be to assay for DNMT1 ONLY but in a different part of the cells such as the animal hemisphere.
Twenty mice in total, multiple pregnancies of 10 stained for Endo16 and DNMT1 in vegetal plate and 10 single pregnancies stained for DNMT within the animal hemisphere. The embryos would need to be sacrificed after archenteron formation so they can be observed. Using stained Endo16 we will find out how methylation affects those cells and what role they play in gastrulation.
If the proposed experiment works then location of genes with DNA methylation at those early stages would be known and from this, those genes can be, reverse derived and direct the mechanism of differential gene expression of embryos with supposed identical genes.
Hypothesis 2: Methylation must occur before any type of gene activation
Before a gene can be expressed in early development methylation of that DNA must occur. For this to be true DNA methylation has to occur before cells begin to become different. After 10 days, the chorion is embedded into the uterus and this gives way to the placenta. HERVWE1 is necessary for proper placenta formation and upkeep and this process is influenced by DNA methylation (Gao et al 2012). Gao and others found that methylation of HERVWE1 promoter resulted in higher expression of smaller twins than in bigger twins for discordant growth, which reveals methylation plays a role in twin differentiation. By doing a knockout of DNMT1, it should reveal how important methylation is to embryonic cells. By knocking out DNMT1 we would expect the cells of the trophoblast to lack the ability to form the placenta and HERVWE1 will not be present. A control for this experiment would be to run CRISPR for some housekeeping gene and knock methylation off of that.
If the results are correct then methylation is a leading factor in more than just epigenetic differences and the mechanism by which HERVWE1 is methylated will become clear.
Hypothesis 3: Twins observe different methylation patterns due to the pathway deriving it, being independent of currently known differently gene expression pathways
Twins share identical DNA and growth environments and yet still can have differences in phenotype. Chromatin structure of identical twins must be different in order them to have these phenotypic differences. How methylated each chromatin is must differ between them. Running an in situ for the genes responsible for chromatin formation, they can then be PCR assayed for DNMTs and this will reveal how methylated each one is in comparison to their twins.
Using mice with many twin embryos, determine the date of the first time methylation is witnessed and in which cells. Assay for DNMTs in those cells and locate the genes being methylated. Using CRISPR removes those genes earlier than in the previous step and see if methylation occurs. A control for this would be to pick known genes without signs of DNMT activity early on and knock them out too. Expected results would be the pathway for DNA methylation as a whole and where it is derived and it would prove the hypothesis to be true.
References
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