The era of genetics began more than a century ago with the findings of an Austrian monk, Gregor Mendel, who worked on the selective breeding of garden peas. Mendel's work demonstrated the predictable heritability of discrete traits and led to the Mendelian Laws of genetic inheritance. However, Mendel's Laws are not always observed in mammals, e.g. genetically identical twins can sometimes have different eye colours.
A variation on Mendel's Laws arose in 1942 with the work of Waddington (Waddington, 1942) who introduced the term epigenetics. He proposed that not only the genes themselves, but the switching on and off of these genes can result in permanent changes to the cellular differentiation path. A few decades later, studies in plants, yeast and mammals (Chandler and Stam, 2004, Grewal and Klar, 1996, Morgan et al., 1999) provided strong evidence for a non-genetic (i.e. epigenetic) basis of phenotype determination.
Epigenetic mechanisms and marks
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Today, the most widely accepted definition of epigenetics is the study of mechanisms that alter phenotypes without a change in DNA sequence. DNA methylation and histone modification are the primary epigenetic modifications involved in the regulation of gene expression in mammals.
DNA methylation, which consists of the addition of a methyl group at cytosine residues, is probably the most extensively studied mechanism of gene repression. In mammals, DNA methylation occurs mainly at CpG dinucleotides and is catalysed by DNA methyltransferases. DNA methylation can be maintained in somatic tissues by the maintenance DNA methyltransferase, DNMT1, making it a highly stable silencing mark that is not easily reversed. Additionally, the de novo DNA methyltransferases, DNMT3a and DNMT3b, establish DNA methylation patterns during early embroygenesis (Law and Jacobsen, 2010).
Modification of the proteins that package the DNA into a highly condensed complex is another way to regulate transcriptional states. The N-terminal tails of histone proteins can be modified in different ways, including acetylation, methylation, phosphorylation and ubiquitylation, thereby influencing chromatin structure and gene function. Repressive histone modifications (e.g., methylation of histone H3 at lysine 9) are typical of silenced chromatin, whereas active histone modifications (e.g., acetylation) are associated with transcriptional activity (Campos and Reinberg, 2009). The opposing actions of histone methyltransferases and histone demethylases as well as histone acetyltransferases and histone deacetylases make these marks reversible and therefore more dynamic.
Recently, another important mechanism for epigenetic regulation of gene expression, which involves RNA, was identified. RNA silencing mechanisms use small RNA molecules, 20-30 nucleotides in length, to regulate gene expression on different levels: from chromatin structure and organization, through mRNA localization and stability, to translational control (Ghildiyal and Zamore, 2009).
Epigenetics and Phenotype
For the whole organism, it is important to ensure that genes are expressed/repressed at the correct time and place throughout development, and it has become clear that epigenetic modifications play a crucial role in the determination of the phenotype. Dysregulation of gene expression (eg. through disruption of epigenetic mechanisms) can lead to developmental defects and disease. For example, the MECP2 gene (methyl-CpG binding protein 2) encodes MeCP2 protein (a 5-methylcytosine binding domain protein, MBD protein), which is essential in forming synapses between nerve cells as well as silencing several other genes. Its silencing effect is exerted through interaction with chromatin remodelling proteins. Mutations in MECP2 lead to Rett syndrome, a neurodevelopmental disorder, which is female-specific and characterised by pleiotropic abnormalities due to over-expression of MECP2-regulated genes (Tao et al., 2009). A deeper understanding of epigenetic mechanisms should lead to improvements in preclinical diagnosis and treatment of patients.
In general, the epigenetic modifications are set up early in development and stably maintained throughout the life of an organism, and these modifications are usually cleared on passage through the germ line (Chong and Whitelaw, 2004). It is known that the establishment and maintenance of the modifications is often stochastic. For example, at any one locus, the precise patterns of DNA methylation vary from nucleus to nucleus even among cells of the same tissue-type (Lorincz et al., 2002, Warnecke and Clark, 1999). In some cases, this difference in the epigenetic state correlates with the locus being active in some cells and inactive in others, resulting in a variegated phenotype. For instance, ivy leaves display a variegated phenotype as different intensities of green colour, including light green and dark green, are shown on individual leaves. These epigenetic states can be inherited through meiosis resulting in transgenerational effects (epigenetic inheritance) (Morgan et al., 1999, Rakyan and Whitelaw, 2003, Sutherland et al., 2000).
Always on Time
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Another example of the stochastic behaviour of epigenetic state is variable expression of phenotype observed in laboratory animals, where genetic variation is reduced through inbreeding and external environmental influence is minimised. For example, genetically identical mice carrying the agouti viable yellow allele (Avy) show a spectrum of coat colour phenotypes from completely yellow, through yellow/agouti patches (mottled) to completely agouti (Morgan et al., 1999). Individuals that have mottled coats are examples of the phenotype variegation discussed above. The agouti gene encodes a paracrine signalling molecule, which causes the production of yellow pigment in the hair. In mice carrying the Avy allele, expression of the agouti coding sequence is controlled by a retrotransponson intra-cisternal A particle (IAP) that has integrated upstream of the agouti promoter. Mice constitutively expressing Agouti display a completely yellow coat colour and develop diabetes and obesity later in life (Duhl et al., 1994). Expression from this retrotransposon can be epigenetically silenced in some cells producing mottled mice. The silencing correlates with different levels of cytosine methylation at the Avy allele (Morgan et al., 1999). The Avy allele is called a metastable epiallele to emphasize the fact that its activity is dependent on epigenetic state and that this state is to some degree unstable (Rakyan and Whitelaw, 2003).
Transgenes are particularly susceptible to variegation, variable expressivity and transgenerational epigenetic inheritance, and can be used to study these mechanisms. For instance, the analysis of transgene expression has contributed to the discovery of parental imprinting in mice (Chaillet, 1994).
An erythrocyte-specific transgene containing a green fluorescent protein (GFP) reporter gene has been used to study epigenetic inheritance in the mouse (Preis et al., 2003). The GFP expression in erythrocytes can be measured by flow cytometry, a fast and extremely accurate method on a cell-by-cell basis. Mouse lines were produced and maintained in the inbred FVB/N strain to ensure that individuals are isogenic. Nine mouse lines were produced with some of them showing variegation of GFP expression. Others expressed the transgene in the majority of cells(Preis et al., 2003). It is likely that these differences are related to the integration site or the copy number of the transgene in the different mouse lines (Garrick et al., 1998).
Development of a random mutagenesis screen
Epigenetics is an emerging field and little is known about the mechanisms by which epigenetic states are established. More studies will help us to get a better understanding of the nature of epigenetic inheritance as well as phenotype determination. The identification of genes involved in the establishment and maintenance of epigenetic states will be a start to the characterisation of the mechanisms involved.
One way to identify the function of genes is by disruption of the normal gene function and observation of the resultant phenotype. There are different approaches to prevent or reduce the expression of a gene, including gene knockdown and gene knockout which require the DNA sequence to be known. To identify novel genes that play a role in a particular mechanism, a forward genetic approach that relies on the generation of mutant phenotypes in order to identify the underlying genes is necessary.
A sensitized screen to identify genes involved in gene silencing, using random N-ethyl-N-nitrosourea (ENU) mutagenesis in an inbred mouse line (FVB/N strain) has been developed in Professor Whitelaw's lab. Similar screens have been conducted in Drosophila to identify genes involved in position effect variegation (PEV) (Schotta et al., 2003).
The Drosophila screen used the white (w) locus that, upon rearrangement and positioning adjacent to pericentric heterochromatin, is expressed in a stochastic and variegated manner. Random mutagenesis produced phenotypes that were classified into two groups; suppressors of variegation (Su(var)), showing an increased proportion of the red eye phenotype due to a loss of silencing, and enhancers of variegation (E(vars)) displaying an increased proportion of the white eye phenotype due to more silencing. The Drosophila screens identified important genes involved in epigenetic regulation, including the gene encoding heterochromatin-associated HP1 protein (Su(var)2-5), and the one encoding histone methyltransferase (Su(var)3-9) (Reuter and Spierer, 1992).
Approximately 150 Su(var) and E(var) genes have been identified of which about a third have so far been characterized at a molecular level. It is expected that an equal number of modifiers will be found in the mouse. In addition, some of the genes identified in the mouse might not be found in the Drosophila screen, because some gene silencing mechanisms, for example, DNA methylation of the adult genome, is unique to mammals. Moreover, the mouse screen can be helpful to understand epigenetic regulation and diseases as some of the mutants might develop phenotypes similar to symptoms observed in human diseases.
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The mouse line subjected to ENU treatment was created by Preis et al and carries a transgene expressing GFP in a variegating manner that, 55% of the erythrocytes express the transgene (Blewitt et al., 2005, Preis et al., 2003). It is assumed that the mutants identified from the screen carry a point mutation, which alters the activity of a gene involved in epigenetic regulation of the transgene. The variegated expression of the transgene itself allows the identification of both suppressors and enhancers of variegation.
Homozygous males of this line, Line3 (L3), are injected with one dose of 100 mg of ENU. ENU is a potent mutagen and introduces point mutations into the DNA. Most commonly, these mutations can be found on A/T base pairs where ENU induces either T/A or G/C substitutions (Justice et al., 1999). The mated mice recover fertility between 15 and 30 weeks later and are then mated with homozygous Line3 (L3) females. GFP expression of the offspring is screened at three weeks of age (weaning) for alteration in the percentage of expressing cells by flow cytometry. Mice showing a change in the percentage of GFP expressing cells, higher or lower than the wild type (WT) by more than two standard deviations, are bred further to test heritability. When heritability is confirmed, the mutants are named and further experiments are carried out to characterise the lines and identify the underlying mutations.
Modifiers of murine metastable epialleles (Mommes)
To date more than 2,500 offspring from an inbred F1 cross have been screened, and around 25 mice carrying mutations that alter either the percentage of GFP-expressing erythrocytes or the mean fluorescence of the GFP-expressing cells have been identified and named Modifiers of Murine Metastable Epialleles (Mommes). All MommeDs display a significantly different GFP expression profile compared to that of their WT littermates. In most of the cases, the mutations are semidominant and several Mommes are found to be homozygous embryonic lethal, with significantly reduced litter size at weaning for each heterozygous intercross. This suggests that the mutated genes play important roles in development (Blewitt et al., 2005, Ashe et al., 2008).
In a number of cases, the underlying mutations have been found, and novel factors as well as novel mutations in known genes have been identified. Examples include DNA methyltransferases (Dnmt1, Dnmt3b) and histone modifying enzymes (Hdac1) as well as chromatin remodelers (Snf2h and Baz1b) (Chong et al., 2007, Ashe et al., 2008).
One mutant line (MommeD1) displayed homozygous embryonic lethality in females and female-specific effects, suggesting that the mutated gene has a role in X-inactivation. A gene called SmcHD1 (structural maintenance of chromosome hinge domain containing 1) has been identified to be mutated in MommeD1 (Blewitt et al., 2008). SmcHD1 encodes a protein that contains a C-terminal hinge domain similar to that of SMC proteins, which are involved in chromosome condensation, chromosome segregation and DNA recombination and repair. This gene was not previously characterised, but has now been found to play a critical role in X inactivation. It is required for gene silencing and DNA methylation of genes on the inactive X chromosome (Xi) (Blewitt et al., 2008).
Another mutant line, MommeD10, has been found to have a mutation in the chromatin remodelling protein Baz1b. The majority of MommeD10 homozygous mice die shortly after weaning. Some homozygous mice survive and they are found to be significantly smaller than heterozygous or WT littermates at weaning. They have widened, bulbous foreheads and shortened snouts (Ashe et al., 2008). Detailed craniofacial analysis of homozygous MommeD10 mice confirmed this phenotype which resembles that of Williams Beuren syndrome (WBS) patients (Perez Jurado, 2003). Therefore, Baz1b may play a role in WBS. Although it is already known that WBS is associated with a hemizygous deletion of approximately 28 genes in humans, it is still unclear which gene is responsible for the craniofacial phenotype. MommeD10, which is the first mouse carrying a mutation at this locus suggests that Baz1b contributes to the WBS phenotype, making it, at least in part, a chromatin-remodeling factor disease (Ashe et al., 2008).
MommeDs have clearly demonstrated that this "sensitized" ENU screen is powerful not only in discovery of genes involved in the target process, but also for the elucidation of that mechanism. SmcHD1/MommeD1 shows the identification of a novel critical gene, which might have been ignored previously, in epigenetic gene silencing. Moreover, Baz1b/MommeD10 has demonstrated the significance of the ENU screen for further understanding of the genes that could play a critical role in genetic diseases.
MommeD24(Clio) and MommeD25(Dagon): Two novel mutant mouse lines
Phenotypic characterisation of MommeD24(Clio) and MommeD25(Dagon).
Identification of the point mutation affecting transgene variegation in MommeD24(Clio) and MommeD25(Dagon) using linkage analysis.
Two novel mutant lines, MommeD24(Clio) and MommeD25(Dagon), are the central focus of my honours project. Both of them were generated by ENU injection of homozygous Line 3 (L3) male mice carrying the GFP transgene as described above and both show a significant alteration in GFP expression compared to WT.
MommeD24(Clio) is a suppressor of variegation (Su(var)), showing an increase in the percentage of GFP expressing cells. MommeD25(Dagon) is an enhancer of variegation (E(var)) displaying a reduction of the percentage of GFP expressing cells. Both mutant lines have distinct expression profiles from WT mice as well as each other (Figure 1). 55% of erythrocytes express the GFP transgene in WT mice, whereas 67.30% and 41.17% of erythrocytes express the GFP in MommeD24(Clio) and MommeD25(Dagon) respectively.
Figure 1. GFP expression profiles in MommeD24(Clio) and MommeD25(Dagon).
Erythrocytes from 3 weeks of age mice were analysed by flow cytometry. The phenotypic WT mice are shown in black, and heterozygous mutant mice are shown in grey. The x axis represents GFP fluorescence on a logarithmic scale. The y axis indicates the numbers of cells present at each fluorescence level.
For both MommeDs, the heritability of the mutation has been tested and confirmed for at least 3 generations. Breeding of these mutant lines will be continued and flow cytometry will be used to monitor whether their expression profiles remain constant. A stable GFP expression profile over multiple generations indicates that the MommeDs carry single causative mutations.
It has been shown for several MommeDs that they are homozygous lethal (Ashe et al., 2008, Blewitt et al., 2005). Heterozygous intercrosses will be performed with the two novel MommeD lines to look for evidence of homozygous lethality. If no viable homozygotes are found at weaning then embryo dissections at an earlier timepoint will be carried out.
Preliminary data from heterozygous intercrosses of MommeD24(Clio) indicates that it is a semidominant and homozygous viable MommeD line as three distinct GFP expression profiles have been observed. Currently more breeding data is being collected to determine how many of the potentially homozygous mice survive to weaning and whether they show or develop any phenotypic abnormalities. Statistical analysis such as Chi-square test and Student t-test will be used to interpret the data.
In addition, progeny testing will be performed by crossing potential homozygous mice to Line 3 (L3) WT mice. In the case that the mutant mouse was homozygous, all offspring are expected to be heterozygous. After the point mutation has been identified, homozygousity can be confirmed by genotyping.
For linkage analysis, MommeD24(Clio) and MommeD25(Dagon) mutants, which are in the FVB/N strain, were crossed to C57BL/6J mice to create genetic heterogeneity. The resultant mice that carried the mutation as determined by Fluorescence-Activated Cell Sorting (FACS) phenotype are being backcrossed to C57BL/6J. The offspring of this cross is termed Backcross1 (BC1) generation and will be used for linkage analysis (Figure 2).
Figure 2. Backcross1 (BC1) mice generation.
Mice used for mapping will be homozygous for the transgene and "heterozygous" or WT for the mutation as determined by FACS phenotype. WT mice are expected to be C57BL/6J at the linked interval whereas the heterozygous mutant mice should display both C57BL/6J and FVB/N chromosome marks at the linked interval. Sequence differences (polymorphisms) in the two strains will be used to identify a linked interval.
Recent advances in high throughput and genome-wide technologies allow to perform initial crude mapping of the new MommeD lines using a relatively low number of BC1 animals. Only 30 mice are required for crude mapping using the Illumina GoldenGate SNP Genotyping Assay.
Using this technology, a 40 Mb interval on Chr 17 has been identified that carries the point mutation affecting transgene variegation in MommeD24(Clio). For MommeD25(Dagon), tails from BC1 mice are being collected to perform the SNP Genotyping Assay. To perform fine mapping, which will confirm the initial interval but also narrow down the interval, more BC1 offspring is being generated and DNA from tails of these mice will be isolated. For fine mapping, microsatellites (simple sequence repeats scattered throughout the genome) and single nucleotide polymorphism (SNP) markers that create restriction sites in one but not the other mouse strain will be used. PCR-based amplification and gel electrophoresis of the products allow to assay the contributions from the different mouse strains relatively easily.
Once the linked interval is found, candidate genes in the interval will be identified based on the information from the Mouse genome databases. Ideal candidate genes are ones that are associated with or serve as regulators of epigenetic state. It is planned to start sequencing when there are approximately 5 to 10 candidate genes in the mapping region.
Figure 3. Process of the sensitized ENU screen for the identification of modifiers of epigenetic regulation.
It is hypothesized that the mutagenized gene in each mutant line, MommeD24(Clio) and MommeD25(Dagon), is strongly associated with epigenetics regulation. We anticipate that these genes will be novel and as such will increase our understanding of the role of epigenetic processes in the determination of phenotype.
Flow cytometry of blood
GFP fluorescence in erythrocytes is measured with flow cytometry. A drop of blood is collected in Osmosol buffer by cutting 1mm tail tip of the mice at weaning (3 weeks of age).The excitation is set at 488mm and 550mm. The 488mm channel measures the target GFP fluorescence, whereas the 550mm channel aims to measure the erythrocyte autofluorescence. The data is being analysed using CELL QUEST software and the GFP-positive gate is set to exclude 99.9% of WT cells.
Heterozygous mice (FVB/N) are mated with WT (C57BL/6J) to generate F1 offspring. The FI individuals (FVB/N/C57BL/6J) are backcrossed with WT (C57BL/6J) to generate backcross 1 (BC1) offspring (Figure 2).
Genomic DNA will be isolated from BC1 tail tips using Phenol/Chloroform extraction and Ethanol precipitation. The DNA will be used for PCR reactions and the resultant products will be analysed using gel electrophoresis.
Heterozygous mice (FVB/N) are being intercrossed to generate homozygous mice. Offspring will be classified as homozygous based on the GFP expression profile, which is expected to show a larger difference in the percentage of expressing cell comparing to the WT GFP expression profile (55%) than that of heterozygous.
Chromatin - A complex of DNA and proteins (histones) that is the component of chromosome. DNA is wrapped around an octamer of histones, forming euchromatin or heterochromatin. Euchromatin is a loosen packed chromatin so that DNA sequence in the region is accessible for transcription factors. Heterochromatin is a tightly packed chromatin and genes located in the region are silenced, since transcription factors cannot access to the genes.
Epigenetics - A study of mechanisms that are able to alter gene expression, either suppressor or enhance, without a change to the DNA sequence.
Flow cytometry/FACS - A high throughput technology that can analyse several thousand particles (eg. cells) according to the set parameters in seconds. Many different types of flow cytometry are available and a specialized type of flow cytometry called Fluorescence-Activated Cell Sorting (FACS) is employed in this study. It separates heterogeneous mixture of cells based on the light scattering or fluorescence characteristics (according to the set parameters) of each cell. Outcome of the analysis is numerical quantification of the set parameters.
Green Fluorescence Protein (GFP) - A protein which emits green fluorescent light that can be observed under UV light.
Metastable epiallele - Endogenous alleles that are susceptible to epigenetic regulation. It can either be actively transcribed or silenced based on epigenetic modification pattern on its promoter sequence.
N-ethyl-N-nitrosourea (ENU) - A chemical that is a potent mutagen. It is an alkylating agent that can transfer the ethyl group of ENU to a nucleotide, resulting in substitution of either A/T or C/G. One of its main targets is spermatogonial stem cells.
Phenotypic variegation - A phenomenon that individuals with the identical genome (eg. homozygous twins) display different phenotypes. The variegation correlates with different epigenetic state patterns in each genome that genes are either transcribed or silenced.