The Most Difficult Definitions in Science:

Nature (2008) 455: 1023-1028

Paradigm shift

EpigeneticComplexityRaceTipping pointStem cellSignificantConsciousness

No one denies that epigenetics is fashionable: its usuage in PubMed papers increased by more than tenfold between 1997 and 2007. And few deny that epigenetics is important. What they do disagree on is what it is.

“Epigenetics is a useful word if you don’t know what’s going on – if you do, you use something else” -- Adrian Bird

The definition of “Epigenetics”

The term 'epigenetics' was introduced by Conrad H. Waddington (1905-1975) in 1942 to describe “the interactions of genes with their environment that bring the phenotype into being”.

The current term of epigenetics: changes in phenotype that are inheritable but do not involve DNA mutation.

Waddington’s classical epigenetic landscape: in 1957, Waddington proposed the concept of an epigenetic landscape to represent the process of cellular decision-making during development. At various points in this dynamic visual metaphor, the cell (the ball) can take specific permitted trajectories, leading to different outcomes of cell fates.

Over the years, numerous biological phenomena, some considered bizarre and inexplicable, have been lumped into the category of epigenetics.

Genetics Epigenetics

mutations

alterations

Position-Effect Variegation (PEV) (H.J. Muller, 1930)

In Drosophila, virtually every gene that has been examined in an appropriate rearrangement has been shown to variegate, and rearrangements involving the pericentromeric heterochromatin of any chromosome can lead to PEV. PEV has also been observed in a variety of organisms.

Genetically defined Su(var) and E(var) genes and their functions (from ~30)

Su(var)/E(var) gene M olecular function

Suv4-20 [su(var)] HKMT, histone H4K20 trimethylationSu(var)2-5 (HP1) Hetr ochromatin protein 1, binding of H3K9me2, H3K9me3, SU(VAR)3-9Su(var)3-9 HKMT, histone H3K9 methylation, interact with HP1Su(var)3-7 Zinc-f inger protein, interact with HP1 and SU(VAR)3-9Su(var)3-64B Histone deacetylase HDAC1, deacetylation of H3K9E(z) [(Suvar)] HKMT, H3K27 mono-, di-, and trimethylationTrl [E(var)] GATA f actor, binding of repetitive DNA sequencesE(var)3-93D Transcription regulatorE(var)3-93E E2F transcription f actor, haplo-enhancer and triplo-suppressor

Paramutation (R.A. Brink, 1958)Brink described his stunning observations of “paramutation” at the R locus in maize in 1958. Several similar loci were later again discovered in maize.

~9000 colorless seeds (B’/B-I) planted1 B’B’ + 2 B’/B-I + 1 B-I’B-’I’

Stam et al. (2002) Genetics

B' plants (light pigmented plant, colorless kernels) were crossed with B-Peru plants (nearly green plant, purple kernels). The resulting seeds (B'/B-Peru; purple kernels) gave rise to B' colored F1 plants. The F1 plants were crossed to B-I plants (dark pigmented plant, colorless kernels), giving rise to an ear segregating colorless (B'/B-I) and purple (B-Peru/B-I) kernels. When the colorless seeds were planted, the vast majority of the resulting plants showed a B' plant phenotype (B'/B-I' plants; the paramutation of B-I in these plants is indicated as B'*). Two dark individuals were isolated in which the B-I allele was not paramutated. The B' allele in these individuals is neutral for paramutation (B'-n).

~6500 B’/B’* plants(100% paramutation)

2 B’-n/B-I plants(No paramutation)

(A) B’ Mop1/mop1Alleman et al. (2006) Nature

The mop1 mutations reactivate silenced Mutator elements. Plants carrying mutations in the mop1 gene also stochastically exhibit pleiotropic developmental phenotypes. Mop1 is an RNA-dependent RNA polymerase gene (RDRP), most similar to the RDRP in plants that is associated with the production of short interfering RNA (siRNA) targeting chromatin. It was proposed the mop1 RDRP is required to maintain a threshold level of repeat RNA, which functions in trans to establish and maintain the heritable chromatin states associated with paramutation.

(B) B’ mop1/mop1

(C) B-I Mop1/Mop1 (D) B’ mop1/mop1 with B’-like sectors

(E) Pl’ Mop1/mop1 (F) Pl’ mop1/mop1

The mop1 (mediator of paramutation1) mutation

RMR6 (Required to Maintain Repression) is Pol IV

Erhard Jr. et al. 2009 Science 323:1201-1205

X-chromosome Inactivation (M.F. Lyon, 1961)

The mottled (Mo) dominant mutant

Mo / Y Mo / +

1. In XY-males, the single X chromosome is active in all cells, while in each cell of the female (XX) one of the two X chromosomes becomes inactivated.

2. Paternal and maternal X chromosomes have an equal chance of being inactivated.

3. Inactivation occurs early in the life of the female embryo.

4. Once it has been decided which X chromosome is activated in a cell, the same X chromosome will always be inactivated in the descendants of that cell.

5. The inactive X chromosome becomes heterochromatinized and forms the sex chromatin.

6. The inactive X chromosome is reactivated in the oocytes some time before meiosis (Gartler and Andina 1976).

The mottled (Mo) dominant mutant

Mo / Y Mo / +

Molecular mechanism of X chromosome inactivation

Chow et al. (2005) Annu. Rev. Genomics Hum. Genet. 6: 69-92.

Human genes escaping from X inactivation

624 genes were tested in nine Xi hybrids. Each gene is linearly displayed. Blue denotes significant Xi gene expression, yellow shows silenced genes, pseudoautosomal genes are purple, and untested hybrids remain white. Positions of the centromere (cen) and XIST are indicated.

Carrel and Willard, 2005, Nature 434: 400-4.

What do we learn from the last three classical epigenetic cases?

Controlled by non-coding RNA and DNA/histone modification

Significant variability/stability (PEV, ina-X)

Reversible and/or transmittable through germ cells

The “histone code” hypothesis by Strahl and Allis (2000)

Histone code hypothesis: A specific histone modification, or combinations thereof, can affect distinct downstream cellular events by altering the structure of chromatin (cis mechanisms) or by generating a binding platform for effector proteins (trans mechanisms).

Strahl and Allis 2000, Nature 403: 41-45

David Allis identified the first transcription-associated histone acetyltransferase (HAT) in Tetrahymena in 1996.

Many of the histone-modifying enzymes have other nonhistone substrates

Kouzarides (2007) Cell 128: 693-705.

There are over 60 different residues on histones where modifications have been detected.

Weaver et al. 2005, Nat. Neurosci. 7: 847-54.

Mothering Style and DNA Methylation: Increased pup licking and grooming (LG) and arched-back nursing (ABN) by rat mothers alter the offspring epigenome at a glucocorticoid receptor (GR) gene promoter in the hippocampus, leading to tighter regulation of stress hormone levels. Offspring of mothers that showed ‘high-LG-ABN’ have differences in DNA methylation in the promoter, as compared to offspring of 'low-LG-ABN' mothers. These differences emerged over the first week of life and persisted into adulthood and were associated with altered histone acetylation and transcription factor (NGFI-A) binding to the GR promoter. Strikingly, pups raised by a high-LG-ABN mother become high-LG-ABN mothers themselves as adults, thereby passing on the traits in a case of multi-generational, non-mendelian inheritance.

Most recently reported epigenetic phenomenon

In humans, childhood abuse alters hypothalamic-pituitary-adrenal (HPA) stress responses and increases the risk of suicide. We examined epigenetic differences in a neuron-specific glucocorticoid receptor (NR3C1) promoter between postmortem hippocampus obtained from suicide victims with a history of childhood abuse and those from either suicide victims with no childhood abuse or controls. Decreased levels of NR3C1 mRNA and increased cytosine methylation of an NR3C1 promoter were found to be associated with suicide victims with a history of childhood abuse.

Methylation of the NR3C1 promoter in the hippocampus. Twenty clones were sequenced for each subject for methylation mapping. (a) Mean plusminus s.e.m. percentage of methylated clones. (b) Methylation of the NR3C1 promoter region, showing the frequency of methylation observed at each CpG site

Epigenetic variation among monozygous twinsMonozygous twins are considered genetically identical, but significant phenotypic discordance between them exist, which is particularly noticeable for psychiatric diseases.

Although MZ twins are epigenetically indistinguishable during early years of life, older MZ twins exhibited remarkable differences in their overall content and genomic distribution of 5-methylcytosine DNA and histone acetylation, affecting their gene expression portrait.

Fraga et al. 2005, PNAS 102: 10604-9

Epigenetic variation and transgenerational inheritance

Whitelaw 2006 Human Mol. Genet. R131-R137.

Model for the regulation of the floral repressor FLC throughout the Arabidopsis life cycle. During seedling growth, a group of genes encode proteins that function as activators of FLC expression (shown in blue). These proteins may maintain FLC chromatin in an active state. The RNA-binding proteins (shown in red) may function post-transcriptionally to achieve this. The FVE/FLD proteins act with a putative histone deacetylase (shown in orange) to promote an inactive FLC chromatin state. FLC is also repressed by exposure to long periods of cold (vernalization). The proteins acting in the vernalization pathway are shown in purple. Prolonged cold induces VIN3 expression, which promotes an inactive FLC chromatin state. Subsequently, the VRN1 and VRN2 proteins are recruited to FLC, and are required for the methylation of FLC histones and the maintenance of silencing. These marks may promote the association of silencing factors with FLC chromatin that reinforce its repression. During meiosis, gametogenesis or early embryogenesis, FLC repression is overcome, thus resetting its expression in the next generation.

Henderson and Dean, 2004, Development 131, 3829-3838.)

3. There is an increasing belief that epigenetic variants and inheritance could provide the missing piece of the puzzle for understanding the basis of many complex phenotypes.

4. Our understanding of epigenetic variation and inheritance is still in its infancy, and it is unclear what proportion of heritable phenotypic variability can be ascribed to epigenetic factors.

Epigenetic Variation - the Excitements and Challenges:

1. Phenotypic variation is traditionally parsed into components that are directed by genetic and environmental variation. Now the line between these two components is blurred by inherited epigenetic variation.

2. How widely exist about the inheritable epigenetic variation in the nature? Could inheritance of epigenetic variants be an important means of adaptive evolution in the face of environmental change, without a permanent alteration in the DNA? What’s the difference between inheritable epigenetic variation and neo-Lamarckian?

A rush to discover more exciting epigenetic phenomena: make sure that you don’t get your head too hot!