1. Epigenetics
Any potentially stable and heritable change in gene expression that occurs without a change in DNA sequence
Epigenetic mechanisms contribute to stable silencing or activation of genes, transposons, centromeres and even whole chromosomes.
Epigenetic controls of DNA activity are indispensible – defects in epigenetic control contribute to developmental defects and disease

2. What does “epigenetics” mean?
Literally, epigenetics means above, or on top of, genetics.
Usually this means information coded beyond the DNA sequence, such as in covalent modifications to the DNA or modifications to the chromatin structure.
Transcription
Epigenetic Silencing
Practically, epigenetics describes phenomena in which genetically identical cells or organisms express their genomes differently, causing phenotypic differences.
Genetically identical cells or individuals
Different epigenetic modifications leading to different expression patterns
Different phenotypes

3. In plants, the activity of many developmental genes and environmentally-regulated genes are epigenetically regulated, to be stably maintained in an ON or OFF position
Holec, S. and Berger, F. (2012). Polycomb group complexes mediate developmental transitions in plants. Plant Physiol. 158:

4. Epigenetic marks: DNA methylation and histone modification
cytosine
5-methylcytosine
Methyltransferase
DNA can be covalently modified by cytosine methylation.
TTCGCCGACTAA
Methyl-cytosine
The histone proteins that DNA is wrapped around can be covalently modified, affecting chromatin structure
NUCLEOSOME
Epigenetic Silencing
DNA
Histone modifications
Histone octamer

5. Epigenetic marks contribute to large-scale chromatin domains
The centromere and regions around it are usually densely packaged with few protein-coding genes
Densely packaged heterochromatin
CENTROMERE
Less densely packaged, gene-rich euchromatin
Euchromatin DAPI DNA stain Merged
Centromeric
heterochromatin
Deal, R.B., Topp, C.N., McKinney, E.C., and Meagher, R.B. (2007) Repression of flowering in Arabidopsis requires activation of FLOWERING LOCUS C expression by the histone variant H2A.Z. Plant Cell 19:

6. Heterochromatin DNA is highly methylated
BLUE = Gene density
RED = Repetitive element density
Plants have different DNA methylases that act on different sequences:
5'-CG-3'
5'-CHG-3‘
5'-CHH-3'
These histograms show the extent of gene density,, repetetive element density and DNA methylation along the length of Arabidopsis chromosome 3. The centromere is shown in pink and the pericentromeric region in dark blue.
Reprinted from Zhang, X., Yazaki, J., Sundaresan, A., Cokus, S., Chan, S.W.-L., Chen, H., Henderson, I.R., Shinn, P., Pellegrini, M., Jacobsen, S.E., and Ecker., J.R. (2006) Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 126: 1189–1201 with permission from Elsevier.; Cokus et al., (2008) Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning Nature 452:

7. Epigenetic programming in plants helps silence transposons and maintain centromere function
Photo by Damon Lisch, in Gross, L. (2006) Transposon silencing keeps jumping genes in their place. PLoS Biology 4(10): e353. Photos courtesy of the Barbara McClintock Papers, American Philosophical Society. Zhang, W., et al. (2008) Plant Cell 20:

8. Transposons can cause inactive or unstable alleles
Transposons are fragments of DNA that can insert into new chromosomal locations. Some transposons copy themselves and increase in number within the genome
Wild-type allele
Pigmented kernel
Mutant allele
Unpigmented kernel
Unstable allele
Partially pigmented kernel
Excision of the transposon causes unstable alleles
Gene required for pigment biosynthesis
Gene interrupted by transposon
Transposons are responsible for large scale chromosome rearrangements and single-gene mutagenic events
Photo by Damon Lisch, in Gross, L. (2006) Transposon silencing keeps jumping genes in their place. PLoS Biology 4(10): e353.

9. Epigenetic marks silence transposons and repetitive elements
Transposons must be tightly controlled to prevent widespread mutagenic activity. Epigenetic controls to maintain silencing include DNA methylation, histone modification and siRNA production.

10. Small interfering RNAs (siRNAs) are preferentially derived from pericentromeric regions
Small RNAs
The density of small RNA-homologous loci is highest in the centromeric and pericentromeric regions which contain a high density of repeat sequence classes, such as transposons.
Kasschau, K.D., Fahlgren, N., Chapman, E.J., Sullivan, C.M., Cumbie, J.S., et al Genome-wide profiling and analysis of Arabidopsis siRNAs. PLoS Biol 5(3): e57.

11. Small interfering RNAs also contribute to transposon silencing
siRNA-mediated silencing
ARGONAUTE (AGO) protein. Guides siRNA to target sites
RNA
DNA
RNA Pol
AGO
AGO
RdRP
RNA-dependent RNA polymerase (RdRP)
DCL
RNA Pol
Double-stranded RNA (dsRNA)
Dicer-like protein (DCL). Cuts dsRNA into smaller pieces including small interfering RNA (siRNA)
siRNA targeting to nascent RNA transcript leads to DNA and histone methylation and silencing

12. Epigenetic control of flowering time
Prolonged cold treatment
Vegetative Development
Reproductive Development
Winter
Spring
Autumn
Some plants require a prolonged cold period (vernalization) - as experienced during winter, before they will flower.

13. FLOWERING LOCUS C (FLC) mutants flower early
Winter
Spring
Autumn
FLC is an inhibitor of flowering; removing FLC removes the vernalization requirement

14. FLC inhibits FT, an activator of flowering
Wild-type plant
FLC
FT gene
Transcription of FT gene repressed by FLC binding
flc mutant plant
FT gene FT

15. FLC is silenced by vernalization
After 40 days at 4ºC, FLC is not expressed. Ten days after return to 22ºC, FLC expression is still off.
Winter
Spring
Autumn
FLC gene transcribed
FLC gene silenced
Reprinted by permission from Macmillan Publishers, Ltd: NATURE Sung, S., and Amasino, R.M. (2004) Vernalization in Arabidopsis thaliana is mediated by the PHD finger protein VIN3. Nature 427: Copyright 2004.

16. FLC is regulated by epigenetic modifications
H2A.Z incorporation
H3K4me, H3K36me
H3K9Ac, H3K14Ac
H3K9me2, H3K27me2
cold
Winter
Spring
Autumn
FLC gene transcribed
FLC gene silenced

17. Many epigenetic marks are reset during reproduction
FLC ON
FLC OFF
Winter
Spring
Autumn
FLC is reset to the ON state during reproduction
Embryo within seed
Female gametophyte, including egg cell

18. Genomic imprinting is regulated by epigenetic processes
The zygote receives two copies of each gene, one from the mother’s genome and one from the father’s.
At most loci, both copies are active.
Some loci, imprinted loci, show a “parent of origin effect”.
Expression of these loci is controlled by epigenetic factors.

19. Imprinting – nuclear transplant experiment
Zygotes that receive only maternal or only paternal nuclei do not survive.
Placing a sperm and an egg nucleus into an enucleated fertilized cell leads to a normal embryo.
The two parental genomes are not equivalent

20. The MEDEA (MEA) gene is imprinted
MEA/MEA x MEA/mea
50% of seeds abort
MEA/mea x MEA/MEA
All seeds viable
In the second cross, 50% of the seeds receive the mutant mea allele from their mother.
These seed abort, even though they also have a wild-type MEA allele inherited from their father; the paternal allele is epigenetically silenced and inactive.
From: Grossniklaus, U., Vielle-Calzada, J.-P., Hoeppner, M.A., Gagliano, W.B. (1998) Maternal control of embryogenesis by MEDEA, a Polycomb Group gene in Arabidopsis. Science 280: Reprinted with permission from AAAS.

21. Angiosperm reproduction: Development of male and female gametophytes
Vegetative nucleus
2-celled male gametophyte
(aka pollen grain) n 2n
Microspore mother cell
meiosis
mitosis
Microspore
8-celled female gametophyte
(aka embryo sac)
Megaspore mother cell
Megaspore
Egg cell
Central cell
Generative cell 2n

22. Angiosperm reproduction: Double Fertilization
Mitotic division of generative cell to two sperm cells
One sperm nucleus fertilizes the egg cell to produce diploid embryo.
The second sperm nucleus fertilizes the polar nuclei to produce triploid endosperm.
Pollen tube growth
2n zygote
3n endosperm

23. Angiosperm reproduction: Imprinting and epigenome resetting
Haploid sperm nuclei
Epigenetic marks can be renewed and reset during angiosperm reproduction
Triploid endosperm
Imprinting may ensure a balance between maternal and paternal genomes, and is necessary for endosperm and seed development
Diploid embryo

24. Summary
Expression of DNA is regulated by epigenetic marks including DNA methylation and histone modifications, with the contributions of siRNAs
Epigenetic programming silences transposons and controls the timing of many genes that control plant development.