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

3. What does “epigenetics” mean?
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

4. Epigenetic marks and their maintenance
Transcription
Epigenetic Silencing

5. Eukaryotic DNA is packaged in nucleosomes
Approximately 147 base pairs of DNA wrapped around a histone octamer
8 histones:
2 each H2A
H2B H3 H4 +
~ 147 bp DNA

6. Epigenetic modifications
Epigenetic modifications include:
- Cytosine methylation of DNA
- Histone modifications
Collectively, these changes contribute to the distribution of DNA into silent, heterochromatin and active euchromatin
euchromatin
heterochromatin

7. DNA methylation
cytosine
5-methylcytosine
Methyltransferase
DNA can be covalently modified by cytosine methylation.
TTCGCCGACTAA
Methyl-cytosine

8. CG methylation can be propagated during DNA replication
5’ A T G C G T A C T
A T G C G T A C T
T A C G C A T G A
REPLICATION
“MAINTENANCE”
METHYLATION
3’ T A C G C A T G A

9. Some sites are maintained by small interfering RNAs (siRNAs)
A T G C A A A C T
T A C G T T T G A
REPLICATION
3’ T A C G T T T G A
DRM1/2
5’ A T G C A A A C T
See “Small RNA” for more slides on RdDm
siRNA

10. Chromosomes consist of heterochromatin and euchromatin
Densely packaged heterochromatin
CENTROMERE
DNA around the centromere is usually packaged as heterochromatin.
Less densely packaged euchromatin

11. Heterochromatin DNA is highly methylated
BLUE = Gene density
RED = Repetitive element density
BROWN = Methylated DNA in a met1 mutant
GREEN =
Methylated DNA
DNA methylation is densest at the repetitive elements around the centromere.
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.,  et al. (2006) Genome-wide high-resolution mapping and functional analysis of DNA methylation in Arabidopsis. Cell 126: 1189–1201 with permission from Elsevier.

12. Histone proteins can be modified to affect chromatin structure
DNA
Histone octamer
The amino terminal regions of the histone monomers extend beyond the nucleosome and are accessible for modification.
NUCLEOSOME

13. The Histone Code Histones can be modified by
Acetylation (Ac)
Ubiquitination (Ub)
Methylation (Me)
Phosphorylation (P)
Sumoylation (Su)
Depending on their position, these can contribute to transcriptional activation or inactivation.

14. Example – H3 modifications
H3 A R T K Q T A R K S T G G K A P R K Q L A T K A A R K S Me
Me P
Me Ac
Me Me P Ac
The amino terminus of H3 is often modified at one or more positions, which can contribute to an activation or inhibition of transcription. +
NH2
CH3 NH N
Lysine can be acetylated, or mono-, di-, or tri-methylated
Methylated lysine
Mono (Kme1)
Di (Kme2)
Tri (Kme3)
NH3
Lysine (K) O
Acetylated lysine
(KAc)

15. Histone modification affects chromatin structure
Open configuration
Me P Ac
K4 S10 K14 H3
Closed configuration
Me Me P
K K27 S28 H3

16. H3K27me3 is associated with genes
GREEN = H3K27me3
PURPLE = methylcytosine
BLUE = Gene density
RED = Repetitive element density
H3K27me3 in Arabidopsis is present within the gene-rich region, not the repeat-rich region.
In animals, H3K27me3 is associated with silenced genes. ~15% of Arabidopsis genes are associated with H3K27me3 – note that it is found throughout the gene-rich region.
Zhang, X., Clarenz, O., Cokus, S., Bernatavichute, Y.V., Pellegrini, M., Goodrich, J., Jacobsen, S.E. (2007) Whole-genome analysis of histone H3 lysine 27 trimethylation in Arabidopsis. PLoS Biol. 5: e129.

17. An H2 variant, H2A.Z is associated with some active genes
Inactive gene
SWR1/
SRCAP complex
H2A
H2A.Z
H2A.Z interferes with DNA methylation and helps to maintain a transcribed state.
The histone variant H2A.Z promotes transcription and is swapped into the nucleosome by the SWR1/SRCAP complex.

18. There are three histone H3 variants: H3.1, H3.3, and CENH3
Nucleosomes at the centromere incorporate an H3 variant called CENH3 that is necessary for centromere maintanence
CENH3 H3
H3.1 and H3.3 differ in only four amino acids, but their distribution is very different. H3.1 is associated with heterochromatin, and H3.3 with transcribed genes
Redrawn from Jiang, J.,  Birchler, J.A.,  Parrott, W.A., and Dawe, R.K. (2003) A molecular view of plant centromeres. Trends Plant Sci. 8: with permission from Elsevier. Stroud, H., Otero, S., Desvoyes, B., Ramírez-Parra, E., Jacobsen, S.E. and Gutierrez, C. (2012). Genome-wide analysis of histone H3.1 and H3.3 variants in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 109: See also Shi, L., Wang, J., Hong, F., Spector, D.L. and Fang, Y. (2011). Four amino acids guide the assembly or disassembly of Arabidopsis histone H3.3-containing nucleosomes. Proc. Natl. Acad. Sci. USA 108:

19. Summary – epigenetic marks
Histone deacetylation
Histone acetylation
Histone (de) methylation
DNA methylation
DNA demethylation
Histone variants

20. Epigenetic controls in whole-plant processes
Transposon silencing
Control of flowering time
Developmental switches and stress responses
Control of imprinted genes

21. Transposons
Fragments of DNA that can insert into new chromosomal locations
Some copy themselves and increase in number within the genome
Responsible for large scale chromosome rearrangements and single-gene mutagenic events
Reprinted by permission from Macmillan Publishers, Ltd: Nature Review Genetics. Surridge, C. (2001) Plant genetics: Turning off transposons. 2: 404. Copyright 2001.

22. Transposons can cause inactive or unstable alleles
Gene required for pigment biosynthesis
Gene interrupted by transposon
Excision of the transposon causes unstable alleles
Wild-type allele
Pigmented kernel
Mutant allele
Unpigmented kernel
Unstable allele
Partially pigmented kernel

23. Naturally occurring transposons are a source of genetic variation
An Antirrhinum transposon that is only active at low temperatures.
15˚
21˚
“Variation is the raw material of evolutionary change”
- Stephen Jay Gould
(1941 – 2002)
Hashida, S.-N. Uchiyama, T., Martin, C., Kishima, Y., Sano, Y., and Mikami, T., (2006) The temperature-dependent change in methylation of the Antirrhinum transposon Tam3 is controlled by the activity of its transposase. Plant Cell 18:

24. Transposon silencing Maize has many active transposons
Epigenetic marks are thought to have evolved to silence foreign DNA (transposons, viruses)
Mutants that interfere with epigenetic silencing release transposons from silencing, and allow mutagenic transposon activity
Photo by Damon Lisch, in Gross, L. (2006) Transposon silencing keeps jumping genes in their place. PLoS Biology 4(10): e353.

25. Activated transposons induce mutations
After DDM inactivation, plants become more and more abnormal as they accumulate transposon-induced mutations.
Wild-type
After one
generation
After three
generations
After five
Kakutani, T., Jeddeloh, J.A., Flowers, S.K., Munakata, K., and Richards, E.J. (1996) Developmental abnormalities and epimutations associated with DNA hypomethylation mutations. PNAS 93: Copyright (1996) National Academy of Sciences, U.S.A.

26. Initiating and maintaining silencing at repetitive DNA and transposons
How does the genome specifically recognize and silence repetitive elements and transposons?
In other words, how does it recognize “self” (genes) from “non-self”? What is the basis for this “genomic immune recognition system”?

27. siRNAs recruit DNA methylases and histone-modifying enzymes to targets
DRM1/2
DCL
Dicer-like (DCL) nuclease produces siRNAs which bind to Argonaute (AGO) proteins to bring modifying enzymes such as DRM1 and DRM2 to DNA targets.
siRNA
More information on the biogenesis and functions of siRNAs can be found in Feb 2010 Teaching Tools in Plant Biology “The Small RNA World”.
AGO

28. 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.

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

30. 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

31. FLC is silenced by vernalization
After 40 days at 4°C, FLC is not expressed. Ten days after return to 22°C 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.

32. 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

33. VIN3 and the PRC2 complex epigenetically silence FLC
(including VIN3)
FLC gene transcribed
FLC gene silenced
Winter
Spring
Autumn

34. Epigenetic control of flowering time
During vernalization, silencing of FLC allows expression of FT and other flowering promoters
VIN3 and PRC2 complex proteins epigenetically modify FLC to silence it
How cold induces VIN3 expression is not currently known

35. Many stress-responsive genes are epigenetically regulated
Biotic or Abiotic Stress
Hormones
Stress signalling may or may not involve hormones
Many stress-responsive genes are epigenetically regulated
The stress-responsive phenotype is usually not transmitted to progeny, but chromatin changes can be heritable
Reprinted from Gutzat, R. and Mittelsten Scheid, O. (2012). Epigenetic responses to stress: triple defense? Curr. Opin. Plant Biol. 15: , with permission from Elsevier.

36. Genomic imprinting
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.

37. Genomic imprinting
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.

38. The MEDEA (MEA) gene is imprinted
MEA/MEA x MEA/mea
50% of seeds abort
MEA/mea x MEA/MEA
All seeds viable
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.

39. 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.
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.

40. The MEDEA (MEA) gene is imprinted
MEA/MEA x MEA/mea
50% of seeds abort
MEA/mea x MEA/MEA
All seeds viable
MEA
mea x
The phenotype of the progeny is based on the maternal genotype only.
The paternal allele is silent.

41. Summary
Expression of DNA is controlled by epigenetic marks including DNA methylation and histone modifications.
siRNAs contribute to epigenetic programming
Epigenetic programming silences transposons and controls the timing of many genes that control plant development.