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 www.plantcell.org/cgi/doi/10.1105/tpc.110.tt0110

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

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

Epigenetic marks and their maintenance Transcription Epigenetic Silencing

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

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

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

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

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

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

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.

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

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.

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 4 9 10 14 1718 23 262728 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)

Histone modification affects chromatin structure Open configuration Me P Ac K4 S10 K14 H3 Closed configuration Me Me P K9 K27 S28 H3

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.

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.

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: 570-575 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: 5370-5375. 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: 10574-10578.

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

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

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.

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

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 18:104-118.

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.

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: 12406-12411. Copyright (1996) National Academy of Sciences, U.S.A.

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”?

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

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.

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

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

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: 159-164. Copyright 2004.

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

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

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

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: 568-573, with permission from Elsevier.

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.

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.

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: 446-450. Reprinted with permission from AAAS.

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: 446-450. Reprinted with permission from AAAS.

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.

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.