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

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

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: 35-43.

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

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: 74-83.

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: 215-219 .

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: 25-34..

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.

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.

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. 2007 Genome-wide profiling and analysis of Arabidopsis siRNAs. PLoS Biol 5(3): e57.

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

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 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: 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

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

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.

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

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: 446-450. Reprinted with permission from AAAS.

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

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

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

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.