a nd Regulation of Developmental Genes

1. Chromatin and Regulation of Developmental Genes Miguel Flores PLB/HRT865 Plant Growth & Development

2. OUTLINE Epigenetics Identical genotype, different phenotypes Eukaryotic DNA is packed into nucleosomes Chromatin organization Epigenetic modifications Euchromatin and Heterochromatin Epigenetic regulation in plants

3. Why a cloned cat isn’t exactly like the original? Rainbow Rainbow’s clone

4. Epigenetics The inheritance of a property (i.e. gene expression) through cell division that does not involve changes to the DNA sequence. Epigenetics Genetics Allis, et al.(2007).Epigenetics, overview and concepts. CSHL.

5. Identical genotype, different phenotypes The cells in a multicellular organism have nominally identical DNA sequences, yet maintain different terminal phenotypes. This non-genetic cellular memory, which records developmental and environmental cues, is the basis of epigenetics.

6. Identical genotype, different phenotypes Practically, epigenetics describes phenomena in which genetically identical cells or organisms express their genomes differently, causing phenotypic differences. Different epigenetic modifications leading to different expression patterns Different phenotypes Genetically identical cells or individuals

7. X chromosome inactivation involves epigenetic silencing In female mammals, one copy of the X chromosome in each cell is epigenetically inactivated. Fur color in cats is determined in part by orange, an X-linked gene. A female cat that is heterozygous for the orange gene shows black and orange patches, corresponding to which X chromosome is active.

8. Epigenetic programming in plants helps silence transposons and maintain centromere function

9. Epigenetic programming in plants helps control developmental transitions Embryonic to vegetative transition Vegetative to reproductive transition Embryonic development Vegetative development Reproductive development

10. Eukaryotic DNA is Packed into Nucleosomes 8 histones: 2 each H2A H2B H3 H4 Gräff and Mansuy (2008).Behav Brain Res.

11. Chromatin Organization Short region of DNA double-helix 2 nm Histones + DNA. “Beads-on-a-string” form of chromatin 11 nm Plant Cell Nucleosome Fibers. 30-nm chromatin fiber of packed nucleosomes 30 nm Chromatin extended scaffold-associated form 300 nm Nuclear Matrix

12. Epigenetic Modifications (the most studied) • Epigenetic modifications include: • Cytosine methylation of DNA. • Histone modifications.

13. DNA Methylation Arabidopsis 46-days old NH2 NH2 CH3 N N N N O O Methyltransferase ~ ~ WT met1/cmt3 cytosine 5-methylcytosine Xiao, et al.(2006). The Plant Cell. 18: 805-814

14. Histone Modifications Biotinylation ADP-ribosylation k6 k11 * * * Gräff and Mansuy (2008).Behav Brain Res.

15. Phenotype of histone methyltransferase mutants Mutants are deficient in methylation of H3K36. Dong, et al. (2008). Biochemical and Biophysical Research Communications. 373: 659-664

16. Chromosomes Consist of Euchromatin and Heterochromatin Euchromatin Heterochromatin • Less condensed • At chromosome arms • Contains unique sequences • Gene-rich • Highly condensed • At centromeres and telomeres • Contains repetitious sequences • Gene-poor http://www.nyas.org/

17. Chromatin organization in different tissues Epiblast Hepatocyte Lymphocyte Open Intermediate Compact Chromatin: Lindsy, et al.(2012). Micron.43:150-158.

18. Heterochromatin Changes During Plant Development 2 days 4 days 3 weeks Arabidopsis Nuclei Exner & Hennig.(2008). Current Opinion in Plant Biology. 11:64-69

19. Epigenetic genome regulation in plants • Transposon silencing • Control of flowering time • Resetting the epigenome

20. 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 chromosomal rearrangements as well as single-gene mutagenic events

21. 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

22. DNA methylation is necessary to silence transposons BLUE = Gene density RED = Repetitive element density Loss-of-function met1 or ddm1 (decrease in DNA methylation1) mutants have hypomethylated DNA GREEN = Methylated DNA BROWN = Methylated DNA in a met1 mutant Zhang, et al. (2006). Cell 126: 1189-1201

23. Activated transposons in ddm mutants induce mutations After one generation After three generations After five generations Wild-type After DDM inactivation, plants become more and more abnormal as they accumulate transposon-induced mutations. Kakutani, et al. (1996). PNAS. 93: 12406-12411

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

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

26. 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

27. 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. Autumn Winter Spring FLC gene transcribed FLC gene silenced Sung, et al. (2004). Nature. 427: 159-164.

28. FLC is regulated by epigenetic modifications H3k4me H3K36me H3K9ac H314ac H3k9me2 H3k27me2 Cold Autumn Winter Spring FLC gene transcribed FLC gene silenced

29. VIN3 and the PRC2 complex epigenetically silence FLC PRC2= PolycombRepresiveComplex 2) VIN3 PRC2 (including VIN3) Autumn Winter Spring FLC gene transcribed FLC gene silenced

30. Henderson & Jacobsen.(2007). Nature.447:418.

31. Resetting of the epigenetic marks (Pollen) In vegetative cells (VCN), TE genes are demethylated and reactivated, and 21-nt small interfering RNAs (siRNAs) are produce. These 21-nt siRNA may be transported to sperm cells (SC) to enhance repression of TE genes.

32. Resetting of the epigenetic marks (Seed) In endosperm, the maternal alleles of TE genes are demethylated and reactivated, producing 24-nt siRNAs. These 24-nt siRNAs may be transported to to the embryo to keep the TE genes silenced and ensured genome stability.

33. The heterochromatin fraction is reduced in endosperm EMBRYO NUCLEI ENDOSPERM NUCLEI H3K9me In endosperm nuclei, heterochromatin marks such as H3K9me and K3K27me2 disperse into euchromatin, suggesting genome-wide epigenetic changes. H3K27me2 DNA DNA Histone mark Histone mark Merged Merged Baroux, et al.(2007). Plant Cell.19:1782-1794.

34. 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.

35. Meristem Identity Changes upon Floral Induction Cold Autumn Winter Spring