1. Moving to the nucleus..... Biased transmission of alleles or entire chromosomes Segregation distortion (meiotic drive, selfish DNA) Gametophytic effects in plants Biased or unusual patterns of gene expression Maternal effect genes Imprinted genes (parent-of-origin expression bias) Paramutation (allelic cross-talk & silencing)

2. Epigenetics – Heritable changes in gene expression that do not involve changes in gene sequence Mitotic (cell to cell) Meiotic (generation to generation) (Grelon & Elgin, Nature 447:399) Mediated by changes in chromatin structure ? How are these changes established how/why are they stably inherited

3. Epigenetics – Mediated by changes in chromatin environment (Grelon & Elgin, Nature 447:399) An inversion placed the white gene close to the pericentric heterochromatin Sectors of drosophila white gene off (white eye color) result from heterochromatic regions expanding to include the white gene e.g. Position effect variegation (PEV) in Drosophila genes or transgenes located near heterochromatic regions undergo mitotically stable changes in expression due to changes in chromatin organization How might this phenotype be used to investigate the mechanisms of PEV and related gene expression phenomena?

4. (Grelon & Elgin, Nature 447:399) Epigenetics - Factors implicated in heterochromatin formation Sp Nc Dm Mm At Repetitive DNAYesYesYesYesYes DNA meNo Yes No* Yes Yes H3K9 meYes Yes Yes Yes Yes HP1 Yes Yes Yes Yes No* Small RNAs Yes No* Yes Yes Yes RDR Yes No* No No Yes Yes - factor implicated in heterochromatin formation No - factor is not present in the organism No* - organism has the factor but no role in heterochromatin formation Sp, Schizosaccharomyces pombe; Nc, Neurospora crassa; Dm, Drosophila melanogaster; Mm, Mus musculus ; At, Arabidopsis thaliana RDR, RNA-dependent RNA polymerase; HP1, Heterochromatin Protein 1; Pol II, RNA polymerase II; H3K9, Histone 3- lysine 9

5. Epigenetics - RNA silencing mechanisms (RNAi) Transcriptional (chromatin changes) Post transcriptional Transcript degradation Translation repression Share common/core mechanistic features Elucidated via Nobel-prize winning work in C. elegans! a) Silencing of a gfp transgene via double- stranded gfp RNA b) Mutant defective in RNAi [Fire et al. Nature 391: 806] [Mello & Conte, Nature 431:338]

6. Epigenetics – RNAi mechanisms Post transcriptional Transcript degradation Translation repression Share common/core mechanistic features Note there are lots of routes to dsRNA [Buchon & Vaury Heredity 96:195] cytosol Nucleus

7. Epigenetics – RNAi Transcriptional (chromatin changes) [Buchon & Vaury Heredity 96:195] Nucleus

8. Epigenetics - RNAi Transcriptional (chromatin changes) Post transcriptional Transcript degradation Translation repression Share common/core mechanistic features Present in a wide array of organisms How are RNAi pathways adaptive or advantageous? (To the organisms not the molecular biologists!)

9. Modification of specific genes during gametogenesis so that only the paternal or maternal allele is expressed after fertilization, i.e. “Parent of origin” gene expression A small subset of the total genes behave in this way! Seen in placental mammals and in plants For some imprinted genes, the maternal allele is silenced & paternal allele expressed For others, the paternal allele is silenced & maternal allele expressed Epigenetics – Genomic imprinting

10. Modification of specific genes during gametogenesis so that only the paternal or maternal allele is expressed after fertilization, i.e. “Parent of origin” gene expression Affects the expression but not transmission of alleles Two gene copies present One gene copy active = functional haploidy Epigenetics – Genomic imprinting

11. Modification of specific genes during gametogenesis so that only the paternal or maternal allele is expressed after fertilization, i.e. “Parent of origin” gene expression A limited number of genes behave in this way ~80 of 30,000 human genes Primarily expressed in embryo and placenta Plant imprinted genes are expressed in the endosperm Almost no plant embryo expressed imprinted genes Epigenetics – Genomic imprinting

12. Early evidence from isoparental embryos in mice created by manipulation of nuclei post fertilization always lethal gynogenotes (2 egg-derived genomes) have under-developed placenta androgenotes (2 sperm-derived genomes) have over-developed placenta What does this say about the contributions of each parental genome with respect to development? Epigenetics – Genomic imprinting [Van Soom et al. Reprod. Dom Anim 49, suppl3:2]

13. [MacDonald Genetics Res Intl doi:10.1155/2012/585024] Insulin growth factor 2 (Igf2) paternally expressed H19 (a non-coding RNA) maternally expressed Regulated by a cis-acting, differentially methylated region (DMR) also called an imprinting control region (ICR) a Genomic imprinting - Human Igf2-H19

14. [MacDonald Genetics Res Intl doi:10.1155/2012/585024] Paternal DMRs methylated - H19 off, Igf2 on DMR1 is a silencer inactivated by methylation DMR2 is an enhancer that is activated by methylation Maternal DMRs hypo-methylated - H19 on, Igf2 off CTCF binds DMR Downstream enhancer engaged for H19 expression DMR1 silencer of Igf2 active DMR2 enhancer of Igf2 is inactive Genomic imprinting - Human Igf2-H19

15. Practice question In mice, complete loss of Igf2 function is viable, the mutant mice are just much smaller than wild-type mice. If a mouse is heterozygous for a loss-of-function mutation at the Igf2 locus (genotype Igf2 – / + ), will this mouse have a mutant or wild-type phenotype? Explain your answer. If an Igf2 – / + male mouse is mated with a wild-type (Igf2 +/+ ) female mouse, what are the expected frequencies of Igf2 genotypes and resulting phenotypes in the offspring? Explain your answer..

16. Genomic imprinting in mammals Parent-of-origin gene expression imprints must be correctly set in the gametes every generation! [Kinoshita et al. Sem Cell Devel Biol 19:574] Maternal & paternal imprints are erased in primordial germ cells (PGCs) Left, reset for a paternal pattern in sperm Right, reset for maternal pattern in eggs Failure to re-set correctly leads to developmental disorders in next generation meiosis Male embryo sperm imprints meiosis egg imprints Female embryo

17. Genomic imprinting - Arabidopsis endosperm (but not embryo) PHE1 transcription factor master regulator of seed development paternally expressed FWA, FIS2, MEA repressive chromatin re-modeling proteins maternally expressed Differential methylation established in gametophytes Paternal PHE1 on via 3’ methylation Paternal FWA, FIS2, MEA off via 5’ methylation Maternal FWA, FIS2, MEA on - demethylated by DME Maternal PHE1 off - repressed by MEA-FIS2 chromatin remodeling complex [Kinoshita et al. Sem Cell Devel Biol 19:574]

18. Epigenetics – Genomic imprinting Parent-of-origin in gene expression Plants and animals: Genes involved in growth and development Differential methylation (paternal vs maternal) Plants: Occurs in endosperm not embryo Endosperm only (genetic dead-end) does not require “re-setting” each generation Genes are not clustered Animals: Occurs in placenta and embryo Must be set for correct sex every generation Complex loci clustering of genes around Cis-acting imprinting control (IC) regions Clusters include paternally and maternally expressed genes

19. A change in expression of one allele brought about by association with another allele How does this violate Mendel’s law of independent segregation? A paramutagenic allele is able to direct change A paramutable allele is susceptible to change (paramutation) After paramutation, the altered allele also becomes paramutagenic Paramutation is stable through at least one (and sometimes many) subsequent generations A neutral allele is neither paramutable nor paramutagenic Most alleles are neutral! Epigenetics – Paramutation

20. (Chandler and Stam, Nature Rev Genet 5:536) Epigenetics – Paramutation at the B locus of maize How does this progeny differ from Mendelian expectation? B’- paramutagenic allele conditions pale pigment B-I paramutable allele conditions intense purple pigment F1 progeny are all pale! BC1 progeny are all pale!

21. Forward genetics provides answers! Screen for mutants that maintain purple pigment in the presence of B’ modifier of paramutation mop1-1 mutation (A) B‘/B’; Mop1+/mop1-1 (B) B‘/B’; mop1-1/mop1-1 should look like A; instead looks like C (C) B-I/B-I; Mop1+/Mop1+ Positional cloning of the affected gene - mop1 encodes an RNA dependent RNA polymerase What mechanism of paramutation is suggested by this finding? Epigenetics – Paramutation at the B locus in maize (Dorweiler et al. Plant Cell 12:2101; Alleman et al. Nature 42:295)

22. [Arteaga-Vazquez & Chandler Curr Opin Genet Dev 20:156] B-I and B’ are identical in sequence B-I and B’ have 7 tandem repeated copies of an 853 bp sequence motif that is single-copy in neutral alleles This motif is 100kbp upstream from the B promoter! B-I has open chromatin conformation and B’ has closed chromatin conformation in the repeat region Small RNAs transcribed from this repeat mediate change in B-I chromatin conformation from open to closed Important note: In paramutation, the direction of the cross makes no difference! Epigenetics – Paramutation at the B locus of maize

23. [Rassoulzadegan et al. Nature 441:469] Epigenetics – Paramutation at the mouse kit (tyrosine kinase) locus Kit site-specific knock-out mutation by lacZ insertion (Kit tm1Alf/tm1Alf ) is homozygous-lethal Heterozygote Kit tm1Alf/+ has white tail and feet

24. [Rassoulzadegan et al. Nature 441:469] Epigenetics – Paramutation at the mouse kit (tyrosine kinase) locus Kit +/+ x Kit tm1Alf/+ All progeny white-tailed! Southern blot confirms recovery of homozygous (Kit +/+ ) and heterozygous (Kit tm1Alf/+ ) progeny Paramutation has altered expression of the Kit + allele, now designated Kit* + Kit +/+ x Kit tm1Alf/+

25. [Rassoulzadegan et al. Nature 441:469] Kit tm1Alf/+ and Kit * +/ * + : Reduced polyA Kit transcript relative to Kit +/+ Increased aberrant transcripts relative to Kit +/+, including small RNAs Epigenetics – Paramutation at the mouse kit (tyrosine kinase) locus

26. Non-Mendelian Genetics What, is any, is the role of epigenetics in heritable adaptation and evolution? How do germ-line processes influence the trans-generational inheritance of epi-alleles?